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MODEL ORGANISMS AND COMPARATIVE FUNCTIONAL GENOMICS
1Department of Physiology, Showa University School of Medicine, Tokyo 142-8555; 2Department of Molecular Genetics, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015; and 3Department of Internal Medicine II, Nara Medical University, Kashihara 634-8522, Japan
Submitted 3 March 2003 ; accepted in final form 21 June 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-globin gene, 108
Asn 
Lys). O2 consumption during normoxia, measured via
open-circuit methods, was significantly higher in the mutant mice than in
wild-type mice. Respiratory measurements were conducted with a whole body,
unrestrained, single-chamber plethysmograph under conscious conditions. During
hypercapnia, there was no difference between the slopes of the hypercapnic
ventilatory responses, whereas minute ventilation at the same levels of
arterial PCO2 was lower in the Presbyterian mice than in
the wild-type mice. During both hypoxic exposures, ventilatory responses were
blunted in the mutant mice compared with responses in the wild-type mice. The
effects of brief hyperoxia exposure (100% O2) after 10% hypoxia on
ventilation were examined in anesthetized, spontaneously breathing mice with a
double-chamber plethysmograph. No significant difference was found in
ventilatory responses to brief hypoxia between both groups of mice, indicating
possible involvement of central mechanisms in blunted ventilatory responses to
hypoxia in Presbyterian mice. We conclude that chronically enhanced
O2 delivery to peripheral tissues can reduce ventilation during
acute hypercapnic and hypoxic exposures. hyperoxia; hypoxia; hypercapnia; tissue; breathing
-and
-globin subunits, has a characteristic affinity for O2 that
is affected by physiological agents, such as 2, 3-diphosphoglycerate,
CO2, and hydrogen ions. A variety of mutations in the genes of
- and -globins has been reported
(2). Among these mutations, Hb
Presbyterian (HbPres) has been characterized biochemically to have
a low Hb-O2 affinity in vitro, and biochemical analysis has
revealed that HbPres carries Lys at Asn-108 of -major globin
(10,
14,
17,
23).
Suzuki et al. (22)
generated mutant mice with the mutation in the -major locus of the mouse
genome by the targeted knock-in strategy. The affinity of the mutant Hb for
O2 is lower than that of the wild-type. The
PO2 at half-saturation is 47.0 mmHg for
HbPres and 43.5 mmHg for wild-type Hb. Theoretically, decreased
Hb-O2 affinity improves tissue oxygenation because of increased
O2 supply to the peripheral tissues as long as the arteriovenous
O2 content difference is maintained. Shirasawa et al.
(21) showed that the
genetically modified Hb chronically improves tissue oxygenation in mutant mice
without changes in arterial PO2 (PaO2) and
that arterial PCO2 (PaCO2) in mutant mice is
unexpectedly elevated compared with that in wild-type mice with increased
CO2 production (
CO2) and
decreased minute ventilation (E),
suggesting that an increase in tissue oxygenation decreases the final output
of the respiratory control system.
The chemical breathing control system, which is composed of central chemoreceptors in the medulla oblongata and peripheral chemoreceptors in the carotid sinus, has been widely studied in terms of hypercapnic and hypoxic ventilatory responses. Prolonged chemical ventilatory stimulation changes these ventilatory responses. Among these changes, the influence of chronic hypoxia has been stressed (1, 19, 20, 25); experimental evidence concerning the influence of chronic hyperoxia on breathing control is limited. Chronic systemic hyperoxia during the perinatal period results in attenuation of hypoxic ventilatory responses (5, 9, 16), which suggests that altered carotid chemoreceptor function occurs under such circumstances. However, the influence of chronically increased O2 delivery, without arterial hyperoxia, on ventilatory responses has not been studied.
We found a human subject with Presbyterian hemoglobinopathy to have attenuated ventilatory responses to hypercapnia and hypoxia compared with normal responses (M Tamaki, M Izumizaki, Y Suzuki, T Shimizu, J Suzuki, M Nakamura, K Ueshima, H Inoue, M Iwase, I Homma, T Kuriyama, and T Shirasawa, unpublished observation). This observation and the fact of elevated PaCO2 in mutant mice with Presbyterian hemoglobinopathy (21) suggest that ventilatory responses to chemical stimulation are attenuated in the mutant mice. Thus we tested the hypothesis that chronically increased O2 delivery can lead to attenuated ventilation in response to acute systemic hypercapnia and hypoxia in mutant mice with Presbyterian hemoglobinopathy.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-globin
genome to generate a mouse model carrying the mutation, Lys at Asn-108 of
-major globin, like that found in human individuals with Presbyterian
hemoglobinopathy (22). The
chimeric mice were cross-bred with C57BL/6CrSlc for five successive
generations. The genotype was confirmed by the HPLC profile of Hb prepared
from mice with the mutation
(21) and by PCR amplification
of the genomic DNAs from tail biopsy specimens. PCR amplification was carried
out with a sense primer (5'-ACC CAG CGG TAC TTT GAT AGC-3') and an
antisense primer (5'-GCT ACT GAA GCT GTC TAA GGC AAC AGG-3').
