Oxygen Affinity of Hemoglobin Regulates O2Consumption, Metabolism, and Physical Activity*

The oxygen affinity of hemoglobin is critical for gas exchange in the lung and O2 delivery in peripheral tissues. In the present study, we generated model mice that carry low affinity hemoglobin with the Titusville mutation in the α-globin gene or Presbyterian mutation in the β-globin gene. The mutant mice showed increased O2 consumption and CO2 production in tissue metabolism, suggesting enhanced O2 delivery by mutant Hbs. The histology of muscle showed a phenotypical conversion from a fast glycolytic to fast oxidative type. Surprisingly, mutant mice spontaneously ran twice as far as controls despite mild anemia. The oxygen affinity of hemoglobin may control the basal level of erythropoiesis, tissue O2 consumption, physical activity, and behavior in mice.

The oxygen affinity of hemoglobin is critical for gas exchange in the lung and O 2 delivery in peripheral tissues. In the present study, we generated model mice that carry low affinity hemoglobin with the Titusville mutation in the ␣-globin gene or Presbyterian mutation in the ␤-globin gene. The mutant mice showed increased O 2 consumption and CO 2 production in tissue metabolism, suggesting enhanced O 2 delivery by mutant Hbs. The histology of muscle showed a phenotypical conversion from a fast glycolytic to fast oxidative type. Surprisingly, mutant mice spontaneously ran twice as far as controls despite mild anemia. The oxygen affinity of hemoglobin may control the basal level of erythropoiesis, tissue O 2 consumption, physical activity, and behavior in mice.
Hemoglobin (Hb), 1 a protein found within erythrocytes, transports oxygen through the vertebrate bloodstream. Hb is a tetrameric protein consisting of ␣and ␤-globin subunits that show a characteristic affinity for oxygen with allosteric effects on various metabolites (1,2). In the literature, more than 1,000 variants of Hb have been reported (3). Some exhibited an altered oxygen affinity, either higher or lower, while maintaining the stability of Hb. Hb Titusville (Hb Titu ) is a low affinity variant of the ␣-globin chain and is well characterized clinically (4,5). Hb Presbyterian (Hb Pres ) is another low affinity variant of ␤-globin chain and is well characterized in vitro (6 -9). Interestingly, individuals with low oxygen affinity Hbs such as Hb Titu or Hb Pres show mild anemia, whereas individuals with high oxygen affinity Hbs such as Hb Malmo or Hb Yakima show symptoms associated with polycythemia (10 -13).
In the present study, we generated mutant mice carrying an homologous mutation with Titusville (Asp ␣94 3 Asn) at the ␣1 locus or with Presbyterian (Asn ␤108 3 Lys) at the ␤-major locus of the mouse genome by a targeted knock-in strategy to generate a murine model of the Titusville and Presbyterian hemoglobinopathies. With the targeted knock-in strategy, the autologous locus control region, as well as the erythropoietin enhancer element, can be kept intact without altering the regulation of endogenous gene expression. Therefore, the knock-in ␣-globin or ␤-globin allele physiologically reacts to stimuli such as hypoxia-inducible factor 1 and erythropoietin. Thus, the model is physiologically relevant and can be used for in vivo physiological analysis of variant Hb. In fact, Titusville heterozygous mice and Presbyterian heterozygous mice both mimic the clinical and laboratory findings of humans with Titusville Hb and Presbyterian Hb, respectively.
We surprisingly found in the present study that Titusville mice, as well as Presbyterian mice, showed enhanced tissue oxygenation, increased O 2 consumption and CO 2 production in tissue metabolism, and an increased running capacity and propensity that resulted in altered behavior with greater physical activity despite mild anemia. Taken together with the human data, the results in the mutant mice implied that Hb determined basic biological parameters such as erythropoiesis, metabolism, physical competence, and behavior.

