From the Department of Molecular Gerontology, Tokyo
Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, ¶ Department of Physiology, Showa University, Shinagawa-ku, Tokyo
142-8555, Departments of
Chest Medicine and
¶¶ Molecular Embryology, Graduate School of Medicine, Chiba
University, Chiba 260-8670, ** Laboratory of Neurochemistry,
Faculty of Integrated Human Studies, Kyoto University, Kyoto 606-8501,
Department of Polymer Chemistry, Waseda
University, Shinjuku-ku, Tokyo 169-8555, §§ Department of Internal Medicine, Iwate
Medical University, Morioka 020-8505, and
Structural
Genomics Group, Japan Biological Information Research Center,
Koto-ku, Tokyo 135-0064, Japan
Received for publication, October 30, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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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 Hemoglobin (Hb),1 a protein found within
erythrocytes, transports oxygen through the vertebrate bloodstream. Hb
is a tetrameric protein consisting of In the present study, we generated mutant mice carrying an homologous
mutation with Titusville (Asp We surprisingly found in the present study that Titusville mice, as
well as Presbyterian mice, showed enhanced tissue oxygenation, increased O2 consumption and CO2 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
Hb 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 × 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 O2 (PtiO2)--
Male
wild-type (n = 8), and Presbyterian (n = 9) mice (12 weeks old) were anesthetized with ether for setting in a
double-chamber plethysmograph and inserting an O2 electrode
with a thermocouple (Clark-type electrode; Unique Medical) to
measure PtiO2. The O2 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 = (273 + Tb)/(273 + Tam) × (760
Each mouse was allowed to acclimate to the chambers (fraction of
inspired O2; FIO2 = 0.21) for at least 60 min
before the hypoxic gas challenge, and a constant level of baseline
PtiO2 was achieved. Subsequently mice were exposed to a
hypoxic gas (FIO2 = 0.15) for 5 min. A gas mixture was
delivered from a respiratory gas circuit consisting of flow meters for
O2 and N2 and a reservoir bottle (2 liters)
connected to the head chamber. FIO2 was altered by mixing
O2 and N2, being continuously monitored by
withdrawing a small fraction of the gas mixture (20 ml/min) with an
O2 and CO2 analyzer (Respina 1H26; NEC
San-ei). Blood Gas Analysis--
Male wild-type (n = 5),
Titusville (n = 5), and Presbyterian (n = 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 Metabolism--
O2 consumption (ml standard
temperature, pressure, dry), CO2 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 = 4), Titusville (n = 5), and Presbyterian
(n = 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 O2,
CO2, and N2 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 (FIO2 = 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.
O2 consumption and CO2 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 µm thick, of the
mid-belly of the muscle, were cut in a cryostat set at Loaded Running-wheel Protocol--
Wild-type (n = 5), Titusville (n = 5), and Presbyterian
(n = 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 × 30.0 × 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 non-smokers and healthy, were analyzed in this study. Genomic DNA
analysis 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
(5'-ACC CAG AGG TTC TTT GAG TC-3') and an antisense primer (5'-TCT GAT
AGG CAG CCT GCA CT-3'). The PCR products were isolated and sequenced
with a nested sense primer (5'-CTG GGT TAA GGC AAT AGC-3') 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 O2, 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 O2 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
As schematized in Fig. 1, the homologous
recombination in the mouse The Mutant Hb Showed Low Affinity for Oxygen in Vitro--
To
characterize the physiochemical properties of HbTitu,
HbPres, and double mutant HbTitu, 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, HbPres showed a
rightward shift in comparison with wild-type Hb (Fig. 2A, P50 = 43.5 versus 47.0 mmHg) whereas HbTitu and
HbTitu, Pres exhibited even more extensive rightward shifts
as shown in Fig. 2 (P50 = 66.0 or 72.0, respectively). Analyses of Hill's plot and the Bohr effect, however,
indicated that HbPres retained all physiochemical
properties (Fig. 2, B and C) whereas HbTitu or HbTitu, 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 HbPres Delivers More Oxygen to Peripheral Tissues under
Moderately Hypoxic Conditions--
Presbyterian mice were exposed to
15% O2 for 5 min to investigate the physiological effects
of HbPres on tissue hypoxia in vivo.