The homozygous and heterozygous mice were born live, grew normally, and
were fertile. Homozygous mice had hemolytic anemia with mutant red blood cells
exclusively composed of HbPres
(22). Complete blood cell
counts of the heterozygous mice showed no significant abnormalities, as judged
by counts in wild-type mice
(21). From the HPLC profile,
30% of Hb in the peripheral blood of heterozygous mice is
HbPres (21,
22). The expression of
HbPres has been reported in 29.9-41.7% of cases of human
Presbyterian hemoglobinopathy
(10,
14,
17,
23). Therefore, the
heterozygous Presbyterian mouse model is an equivalent animal model for human
Presbyterian hemoglobinopathy. We used the heterozygous mouse model in the
present study as Presbyterian mice to analyze the physiological properties of
HbPres. Male mice used in the experiments were provided with food
and water ad libitum, housed at a controlled temperature (22 ±
1°C), and exposed to a daily 12:12-h light-dark cycle under normoxic
conditions. All experiments were conducted in an environmentally controlled
room at 22 ± 1°C between 10:00 AM and 5:00 PM.
Measurement of metabolism. O2 consumption
(O2, ml/min STPD) and
CO2 (ml/min STPD) were
measured during normoxia (21% O2 in N2) and two levels
of hypoxia (10 and 15% O2 in N2) with an open-circuit
system (ARCO-1000, ARCO system, Kashiwa, Japan) in wild-type (n = 4,
10%; n = 5, 15%) and Presbyterian (n = 4, 10%; n =
7, 15%) mice (age 12 wk). Each mouse was placed in a chamber where a steady
flow of air was delivered by a vacuum pump. The ARCO system measured the
fractions of O2, CO2, and N2 at the inflow
and outflow of the chamber with a mass spectrometer and measured the flow rate
with a pneumotachograph. The mouse was placed in the chamber for 1 h to
acclimatize to the surroundings before the experiments. After equilibration
was achieved, the metabolic factors in the normoxic condition were measured,
and 10 or 15% hypoxic gas was delivered to the metabolic chamber from a
respiratory gas circuit. The steady state of O2 gas concentration
in the chamber was obtained within 5 min. Measurements were made 20 min after
the onset of hypoxic gas exposure during a 5-min period of the steady state.
O2 and
CO2 were normalized by body weight
(kg).
Ventilatory measurements. Ventilation was measured under conscious
conditions via a whole body, unrestrained, single-chamber plethysmograph
(PLY3211, Buxco Electronics, Sharon, CT). The system consisted of a Plexiglas
experimental chamber equipped with two pneumotachographs. The chamber was
provided a continuous airflow at 1 l/min via a flow pump-reservoir system
(PLY1020, Buxco Electronics). Differential pressure between the experimental
and reference chambers was measured with a differential pressure transducer.
The pressure signal was amplified and then integrated by data analysis
software (Biosystem XA for Windows, Buxco Electronics). A calibration volume
of 0.5 ml of ambient air was introduced into the chamber with a syringe before
recordings. Barometric pressure, body weight, and rectal temperature were
measured routinely before experiments to express tidal volume (VT,
ml BTPS) per 10 g. Respiratory rate (RR, breaths/min),
VT, and E (ml/min
BTPS) were computed breath-by-breath throughout all baseline and
experimental periods and were stored for offline analysis. A 1-min average was
taken for each variable during hypercapnic or hypoxic exposure. Each animal
was placed in the chamber and allowed at least 60 min to acclimatize before
the assessment of ventilation. Baseline measurements were made when the animal
was quiet but awake and were averaged.
Hypercapnic ventilatory responses. Hypercapnic ventilatory responses were estimated in wild-type (n = 6) and Presbyterian (n = 9) mice (age 12 wk). The hypercapnic challenges were given on a hyperoxic background to exclude the possibility of an arterial O2 content difference between wild-type and Presbyterian mice. Baseline ventilation was measured during 100% O2 inhalation. Each animal received a series of three hypercapnic gas exposures (5, 7, and 9% CO2 in O2). Respiratory measurements were obtained after a period of 10 min at each exposure. Gases were delivered to the chamber with the flow pump at a rate of 1 l/min.