Generation of Titusville and Presbyterian Mutant
Mice-Hb ␣1 globin gene knock-in mice with the Titusville mutation were obtained by replacing Asp-94 of the ␣1 globin gene with Asn as described below. The 129-mouse genomic library in FIXII (Stratagene, CA) was screened with the 372-bp 5Ј flanking sequence of the murine ␣1 globin gene (nucleotides 1-372; GenBank TM accession number V00714) as a probe. Two overlapping clones covered all exons of the gene. The 1.0-kb fragment containing all ␣1 globin exons was amplified with a SpeI/AflIIanchored primer (5Ј-GGA CTA GTC TTA AGA GAC TCA GGA AGA AAC C-3Ј) and XhoI/AflII-anchored primer (5Ј-CCT CTA GAC TCG  AGC TTA AGG TAG GCA TCC AAT TAT GCT T-3Ј). The SpeI/XhoIrestricted ␣1 globin fragment was mutagenized with a 21-bp mutagenic oligonucleotide (5Ј-GCT GCG TGT GAA TCC CGT CAA-3Ј) using the pALTER system (Promega, Madison, Wisconsin). The introduced mutation, D94N, was confirmed by sequencing. The 2.2-kb short homologous fragment was PCR-amplified with a XhoI-anchored primer (5Ј-CCG CTC GAG TCC TTG AGC CAA AGA AGC CA-3Ј) and ApaI/SalI-anchored primer (5Ј-TTG GGC CCG TCG ACT CTG CCC GCT GGC TGA * This work was supported in part by a research grant for chronic respiratory failure from the Japanese Ministry of Health and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  GCT C-3Ј). The SpeI/XhoI-restricted ␣1 globin fragment and XhoI/ApaIrestricted short 3Ј homologous fragment were sequentially inserted into a SpeI/ApaI-restricted pBSII SK vector. The long 6.8-kb homologous fragment was amplified with a NotI-anchored primer (5Ј-ATT TGC  GGC CGC TGG CAT TCA CAG AGC TCA CCA-3Ј) and SpeI-anchored  primer (5Ј-GGA CTA GTG TCA GAA TCA GAA GTG TCT TGG-3Ј). The NotI/SpeI-restricted 5Ј long homologous fragment was inserted into the targeting construct. The 1.3-kb MC1neo cassette flanked by loxp sequences was PCR-amplified from pMC1neo-loxp vector (14) with a SalI-anchored primer (5Ј-CGC GTC GAC ATA ACT TCG TAT AAT G-3Ј) and SalI/EcoRI-anchored primer (5Ј-CGC GTC GAC GAA TTC ATC GAT ACC GGC GAC ATA-3Ј). The SalI-restricted MC1neo-loxp cassette was inserted into the XhoI-restricted targeting construct. The resulting construct containing the short and long homologous fragments, the mutagenized ␣1 globin gene, and the neomycin gene was restricted with NotI/SalI and recloned into the targeting vector, pMC1DT-A (B) (Oriental Koubo). The vector was then linearized with NotI and used for the electroporation of embryonic stem cells. Genomic DNA from each of 240 G418-resistant embryonic stem clones was digested with EcoRI and screened for the homologous recombination by Southern blot analysis using an 800-bp 3Ј probe. One embryonic stem clone with the expected homologous recombination was used for generating chimeric mice by the aggregation method as described (15). The chimeric mice were cross-bred with C57BL/6CrSlc (Nihon SLC), and the germline transmission was confirmed by PCR amplification with the primers p1 (5Ј-TTC CTT GCC TCT GTG AGC-3Ј) and p2 (5Ј-TGG GAC CGA GCC ATC TTC-3Ј) in agouti offspring.
Hb ␤-globin gene knock-in mice with the Presbyterian mutation were obtained by replacing Asn-108 of the ␤-globin gene with Lys as described previously (16). The chimeric mice were cross-bred with C57BL/ 6CrSlc, and the germline transmission was confirmed by PCR amplification with the primers p3 (5Ј-ACC CAG CGG TAC TTT GAT AGC-3Ј) and p4 (5Ј-GCT ACT GAA GCT GTC TAA GGC AAC AGG-3Ј) in agouti offspring.
Biochemical Analyses of Mutant Hb-Blood samples from mice and humans were collected into EDTA-treated tubes. Erythrocytes were washed in cold 0.85% NaCl, collected by centrifugation, and lysed in 4 volumes of distilled water. The hemolysate was then collected by centrifugation at 15,000 rpm for 30 min. The hemoglobin concentration was determined with a Wako hemoglobin test (Wako Chemicals). The hemolysate was then separated by reversed-phase high performance liquid chromatography (RP-HPLC) using a Develosil ODS 300C4-HG-5 column (4.6 ϫ 150 mm; Nomura Chemicals). Globin peaks were eluted at a flow rate of 1 ml/min with a liner gradient of 36 -52% acetonitrile (0.4%/min) in 0.1% trifluoroacetic acid as described previously (16,17). The absorbance was monitored at 214 nm.
Physiochemical Analysis of Mutant Hb-Oxygen equilibrium studies of washed erythrocytes and Hb solution were carried out using a Hemox analyzer (TCS Products, Southampton, PA) at 37°C. For examination of the Bohr effect, hemolysates were concentrated with Ultrafree 30,000 (Millipore) and dialyzed in the same 50 mM HEPES buffer, containing 100 mM NaCl, at pH 7.0, 7.4, and 7.8. For examination of the chloride effect, concentrated hemolysates prepared as described above were dialyzed in 50 mM HEPES, pH 7.4, containing a chloride ion concentration of 0, 50, 150, and 500 mM NaCl.
Analysis of Tissue O 2 (PtiO 2 )-Male wild-type (n ϭ 8), and Presbyterian (n ϭ 9) mice (12 weeks old) were anesthetized with ether for setting in a double-chamber plethysmograph and inserting an O 2 electrode with a thermocouple (Clark-type electrode; Unique Medical) to measure PtiO 2 . The O 2 electrode was inserted through a guide tube into the left gastrocnemius muscle. The output from the electrode was adjusted to the muscle temperature and continuously displayed on a digital monitor (POG-203; Unique Medical, Japan), being recorded on an analogue tape recorder. The electrode was calibrated with room air before and after each experiment.