We simultaneously monitored Ve to elucidate whether the
increased tissue oxygenation was attributable to the increased
ventilation of Presbyterian mice or an efficient O2
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 O2 delivery by
HbPres.
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, PaCO2, and PaO2
level of arterial blood in Titusville and Presbyterian mice (Table
II). Titusville mice showed a normal
PaO2, PaCO2, and pH in room air and during
hypoxia whereas Presbyterian mice showed a low pH associated with an
elevated PaCO2 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
PaO2 level (Table II). Given the normal respiratory
functions and reduced ventilation (Ve) (Fig. 3B),
the primary cause of elevated PaCO2 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 PaCO2 by reducing the ventilation. It
is still surprising that Presbyterian mice consumed more O2
and produced more CO2 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 PaCO2 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 Titusville and Presbyterian Mice Consume More O2 and
Produce More CO2--
To investigate whether enhanced
tissue oxygenation alters the basic metabolism of mutant mice, we
measured metabolic parameters such as O2 consumption,
CO2 production, and the respiratory ratio in Titusville and
Presbyterian mice (Table III). The
metabolic analyses showed that both mutant mice consumed more
O2 and produced more CO2 in room air and
hypoxic conditions (Table III), implying that the low affinity of
mutant Hbs drives the mice to consume more O2 to exclude
the excess tissue O2 delivered by mutant Hbs. The increase
in O2 consumption may then lead to the increased production
of CO2 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 O2 delivery by
HbPres 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 HbTitu and HbPres 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 ~2 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 ~2.5 times longer distance than wild-type mice
whereas Presbyterian mice showed an ~2 times longer distance. Mean
running distances of Titusville and Presbyterian mice versus
wild-type mice were 9539 versus 4613 m
day A Human with Presbyterian Hb Showed an Altered Ventilatory Response
to CO2 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 Titusville and Presbyterian Mice May be Gain-of-function Mutations
in Mouse and Human--
Individuals who carry variant Hbs with low
O2 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 O2
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 O2 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 O2 affinity, Hb Titusville as
a mutant of
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 Titusville and Presbyterian Mice May Compensate for Tissue
Hyperoxia through Anemia, Increased Tissue O2 Consumption,
and Increased Spontaneous Exercise--
This is the first report that
Hb determines or controls the basal level of erythropoiesis, tissue
O2 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 O2 as shown in Fig. 7.
In Titusville mice, the introduced Asn residue locates at the interface of the
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
down-regulating 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 Presbyterian mice. Because the
amount of hemoglobin contained in the peripheral blood directly
correlates with the efficiency of O2 transport in tissues,
one of the determinants of the hemoglobin concentration may be
O2 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, O2 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.
-globin gene or Presbyterian mutation in the
-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- 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 (HbTitu) is
a low affinity variant of the
-globin chain and is well characterized clinically (4, 5). Hb Presbyterian (HbPres)
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 HbTitu or
HbPres show mild anemia, whereas individuals with high
oxygen affinity Hbs such as HbMalmo or HbYakima
show symptoms associated with polycythemia (10-13).
94
Asn) at the
1
locus or with Presbyterian (Asn
108
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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 5' flanking sequence of the murine
1 globin gene (nucleotides 1-372; GenBankTM 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/AflII-anchored primer (5'-GGA CTA GTC TTA AGA GAC TCA GGA AGA AAC C-3') and
XhoI/AflII-anchored primer (5'-CCT CTA GAC TCG
AGC TTA AGG TAG GCA TCC AAT TAT GCT T-3'). The
SpeI/XhoI-restricted
1 globin fragment was
mutagenized with a 21-bp mutagenic oligonucleotide (5'-GCT GCG TGT GAA
TCC CGT CAA-3') 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 (5'-CCG CTC GAG TCC TTG AGC CAA AGA AGC
CA-3') and ApaI/SalI-anchored primer (5'-TTG GGC
CCG TCG ACT CTG CCC GCT GGC TGA GCT C-3'). The
SpeI/XhoI-restricted
1 globin fragment and
XhoI/ApaI-restricted short 3' 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 (5'-ATT TGC GGC CGC TGG CAT TCA CAG AGC
TCA CCA-3') and SpeI-anchored primer (5'-GGA CTA GTG TCA GAA
TCA GAA GTG TCT TGG-3'). The NotI/SpeI-restricted 5' 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 (5'-CGC GTC GAC ATA ACT TCG TAT AAT
G-3') and SalI/EcoRI-anchored primer (5'-CGC GTC
GAC GAA TTC ATC GAT ACC GGC GAC ATA-3'). 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 3' 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 (5'-TTC CTT GCC
TCT GTG AGC-3') and p2 (5'-TGG GAC CGA GCC ATC TTC-3') in agouti offspring.