Blood gas analysis during hypercapnia. We measured PaCO2 in conscious Presbyterian and wild-type mice exposed to 100% O2 and hypercapnic gas (5% CO2 in O2) independently of measurements of hypercapnic ventilatory responses described above. Wild-type (n = 5) and Presbyterian (n = 6) mice (age 12 wk) were the subjects for arterial blood gas analysis. An arterial catheter was implanted in the left carotid artery under anesthesia (pentobarbital sodium, 25 mg/kg ip). Mice were placed in a plastic chamber (90 x 120 x 50 mm) for 6 h into which a normoxic gas mixture (21% O2 in N2) was delivered. After mice recovered from the anesthesia, the airflow through the chamber was replaced with pure O2 for baseline analysis. Arterial blood (120 µl) was collected into a heparinized glass sampling tube (MC0020, AVL Scientific, Roswell, GA) 15 min after introduction of pure O2 and analyzed immediately with a blood gas analyzer (OPTI CCA, AVL Scientific) for pH, PaCO2, and PaO2. The blood loss was compensated for by an infusion of 200 µl of saline. Subsequently, mice were exposed to a hypercapnic gas mixture (5% CO2 in O2), and blood gas analysis was carried out 10 min after introduction of the hypercapnic gas in a similar way. Ventilatory variables were not monitored in this experiment.
Hypoxic ventilatory responses. Acute hypoxic ventilatory responses
were measured during two levels of hypoxia (10 or 15% O2 in
N2) in wild-type (n = 7, 10%; n = 7, 15%) and
Presbyterian (n = 7, 10%; n = 6, 15%) mice (age 12 wk).
After baseline ventilation was measured during the normoxic condition (21%
O2 in N2), a hypoxic gas mixture (10 or 15%
O2 in N2) was delivered to the chamber for 15 min at a
flow rate of 1 l/min. RR, VT, and
E measurements were made at 1-min
intervals during the 15-min exposure to hypoxia.
Brief hyperoxic ventilatory response. The effects of brief hyperoxia exposure on ventilation were examined in male, spontaneously breathing wild-type (n = 6) and Presbyterian (n =5) mice (12-18 wk old). A reduction in respiration in response to brief hyperoxia is used as an index of peripheral chemoreceptor sensitivity (4, 13). The blunted hypoxic ventilatory response in Presbyterian mice may be due to changes in peripheral chemoreceptor sensitivity.
Ventilation was measured with double-chamber plethysmography
(12). Changes in the
fractional concentration of O2 inside the head-chamber were
measured before experiments with an O2 analyzer (Respina 1H26
[PDB]
, NEC
San-ei, Tokyo, Japan), which showed that a quick change in inspired air was
possible within 4 s. Mice were anesthetized with urethane (1.2 g/kg ip) 20 min
before being placed in the plethysmograph. Baseline ventilation was recorded
while the animal breathed a normoxic gas mixture (21% O2 in
N2). Subsequently, the mouse inhaled a hypoxic gas mixture (10%
O2 in N2) for 45 s, and then inspired gas was switched
to 100% O2 for 20 s. E
was analyzed for the last 15 s of each exposure.
Statistical analysis. Results are expressed as means ± SE. We used a commercially available software package (SPSS, SPSS Japan, Tokyo, Japan). Data were analyzed by two-way repeated-measures ANOVA to test for between-factor (genotype) effects, within-factor (CO2 or time) effects, and any interaction between these two effects. Blood gas and metabolic values were analyzed statistically by the t-test. A statistical significance was accepted at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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O2 and
CO2 during
normoxia and hypoxia. We measured systemic
O2 and
CO2 with the open-circuit system to
confirm increased O2 supply from Hb to peripheral tissues in
Presbyterian mice (Fig. 1).
During 21 and 15% O2, both
O2 and
CO2 were significantly higher in
Presbyterian mice than in wild-type mice (all P < 0.05, unpaired
t-test). However, there were no significant differences between
groups of mice in O2 or
CO2 values during 10% O2
inhalation.
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Hypercapnic ventilatory responses. Ventilatory responses to three
levels of inspired CO2 (5, 7, and 9% CO2 in
O2) were recorded in wild-type and Presbyterian mice under
conscious conditions (Fig. 2).
There were significant effects of CO2 inhalation on all variables
(main effect for CO2, all P < 0.05, 2-way ANOVA);
CO2 produced significant increases in RR, VT, and
E in both groups.
E values at the same levels of
inspired CO2 were similar in the groups of mice. There were no
significant differences between genotypes in RR, VT, or
E.