Respiratory variables were also measured with a double-chamber plethysmograph as described previously (18). Respiratory frequency (f; breaths/min) was determined as 60/total breath duration. Tidal volume (VT) was calculated using the equation VT ϭ (273 ϩ Tb)/(273 ϩ Tam) ϫ (760 Ϫ PamH 2 O)/(760 Ϫ PbH 2 O) ϫ 0.5/Vcal ϫ VT ATPS , where Tb is rectal temperature (°C); Tam is ambient temperature (°C); PamH 2 O and PbH 2 O are the water vapor pressures (mmHg) in the ambient air and the alveoli, respectively; and VT ATPS is VT at ambient temperature, pressure, saturated (ATPS) without calibration (ml). The volume injected into the head chamber was 0.5 (ml ATPS) for calibration, recorded as Vcal (ml ATPS) on the personal computer. Minute lung ventilation (V ខ E; ml body temperature, pressure, saturated) was determined as f ϫ ខVT and normalized with respect to body weight per 10 g.
Each mouse was allowed to acclimate to the chambers (fraction of inspired O 2 ; FIO 2 ϭ 0.21) for at least 60 min before the hypoxic gas challenge, and a constant level of baseline PtiO 2 was achieved. Subsequently mice were exposed to a hypoxic gas (FIO 2 ϭ 0.15) for 5 min. A gas mixture was delivered from a respiratory gas circuit consisting of flow meters for O 2 and N 2 and a reservoir bottle (2 liters) connected to the head chamber. FIO 2 was altered by mixing O 2 and N 2 , being continuously monitored by withdrawing a small fraction of the gas mixture (20 ml/min) with an O 2 and CO 2 analyzer (Respina 1H26; NEC San-ei). V ខ E and PtiO 2 were measured at 0, 0.5, 1, 2, 3, 4, and 5 min. ⌬PtiO 2 (mmHg) was calculated as the difference between baseline PtiO 2 and PtiO 2 at each time point.
Blood Gas Analysis-Male wild-type (n ϭ 5), Titusville (n ϭ 5), and Presbyterian (n ϭ 5) mice (12 weeks old) were used. An arterial catheter (BC-1P; Access Technology) was implanted in the left carotid artery under anesthesia (sodium pentobarbital; 25 mg/kg, intraperitoneally) for blood gas analysis. Mice were placed in the plethysmograph for 2 h to recover from the anesthesia and subjected to a hypoxic challenge comparable with that used to obtain V ខ E and PtiO 2 . Arterial blood (120 l) was sampled with a heparinized sampling glass tube (MC0020; AVL Scientific Corporation) and immediately analyzed by a blood gas analyzer (OPTI CCA; AVL Scientific Corporation) for pH, partial pressure of arterial CO 2 (PaCO 2 ), and partial pressure of arterial O 2 (PaO 2 ). Arterial blood was sampled before and at the end of hypoxic gas inhalation.
Metabolism-O 2 consumption (ml standard temperature, pressure, dry), CO 2 production (ml standard temperature, pressure, dry), and respiratory exchange rate were measured during normoxia and hypoxia with an open circuit system (ARCO-1000; ARCO Systems) in male wild-type (n ϭ 4), Titusville (n ϭ 5), and Presbyterian (n ϭ 5) mice. Each mouse was set in a chamber where a steady flow of air was delivered continuously by a vacuum pump for the assessment. The system measured the fractions of O 2 , CO 2 , and N 2 in the in-flow and out-flow of the chamber with a mass spectrometer and the flow rate with a pneumotachograph. The mouse was placed in the chamber for 60 min to acclimatize to the surroundings before the experiments. The metabolic factors were measured under normoxic conditions and then the hypoxic gas (FIO 2 ϭ 0.15) was delivered from the respiratory gas circuit to the metabolic chamber. Measurements were made during a 5-min steady state period 20 min after the onset of hypoxic gas exposure. O 2 consumption and CO 2 production were normalized with respect to body weight per kg.
Muscle Fiber Type Classification and Succinate Dehydrogenase (SDH) Activity-The right tibialis anterior muscle was removed under sodium pentobarbital anesthesia (50 mg/kg body weight, intraperitoneally). The muscle was placed on cork, stretched to its in vivo length, and quickly frozen in isopentane cooled with liquid nitrogen. Serial transverse sections, 10 m thick, of the mid-belly of the muscle, were cut in a cryostat set at Ϫ20°C. The sections were brought to room temperature and air-dried for 30 min. The sections were stained for ATPase activity following acid (pH 4.5) preincubation for fiber typing. The muscle fibers were classified as type IIA and type IIB (19). SDH activities were used for comparisons among fibers of different types (19). The cross-sectional areas and SDH activities of ϳ50 fibers from each of a deep (close to the bone), middle (between the deep and superficial), and superficial (near the surface of the muscle) regions of the muscle were determined using a computer-assisted image processing system. These regions were selected for analysis, because the tibialis anterior muscle shows an increasing gradient of fibers having high oxidative enzymatic activity proceeding from the superficial to the deep aspect of the muscle. The sections were digitized as gray scale images and quantified as one of 256 gray levels (20). A gray level value of 0 was equivalent to a 100% transmission of light whereas that of 255 was equivalent to 0% transmission. The mean optical density value within a fiber was determined using a calibration tablet that has 21 steps of gradient density ranges and corresponding diffused density values.