-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 (5'-ACC CAG CGG TAC TTT GAT AGC-3')
and p4 (5'-GCT ACT GAA GCT GTC TAA GGC AAC AGG-3') in agouti offspring.
PamH2O)/(760
PbH2O) × 0.5/Vcal × VTATPS, where Tb is rectal
temperature (°C); Tam is ambient temperature (°C);
PamH2O and PbH2O are the water vapor pressures
(mmHg) in the ambient air and the alveoli, respectively; and
VTATPS 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 (
E;
ml body temperature, pressure, saturated) was determined as
E and PtiO2 were
measured at 0, 0.5, 1, 2, 3, 4, and 5 min.
PtiO2 (mmHg)
was calculated as the difference between baseline PtiO2 and
PtiO2 at each time point.
E and
PtiO2. Arterial blood (120 µl) 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
CO2 (PaCO2), and partial pressure of arterial
O2 (PaO2). Arterial blood was sampled before and at the end of hypoxic gas inhalation.
20 °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 ~50 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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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, O2 delivery in the peripheral tissues. In
addition, Presbyterian Hb confers a novel allosteric effect with the
variant Lys residue interacting with the Cl
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 O2 delivery either by manipulating
-globin and
-globin or by using a novel allosteric effect.
-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 ~15% of the Hb in the peripheral blood of Titusville mice consists of HbTitu whereas ~30% of that in
Presbyterian mice consists of HbPres. The medical
literature on individuals with mutant hemoglobinopathies revealed the
expression level of HbTitu to be 34.7% (4) and
HbPres 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 ± 0.8 versus
12.9 ± 1.1 g/dl, p < 0.05; see Table I)
without signs of hemolysis (reticulocytes 1.7 ± 0.2%
versus 1.6 ± 0.7%; see Table I), suggesting that
these model mice mimic the human cases well (4-9).
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Fig. 1.
Generation of Hb mutant mice.
A, strategy used to knock-in the Titusville mutation (D94N)
in the murine 1-globin gene locus. Diagrams of the mouse
1-globin
locus (upper) and predicted knock-in allele
(lower) are presented. B, strategy used to
knock-in the Presbyterian mutation (N108K) in the murine
-globin
gene major locus. Diagrams of the mouse
-globin major locus
(upper) and predicted knock-in allele (lower) are
presented. E, EcoRI; neor,
neomycin resistance gene. C and D, the introduced
mutation replaces
94Asp with Asn in knock-in allele (C)
and
108Asn with Lys in knock-in allele (D). E,
genotype analysis of Titusville mice by PCR. The wild-type allele of
the
Wt-globin gene or knock-in allele of the
Titu-globin gene was amplified by PCR with primers P1
and P2. F, genotype analysis of Presbyterian mice by PCR.
The wild-type allele of the
Wt-globin gene or knock-in
allele of the
Pres-globin gene was amplified by PCR with
primers P3 and P4. G, RP-HPLC profiles of hemolysate
prepared from a wild-type mouse (Wt/Wt;
upper), a Titusville mouse (Titu/Titu;
middle), or a Presbyterian mouse
(Pres/Wt; lower). 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).
Complete blood cell count of Titusville and Presbyterian mice
.
Interestingly, Cl
stabilized the deoxy state of
HbPres in a dose-dependent manner, suggesting
that the introduced Lys residue protrudes into the central cavity to
bind to Cl
ion as suggested in the previous model (Fig.
2D).
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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 concentration on oxygen dissociation in hemolysates
from Presbyterian mice and wild-type mice. An enhanced
dose-dependent Cl
effect was seen in
Presbyterian mice.