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Respiratory variables obtained during 0 and 5% CO2 exposures
were plotted as a function of PaCO2 because Presbyterian mice had a
significantly higher PaCO2 during both normocapnic and hypercapnic
conditions than that of wild-type mice
(Table 1, P < 0.05,
unpaired t-test). The slopes of the hypercapnic ventilatory response
lines were the same, but E was lower
in Presbyterian mice than in wild-type mice at the same levels of
PaCO2 (Fig. 3).
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Hypoxic ventilatory responses. Hypoxic ventilatory responses were
attenuated in Presbyterian mice relative to the wild-type mice during hypoxia
(Fig. 4). Ventilatory responses
to hypoxia were recorded for 15 min in Presbyterian and wild-type mice under
poikilocapnic conditions. During the basal period (21% O2 in
N2), there were no significant differences in RR, VT, or
E values between groups (unpaired
t-test). However, during both 10 and 15% hypoxic conditions, the
initial hypoxic response in Presbyterian mice was low compared with that in
wild-type mice. After the peak E
response, there was a decline, and the difference in
E between groups diminished. The
main effect of time was significant for all variables during both hypoxic gas
exposures (all P < 0.05, 2-way ANOVA). A significant genotype
effect was also found for RR and E
during both hypoxic conditions (all P < 0.05, 2-way ANOVA).
Although a comparable tendency was observed in VT, no statistical
difference was found between groups.
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Effects of brief hyperoxia on
E. We compared
E depression, expressed as
percentage of baseline E, in
response to brief hyperoxia (100% O2) after hypoxic gas inhalation
(10% O2) between wild-type and Presbyterian mice. Mean
E values in response to hyperoxia
decreased by 43.5 ± 7.8% in wild-type mice and 40. 1 ± 7.8% in
Presbyterian mice; no significant difference was found in
E depression
(Fig. 5).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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O2 and
CO2 in mutant mice with
HbPres were higher than those in wild-type mice under normoxic
conditions and that the mutant mice showed lower ventilation during acute
systemic hypercapnia and hypoxia, suggesting that chronically increased tissue
oxygenation affects control of breathing.
Increased O2 consumption in Presbyterian
mice during normoxia. O2 was
higher in Presbyterian mice than in wild-type mice during normoxia and 15%
O2, which was consistent with our previous study
(21). In contrast, there were
no differences between genotypes in
O2 in 10% O2. Although
higher O2 in Presbyterian mice was
maintained during 15% O2 exposure, the beneficial effect of
HbPres on O2 was absent
during 10% O2 exposure. These results indicate that Presbyterian
mice have chronically elevated O2 delivery to peripheral tissues
during normoxia and mild hypoxia and that as the intensity of hypoxia
increases, a reduction in O2 uptake in the lungs due to decreased
O2 affinity offsets the increase in O2 release from
HbPres in the tissues.
Blunted ventilation in response to hypercapnia in Presbyterian
mice. Ventilatory responses to hypercapnia were assessed in
unanesthetized Presbyterian and wild-type mice. In conscious small animals,
ventilatory responses to hypercapnia have been evaluated most commonly by the
relation between E and the level of
inspired CO2. However, hypercapnic responses only to various levels
of inspired CO2 are not suitable for estimation of breathing
control in Presbyterian mice because PaCO2 is higher in
Presbyterian mice than in wild-type mice at the same level of inspired
CO2 (21).
E responses to inspired
CO2 in Presbyterian mice were comparable to those in wild-type
mice. However, E plotted as a
function of PaCO2 showed that
E was lower in Presbyterian mice
than in wild-type mice at the same PaCO2. Accordingly, the apneic
threshold (extrapolated PaCO2 at zero ventilation in the steady
state) was higher in Presbyterian mice than in wild-type mice, whereas
ventilatory CO2 sensitivity, defined as the slope of the
ventilatory response, was similar in Presbyterian mice and wild-type mice. The
consensus among published studies is that the ventilatory response to
CO2 is enhanced by chronic systemic hypoxia
(7,
8,
25), with the increased
CO2 sensitivity mediated by peripheral chemoreceptors
(25). Decreased CO2
sensitivity of peripheral chemoreceptors may account for blunted ventilation
during hypercapnia in Presbyterian mice. Although we are uncertain as to the
underlying mechanisms of decreased ventilation, we believe that decreased
ventilation in response to hypercapnia plays a major role in the chronic
hypercapnia of Presbyterian mice.