Loaded Running-wheel Protocol-Wild-type (n ϭ 5), Titusville (n ϭ 5), and Presbyterian (n ϭ 5) male mice (15 weeks old) were voluntarily exercised for 28 days using a running wheel apparatus in which distance can be monitored electronically (21). This apparatus includes a standard plastic cage (20.0 ϫ 30.0 ϫ 12.0 cm) and a running wheel (width 5.0 cm, diameter 25.5 cm) attached vertically to a freely rotating shaft inserted into a metal controller box that is supported on a metal base. The running wheel rotates on the shaft whenever the mouse walks or runs in either direction, and the number of revolutions of the running wheel is recorded continuously.
Human Study-Two females, 31 and 29 years old, who were nonsmokers and healthy, were analyzed in this study. Genomic DNA anal-ysis was approved by the ethical committee of Tokyo Metropolitan Institute of Gerontology, and written informed consent was obtained. Genomic DNAs were prepared from whole blood by GenTLE (Takara, Kyoto, Japan) for PCR. 1,118-bp fragments were amplified using a sense primer (5Ј-ACC CAG AGG TTC TTT GAG TC-3Ј) and an antisense primer (5Ј-TCT GAT AGG CAG CCT GCA CT-3Ј). The PCR products were isolated and sequenced with a nested sense primer (5Ј-CTG GGT TAA GGC AAT AGC-3Ј) by the dye terminator method. Arterial blood gas analysis was carried out with a pH-blood gas analyzer (Bayer medical 860).

Generation of Mutant Mice Expressing Mutant Hb with Altered Oxygen
Affinity-To generate mutant mice with a greater capacity to deliver O 2 , we first searched for mutant Hbs with altered oxygen affinity in the medical literature. We found that individuals with a variant Hb of higher affinity such as Yakima Hb and Malmo Hb usually manifested polycythemia (10 -13) whereas individuals with a variant Hb of lower affinity such as Kansas Hb, Titusville Hb, or Presbyterian Hb showed mild asymptomatic anemia without any medical complications (4 -9, 22-24). These medical profiles prompted us to explore the possibility that the variant Hbs with lower affinity improve O 2 delivery to the peripheral tissues in the physiological state. To test this hypothesis, we generated two distinct models, Titusville Hb mice and Presbyterian Hb mice. Titusville Hb is composed of a variant ␣ chain with Asn-94, an amino acid substitution in the ␣/␤ interfaces, whereas Presbyterian Hb is composed of a variant ␤ chain with Lys-108 protruding into the central cavity of the Hb molecule. These two hemoglobinopathies thus have distinct mechanisms for altering the affinity of Hb for oxygen but a common clinical phenotype such as anemia, suggesting that the lowered affinity of Hb generally enhances, whereas the raised affinity generally suppresses, O 2 delivery in the peripheral tissues. In addition, Presbyterian Hb confers a novel allosteric effect with the variant Lys residue interacting with the Cl Ϫ ion in the central cavity, but Titusville Hb showed no allosteric effect. Therefore, by means of these two models, we can devise multiple strategies to enhance O 2 delivery either by manipulating ␣-globin and ␤-globin or by using a novel allosteric effect.
As schematized in Fig. 1, the homologous recombination in the mouse ␣-globin or ␤-globin genome with target vectors replaced the ␣1 exon or ␤ major exon with a modified ␣1 carrying Asn-94 (Fig. 1A) or ␤ major carrying Lys-108 (Fig. 1B), respectively. The intercrossing of heterozygous mice successfully generated fertile homozygous mice. Southern blot analyses (data not shown) and PCR amplification of ␣-globin or ␤-globin genomes (Fig. 1, E and F) confirmed the expected homologous recombination. The homozygous and heterozygous mice were born viable, grew normally, and were fertile. Sequence analyses of PCR products from homozygous mice further confirmed the expected ␣D94N mutation in Titusville mice (Fig. 1C) and ␤N108K mutation in Presbyterian mice (Fig. 1D). To confirm whether the knock-in allele productively expressed the mutated ␣or ␤-chain, we biochemically characterized the hemoglobin prepared from mutant mice. To separate ␣and ␤-globin chains, purified hemoglobins were applied to an RP-HPLC column under acidic conditions. HPLC profiles of Hb prepared from Titusville mice or Presbyterian mice showed double peaks for the ␣ chain or ␤ chain (Fig. 1G, middle and  lower). Based on the profiles, we estimated that ϳ15% of the Hb in the peripheral blood of Titusville mice consists of Hb Titu whereas ϳ30% of that in Presbyterian mice consists of Hb Pres . The medical literature on individuals with mutant hemoglobinopathies revealed the expression level of Hb Titu to be 34.7% (4) and Hb Pres to be 29.9 -41.7% (6 -9) in human cases. We therefore characterized heterozygous Titusville mice and heterozygous Presbyterian mice in this study as animal models for variant hemoglobinopathies with lower oxygen affinity. In the peripheral blood, red blood cells of Titusville mice showed normal hemograms whereas Presbyterian mice showed mild anemia (Hb 14.8 Ϯ 0.8 versus 12.9 Ϯ 1.1 g/dl, p Ͻ 0.05; see Table  I) without signs of hemolysis (reticulocytes 1.7 Ϯ 0.2% versus 1.6 Ϯ 0.7%; see Table I), suggesting that these model mice mimic the human cases well (4 -9). . The peaks of ␣ Wt -globin, ␣ Titu -globin, ␤ Wt -globin, and ␤ Pres -globin are indicated in HPLC profiles. The peak of ␣ Titu -globin was eluted earlier than the peak of ␣ Wt -globin (an arrow in the middle panel). The peak of ␤ Pres -globin was also eluted earlier than the peak of ␤ Wt -globin (an arrow in the lower panel).