PtiO2 and Ve values during hypoxia are shown
in Fig. 3, A and B,
respectively. After 5 min of hypoxia, the tissue O2 of
Presbyterian mice was significantly retained and sustained a higher
level than in wild mice (p < 0.05, a two-way analysis
of variance for repeated measures). In the course of hypoxia,
Presbyterian mice showed a similar decline in tissue O2 to
wild-type mice within 1 min of hypoxia whereas tissue O2 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 wild-type mice over a certain range of
hypoxic conditions. In fact the benefit of changes in the affinity of
HbPres in vitro is greatest at a
PaO2 concentration of ~50 mmHg as shown in Fig.
2A. Therefore, it is reasonable that the beneficial effect of HbPres is more remarkable in vivo in advanced
tissue hypoxia as shown in Fig. 3A.
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Fig. 3.
Tissue O2
in Presbyterian mice was a significantly higher level than that
in wild-type mice during mild hypoxia. A, tissue
O2 ( PtiO2) response to hypoxia in wild-type
(filled circle; n = 8) and Presbyterian mice
(filled triangle; n = 9). B,
changes in ventilation (
E) to hypoxia. Data
are the mean ± S.E. and were analyzed by a two-way analysis of
variance for repeated measures. Statistical interaction between time
and group is shown. *, p < 0.05.
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 HbPres by reducing ventilation.
Alternatively, HbPres may modulate the respiratory center,
especially the ventilatory response to CO2 in the brain of
mutant mice, in a Presbyterian-specific manner.
Blood gas analyses of Titusville and Presbyterian mice
Metabolic analyses of Titusville and Presbyterian mice
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Fig. 4.
Histochemical and enzymological analyses in
muscles of mutant mice. Transverse sections of the deep region in
tibialis anterior muscle of Titusville (A) and Presbyterian
(D) mice stained for ATPase activity on preincubation at pH
4.5 (upper) and for succinate dehydrogenase
(lower) activity. IIA, type IIA fiber;
IIB, type IIB fiber. Scale bars indicate 50 µm.
Shown are fiber type distributions (B and E) and
succinate dehydrogenase activities (C and F) in
tibialis anterior muscle of wild-type and Titusville or Presbyterian
mice. Significant differences between wild-type and Titusville or
Presbyterian mice are shown (*, p < 0.001; unpaired
Student's t test). Data are means ± S.D.
(n = 5). *, p < 0.001.
1 and 7580 versus 3732 m
day
1, 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 O2 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.
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Fig. 5.
Physical activity of mutant mice on the
running-wheel apparatus. Daily running distances of Titusville
(A) and Presbyterian (B) mice during 28 days of
exercise on the running wheel apparatus. Data are means ± S.D.
(n = 5).
-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 HbPres (data 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 of ventilatory dysregulation by the Presbyterian mutation in the
-globin gene between humans and mice.
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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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin, and Hb Presbyterian as a mutant of
-globin.
Using these models, we addressed whether low affinity Hb actually
releases more O2 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 O2 consumption in tissues, and an increased
running capacity and propensity that resulted in altered behavior with
greater physical activities.
-globin locus.
Because neither of these gain-of-function mutations (
94Asp
Asn
and
108 Asn
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.
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.
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Fig. 7.
Molecular physiology of low affinity
Hbs. A, molecular modeling of Hb Titusville (D94N);
B, molecular modeling of Hb Presbyterian
(N108K);
C, physiological implications of low oxygen affinity
Hbs.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Nakajima, Ogawara, Takahashi, Moriizumi, Ikegami, Baba, Nojiri, Kuroyanagi, Nakai, Morio, and Asaumi for technical assistance.
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FOOTNOTES |
---|
* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Molecular Gerontology, Tokyo Metropolitan Inst. of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan. Fax: 81-3-3579-4776; E-mail: sirasawa@tmig.or.jp.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M211110200
2 M. Tamaki, M. Izumizaki, Y. Suzuki, T. Shimizu, J. Suzuki, M. Nakamura, K. Ueshima, H. Inoue, M. Iwase, T. Kuriyama, I. Homma, and T. Shirasawa, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: Hb, hemoglobin; HbTitu, Hb Titusville; HbPres, Hb Presbyterian; RP-HPLC, reversed-phase high performance liquid chromatography; PtiO2, tissue O2; FIO2, fraction of inspired O2; PaCO2, partial pressure of arterial CO2; PaO2, partial pressure of arterial O2; SDH, succinate dehydrogenase; wt, wild-type; ATPS, ambient temperature, pressure, saturated.
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