Blunted ventilatory responses to hypoxia in Presbyterian mice. When acute hypoxia is imposed on mammals, there is a biphasic ventilatory response: an initial rise in ventilation followed by a reduction (18, 24). The initial hypoxic response is attributed to the afferent inputs originating in peripheral chemoreceptors and the reflex of central neural mechanisms. In our Presbyterian mice, the initial rise in ventilation in response to hypoxia was significantly lower than that in wild-type mice. Shirasawa et al. (21) showed that PaO2 in Presbyterian mice was similar to that in wild-type mice during normoxia and 15% O2 after 5 min of each gas exposure, suggesting changes in ventilation occurred at comparable levels of PaO2 in Presbyterian and wild-type mice.
The effect of chronically increased tissue oxygenation on respiration, especially independent of arterial hyperoxia, has not been studied because no relevant animal model was available until the Presbyterian mouse model. In contrast, hypoxia has been investigated extensively in laboratory animals. A chronic exposure to hypoxia increases the hypoxic ventilatory response, a phenomenon termed ventilatory acclimatization to hypoxia (VAH) (1, 19, 20, 25). The primary physiological significance of VAH is that it counterbalances decreased arterial O2 delivery. However, it is not always associated with an enhanced response to hypoxia, especially in mice (11). Although many questions remain concerning VAH, it is generally accepted that changes in carotid body O2 sensitivity contribute to VAH (19). On the other hand, Dwinell and Powell (6) showed that chronic exposure to hypoxia increases the central nervous system gain of hypoxic ventilatory responses in adult rats. Brief hyperoxic tests to assess the sensitivity of the peripheral chemoreceptors indicate possible involvement of central mechanisms, rather than carotid body O2 sensitivity, in blunted ventilatory responses to hypoxia in Presbyterian mice. It was also of interest that hypoxic ventilatory responses were low despite the elevated PaCO2 in Presbyterian mice. A combination of hypoxia and hypercapnia has a multiplicative effect on ventilatory responses (15, 26), and hypoxic ventilatory responses are generally enhanced in the presence of hypercapnia (3). There may be a reduction in O2-CO2 interaction in Presbyterian mice.
The significance of depressed ventilation. Attenuated ventilatory responses to hypercapnia and hypoxia could be interpreted as compensations for increased O2 delivery due to a low O2 affinity of HbPres. Although O2 is required for tissue survival, an overabundance of O2 generally tends to produce more free radicals detrimental to the cellular redox state. From an oxygen supply perspective, blunted ventilation in Presbyterian mice is useful to counterbalance elevated tissue oxygenation resulting from decreased Hb-O2 affinity. From an oxygen demand perspective, this animal model with HbPres has three other metabolic characteristics that contribute to control of the cellular O2 content: 1) skeletal muscle fibers become more oxidative; 2) mitochondrial succinate dehydrogenase activity, which acts in the citric acid cycle, is enhanced in skeletal muscle fibers; and 3) spontaneous exercise is increased (21). Thus Presbyterian mice compensate for higher O2 delivery not only by means of an increase in metabolic capacity but also by means of a reduction in ventilation. These findings suggest that the overall goal of our biological program includes the control of cellular O2 content in an optimal state and that the breathing control system is adjusted in conformity to the program. We have reported that mutant mice with another low-affinity variant, Hb Titusville (HbTitu), showed normal PaCO2 during normoxic conditions (21), which may raise a question that decreased ventilation is not a general physiological response to low affinity of Hb for O2. Although further research is needed to answer the question fully, in this context, this might be accounted for by the difference in the PO2 at half-saturation between HbTitu (66.0 mmHg) and HbPres (47.0 mmHg). Normal ventilation may therefore be required to maintain O2 loading in the lungs in mutant mice with HbTitu.
In summary, we found that mutant mice with Presbyterian hemoglobinopathy show attenuated ventilation in response to systemic hypercapnia and hypoxia. These results support the hypothesis that chronically increased tissue oxygenation can lead to attenuated ventilation in the presence of chemical ventilatory stimulation.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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108(G10) Asn Lys] found in Japan.
Hemoglobin 8:
407-411, 1984.[Medline]
108 (G10) Asn Lys] in a German family.
Hemoglobin 3:
365-370, 1979.[Medline]
108 (G10) asparagine lysine. A hemoglobin variant
with low oxygen affinity. FEBS Lett
92: 53-56,
1978.[ISI][Medline]
108 Lys) confer hemolytic anemia with altered
oxygen affinity and instability of Hb. Biochem Biophys Res
Commun 295:
869-876, 2002.[ISI][Medline]
108(G 10) Asn Lys] in a Spanish family.
Acta Haematol 76:
161-163, 1986.[Medline]
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