The Mutant Hb Showed Low Affinity for Oxygen in Vitro-To characterize the physiochemical properties of Hb Titu , Hb Pres , and double mutant Hb Titu, Pres , we assessed the oxygen dissociation of red blood cells prepared from Titusville mice, Presbyterian mice, and Titusville/Presbyterian double mutant mice. In oxygen dissociation plots, Hb Pres showed a rightward shift in comparison with wild-type Hb ( Fig. 2A, P 50 ϭ 43.5 versus 47.0 mmHg) whereas Hb Titu and Hb Titu, Pres exhibited even more extensive rightward shifts as shown in Fig. 2 (P 50 ϭ 66.0 or 72.0, respectively). Analyses of Hill's plot and the Bohr effect, however, indicated that Hb Pres retained all physiochemical properties (Fig. 2, B and C) whereas Hb Titu or Hb Titu, Pres showed a reduced Hill's coefficient, suggesting that the Titusville, but not Presbyterian, mutation conferred the reduced incorporation of Hb as reported previously in human cases (4,25). As for de novo allosteric effects, we investigated the influence of Cl Ϫ . Interestingly, Cl Ϫ stabilized the deoxy state of Hb Pres in a dose-dependent manner, suggesting that the introduced Lys residue protrudes into the central cavity to bind to Cl Ϫ ion as suggested in the previous model (Fig. 2D).
Hb Pres Delivers More Oxygen to Peripheral Tissues under Moderately Hypoxic Conditions-Presbyterian mice were exposed to 15% O 2 for 5 min to investigate the physiological effects of Hb Pres on tissue hypoxia in vivo. ⌬PtiO 2 and Ve values during hypoxia are shown in Fig. 3, A and B, respectively. After 5 min of hypoxia, the tissue O 2 of Presbyterian mice was significantly retained and sustained a higher level than in wild mice (p Ͻ 0.05, a two-way analysis of variance for repeated measures). In the course of hypoxia, Presbyterian mice showed a similar decline in tissue O 2 to wild-type mice within 1 min of hypoxia whereas tissue O 2 started to dissociate in the hypoxic phase that followed (Fig. 3A). The result suggested that more oxygen is delivered to the tissues in Presbyterian than wildtype mice over a certain range of hypoxic conditions. In fact the benefit of changes in the affinity of Hb Pres in vitro is greatest at a PaO 2 concentration of ϳ50 mmHg as shown in Fig. 2A. Therefore, it is reasonable that the beneficial effect of Hb Pres is more remarkable in vivo in advanced tissue hypoxia as shown in Fig. 3A.
We simultaneously monitored Ve to elucidate whether the increased tissue oxygenation was attributable to the increased ventilation of Presbyterian mice or an efficient O 2 delivery by the mutant Hb. The results revealed that Presbyterian mice had a consistent depressed ventilation before and during hypoxia, although they showed a similar pattern of ventilatory responses to wild-type mice, such as the initial hypoxic response and subsequent depression (Fig. 3B). The data clearly suggested that in Presbyterian mice, more oxygen was delivered to tissues not by a ventilatory increase but by increased O 2 delivery by Hb Pres .
Presbyterian Mice Showed an Altered Set Point of the Acid-Base Balance-To investigate whether mutant Hbs influence the acid-base balance, we measured the pH, PaCO 2 , and PaO 2 level of arterial blood in Titusville and Presbyterian mice (Table II). Titusville mice showed a normal PaO 2 , PaCO 2 , and pH in room air and during hypoxia whereas Presbyterian mice showed a low pH associated with an elevated PaCO 2 both in room air and under hypoxic conditions (Table II). The results may simply indicate that Presbyterian mice developed chronic respiratory acidosis because of hypoventilation. However, such a pathophysiological explanation is unlikely because, (i) the acidosis is not compensated by metabolic alkalosis (Table II), (ii) Presbyterian mice showed no lung disease causing alveolar hypoventilation, and (iii) they showed a normal PaO 2 level (Table II). Given the normal respiratory functions and reduced ventilation (Ve) (Fig. 3B), the primary cause of elevated PaCO 2 levels in Presbyterian mice may be attributable to central hypoventilation. In this context, we speculate that Presbyterian mice set their acid-base balance to a lower pH and higher PaCO 2 by reducing the ventilation. It is still surprising that Presbyterian mice consumed more O 2 and produced more CO 2 in room air, as well as during hypoxia (see below), despite the fact that they manifest signs of hypoventilation (Fig. 3B) and mild anemia (Table I). Given the fact that a human with Presbyterian Hb also showed a lower pH and higher PaCO 2 level on exercising (see Fig. 6D) and that an acid-base imbalance was not observed in Titusville mice, this imbalance may be a Presbyterian-specific phenotype associated with the ␤108Lys residue. Because primary genetic mutations theoretically confer increased oxygen delivery in peripheral tissues, one explanation for these abnormalities is that Presbyterian mice compensate for tissue hyperoxia caused by Hb Pres by reducing ventilation. Alternatively, Hb Pres may modulate the respiratory center, especially the ventilatory response to CO 2 in the brain of mutant mice, in a Presbyterian-specific manner. Titusville and Presbyterian Mice Consume More O 2 and Produce More CO 2 -To investigate whether enhanced tissue oxygenation alters the basic metabolism of mutant mice, we measured metabolic parameters such as O 2 consumption, CO 2 production, and the respiratory ratio in Titusville and Presbyterian mice (Table III). The metabolic analyses showed that both mutant mice consumed more O 2 and produced more CO 2 in room air and hypoxic conditions (Table III), implying that the low affinity of mutant Hbs drives the mice to consume more O 2 to exclude the excess tissue O 2 delivered by mutant Hbs. The increase in O 2 consumption may then lead to the increased production of CO 2 in the tissue, albeit the respiratory ratio being slightly higher in room air in both mutant mice. This is the first report, to our knowledge, that Hb regulates the basic metabolism of the body by regulating the tissue oxygenation.
Muscle Fiber Distribution and Mitochondrial SDH Activity Were Altered in Titusville and Presbyterian Mice-To clarify whether a relationship exists between increased O 2 delivery by Hb Pres and skeletal muscle properties, we determined the fiber type distributions, fiber cross-sectional areas, and fiber mitochondrial SDH activities in tibialis anterior muscle of Presbyterian mice and compared the results with those for wild-type mice. In cross-sections from deep, middle, and superficial regions of the tibialis anterior muscle, Titusville and Presbyterian mice showed no fiber hypertrophy or atrophy, regardless of the muscle region (data not shown). However, on histochemical staining for ATPase activity, both mice showed a higher percentage of type IIA fibers and a lower percentage of type IIB fibers in deep regions of the muscle compared with wild-type mice (Fig. 4, A and D). The fiber type distribution analyzed in the present study indicated that the tibialis anterior muscle of Titusville mice was composed of 51.4% type IIA and 48.6% type IIB fibers compared with 39.5 and 60.5%, respectively, in the wild-type (Fig. 4B), whereas Presbyterian mice contained 49.8% type IIA and 50.2% type IIB fibers compared with 41.0 and 58.9% (Fig. 4E). The results indicate that Titusville and Presbyterian mice have a higher ratio of type IIA/IIB fibers than do wild-type mice. Interestingly, a higher ratio favors oxidative energy metabolism in skeletal muscles, supporting the idea that Titusville mice, as well as Presbyterian mice, genetically alternate energy expenditure to favor the high oxidative type of metabolism in muscle. To confirm this hypothesis, we analyzed fiber mitochondrial SDH activity in the same sections (Fig. 4, C and F). Surprisingly, SDH activities in both type IIA and type IIB fibers were greater in deep regions of the tibialis anterior compared with those of wild-type mice. Type IIB fibers are characterized as being fast contracting, high glycolytic in their enzymatic activity, and easily fatiguable. Because this type of fiber is only supposed to increase SDH activity with physical exercise, it is worth speculating that the genetic alteration in Hb Titu and Hb Pres also converts the propensity of type IIB fibers favoring glycolytic ATP production over oxidative phosphorylation by increasing SDH activity in mitochondria.

Titusville and Presbyterian Mice Spontaneously Run ϳ2
Times Further on a Running-wheel Apparatus-To clarify whether mutant Hbs influence behavior such as spontaneous physical activity, i.e. running, we monitored the running distances of Titusville and Presbyterian mice during a 28-day exercise period. Surprisingly, both mutant mice ran more than twice as far as wild-type mice (Fig. 5). In the initial training phase of the exercise, both wild-type and mutant mice extended their running distances, but the increase was more remarkable in the Titusville and Presbyterian mice (day 0 -14 in Fig. 5). In the second phase of exercise, Titusville mice showed a steady state of daily running with an ϳ2.5 times longer distance than wild-type mice whereas Presbyterian mice showed an ϳ2 times longer distance. Mean running distances of Titusville and Presbyterian mice versus wild-type mice were 9539 versus 4613 m day Ϫ1 and 7580 versus 3732 m day Ϫ1 , respectively. These results strongly suggested that Titusville and Presbyterian mice have a propensity to run spontaneously with or without a running apparatus. The results also showed an enhanced steady state capacity for running in mutant mice. Taken together with the histochemical findings, Titusville and Presbyterian mice consume more O 2 in skeletal muscles by oxidative phosphorylation. It is therefore speculated that the excessive ATP produced by oxidative phosphorylation with increased SDH activity in mitochondria of mutant mice is consumed by spontaneous running.
A Human with Presbyterian Hb Showed an Altered Ventilatory Response to CO 2 during Exercise-A 29-year old female (case 2 in Fig. 6, A and B) inherited the Presbyterian mutation, A for C at nucleotide 1,357 of human ␤-globin exon 3 (case 2 in Fig. 6, A and B), from her father and grandmother whereas her 31-year-old sister did not (case 1 in Fig. 6, A and B). The mutation was also confirmed in a chromatographic study in which Presbyterian ␤-globin (␤ Pres -globin) was specifically detected in the hemolysate from case 2 but not detected in case 1 (Fig. 6C). This mutant peak in HPLC was detected in the hemolysate from the subject's father who carries Hb Pres (data FIG. 2. Physiological properties of mutant Hb. A, oxygen dissociation curves of red blood cells from wild-type, Titusville, Presbyterian, and double heterozygous Titusville/Presbyterian mice. Mutant red blood cells showed a rightward shift in the oxygen dissociation curve. B, Hill's plots of wild-type, Titusville, Presbyterian, and double heterozygous Titusville/Presbyterian red blood cells. Hill's coefficient was conserved in Presbyterian mice but not in Titusville mice or heterozygous Titusville/Presbyterian mice. C, Bohr effect of hemolysates from Presbyterian mice and wild-type mice. The Bohr effect was also conserved in Presbyterian mice. D, the effect of Cl Ϫ concentration on oxygen dissociation in hemolysates from Presbyterian mice and wild-type mice. An enhanced dose-dependent Cl Ϫ effect was seen in Presbyterian mice. FIG. 3. Tissue O 2 in Presbyterian mice was a significantly higher level than that in wild-type mice during mild hypoxia. A, tissue O 2 (⌬PtiO 2 ) response to hypoxia in wild-type (filled circle; n ϭ 8) and Presbyterian mice (filled triangle; n ϭ 9). B, changes in ventilation (V ខ E) to hypoxia. Data are the mean Ϯ S.E. and were analyzed by a two-way analysis of variance for repeated measures. Statistical interaction between time and group is shown. *, p Ͻ 0.05. not shown). Metabolism and respiration were then assessed in these sisters with a bicycle ergometer. During the exercise, the Presbyterian individual showed a depressed ventilatory response with a respiratory ratio below 1.0 throughout the test whereas the control sister showed a normal ventilatory response (data not shown). Blood gas analysis on moderate exercise (100 watts) showed a severe respiratory acidosis in the younger sister, suggesting that the Presbyterian individual failed to compensate for the metabolic acidosis by inducing ventilation; instead, the depressed ventilation exacerbated the metabolic acidosis (Fig. 6D). The dysregulation of ventilatory response on exercise observed in the Presbyterian individual is consistent with the impaired acid-base balance observed in Presbyterian mice (Table II), suggesting a common mechanism

Titusville and Presbyterian Mice May be Gain-of-function Mutations in Mouse and
Human-Individuals who carry variant Hbs with low O 2 affinity such as Hb Titusville, Hb Presbyterian, and Hb Kansas (4 -9, 22-24) manifest asymptomatic anemia, irrespective of the mutations, whereas individuals who carry Hbs with high O 2 affinity such as Hb Malmo (10,13) and Hb Yakina (11,12) generally show polycythemia. It is therefore speculated that Hbs with low oxygen affinity can dissociate more O 2 in the peripheral tissues whereas the other variant Hbs proceed with normal gas exchange in the lung. To test this hypothesis, in the present study, we generated mice carrying mutant Hbs with low O 2 affinity, Hb Titusville as a mutant of ␣-globin, and Hb Presbyterian as a mutant of ␤-globin. Using these models, we addressed whether low affinity Hb actually releases more O 2 in the tissue in vivo and investigated the various physiological advantages attributed to mutant Hbs in vivo. We surprisingly found that Titusville mice, as well as Presbyterian mice, showed enhanced tissue oxygenation, increased O 2 consumption in tissues, and an increased running capacity and propensity that resulted in altered behavior with greater physical activities.
From an evolutional point, the primary structure of Hb is closely associated with the life and behavior of animals. For example, crocodiles and alligators can hold their breath under water for 30 min, because their Hb has an allosteric effect on bicarbonates produced in the tissues (26). Thus, it is intriguing that Titusville and Presbyterian mutations enable mice to run twice as long as wild-type mice. Because running is a vital form of mouse behavior, the increased running ability of mutant mice is obviously a gain-of-function phenotype in the context of animal evolution. It is also noteworthy that this phenotype may be conserved in mouse and human, although they only share 80% amino acid sequence homology in the ␤-globin locus. Because neither of these gain-of-function mutations (␣94Asp 3 Asn and ␤108 Asn 3 Lys) has been accumulated in the genome of mouse or human as a dominant allele, an as-yet unidentified deleterious effect may exist that prevents the mutation from prevailing in the genome.  FIG. 6. Characterization of a patient with Presbyterian Hb. A, a pedigree of Presbyterian Hb. B, the mutation (N108K) in the ␤-globin gene was confirmed by DNA sequencing in case 2. C, RP-HPLC profiles of hemolysate prepared from a normal individual (top), case 1 (middle), and case 2 (bottom). The peaks of ␤ Pres -globin, ␤ Wt -globin, and ␣-globin are indicated in the profiles. The peak of human ␤ Pres -globin was eluted earlier than the peak of ␤ Wt -globin as shown in the profile of Presbyterian mice (Fig. 1G). D, blood gas analyses for case 2 in a graded exercise test.
and Increased Spontaneous Exercise-This is the first report that Hb determines or controls the basal level of erythropoiesis, tissue O 2 consumption, physical activity, and behavior. Although we could not explain all abnormal phenotypes of Titusville and Presbyterian mice at the molecular level, it is obvious that the initial event is an introduced mutation that modulates the affinity of Hb for O 2 as shown in Fig. 7. In Titusville mice, the introduced Asn residue locates at the interface of the ␣1␤2 subunit as shown in Fig. 7A, causing the subunit to be stabilized in a deoxy state. In Presbyterian mice, however, the introduced Lysine residue protrudes into the central cavity to bind metabolites such as a phosphate or a chloride ion as illustrated in Fig. 7B, generating a novel allosteric effect that favors the deoxy state. Tissue oxygenation, i.e. the supply of oxygen to tissues, is an essential biological reaction on which every animal cell, tissue, and organ is energetically based. Therefore, tissue hypoxia, the lack of oxygen, is the most dangerous insult for an animal and has been investigated extensively in laboratory animals and in vitro studies. Tissue hyperoxia, however, has yet to be studied extensively, because no relevant animal model has been available. We presented here the first relevant animal model for the study of tissue hyperoxia.
The primary function of mitochondria is ATP production in the use of oxygen. In this context, the cell depends on mitochondria to generate energy, but at the same time, mitochondria play a biological role in the reduction of oxygen inside the cell. It is thus important to control the redox state in various organelles including mitochondria, because disruption of the cellular redox state can often result in apoptosis in animal cells (27). From this viewpoint, another important function of mitochondria is to regulate the cellular oxygen concentration by producing ATP or heat (28). It is then interesting that SDH activity is up-regulated in both IIA and IIB type fibers of Titusville and Presbyterian mice, suggesting that the primary sequence of alteration in muscle may be the compensatory reaction for the increased consumption of excess oxygen delivered by mutant Hbs.
It is difficult to judge whether the mutant mice run twice as far to consume more oxygen in the muscle or voluntarily as a result of altered behavior. Because the Titusville and Presbyterian mutations may influence the development of the brain after birth, the propensity to run spontaneously may be attributed to the altered behavioral pattern caused by the mutations. Alternatively, an unidentified signal sensing the cellular redox state, tissue oxygenating state, or hyperoxic state in the peripheral tissues may trigger the central nervous system to partake more actively in running than is the case for wild-type mice.
It is also difficult to clarify the molecular mechanisms downregulating the ventilation in Presbyterian mice. Hypoxia positively drives the ventilation by neuronal signaling via the carotid body (29), whereas hyperoxia may negatively regulate the respiratory center in the central nervous system. Interestingly, the individual with Presbyterian Hb (case 2 in Fig. 6) showed an impaired hyperoxic suppression by the carotid body, 2 indicating the impaired regulatory mechanism in Presbyterian mice. It is also noteworthy that down-regulation of erythropoiesis is one strategy to compensate for tissue hyperoxia in Pres- byterian mice. Because the amount of hemoglobin contained in the peripheral blood directly correlates with the efficiency of O 2 transport in tissues, one of the determinants of the hemoglobin concentration may be O 2 delivered in the tissues as suggested in this study.
Perspective of Clinical Applications of Presbyterian Hb-Recombinant human Presbyterian Hb has been developed as a blood substitute (25,30). In the present study, we investigated the physiological advantages of Titusville Hb or Presbyterian Hb in vivo, demonstrating that in these mutant mice more oxygen is released under hypoxic conditions. Patients with chronic respiratory failure because of lung diseases show tissue hypoxia. However, O 2 therapy largely restricts a patient's daily life. Titusville Hb or Presbyterian Hb can release more oxygen in the peripheral tissues under hypoxic conditions, suggesting that recombinant Hbs or erythrocytes containing mutant Hbs could improve the symptoms of chronic respiratory failure when transfused or introduced by gene therapy.
Moreover, mutant Hb releases more oxygen in anemic conditions, suggesting that recombinant Hb would also benefit ischemic heart diseases or ischemic cerebrovascular disorders. A synthetic allosteric modifier such as RSR13 that induces a rightward shift in hemoglobin improved cardiac metabolism under ischemic cardiac conditions in experimental animals (31). A synthetic chemical is versatile in clinical situations, in which the temporal supply of oxygen is emergently indicated. Because the allosteric effectors of tissue metabolites such as 2,3-diphosphoglycerate were often increased in ischemic tissues, a variant Hb with a novel allosteric effect such as Presbyterian Hb may be more advantageous for chronic ischemic conditions especially associated with impaired respiratory functions. Further animal experiments should be explored to determine the clinical applications of Titusville and Presbyterian mice.