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1 Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205; and 2 Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, Ohio 44109
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Acutely lowering ambient
O2 tension increases ventilation in many mammalian species,
including humans and mice.
Inheritance patterns among
kinships and between mouse strains suggest that a robust genetic
influence determines individual hypoxic ventilatory responses (HVR).
Here, we tested specific genetic hypotheses to describe the inheritance
patterns of HVR phenotypes among two inbred mouse strains and their
segregant and nonsegregant progeny. Using whole body plethysmography,
we assessed the magnitude and pattern of ventilation in C3H/HeJ (C3)
and C57BL/6J (B6) progenitor strains at baseline and during acute
(3-5 min) hypoxic [mild hypercapnic hypoxia, inspired
O2 fraction
(FIO2) = 0.10] and normoxic (mild hypercapnic normoxia,
FIO2 = 0.21) inspirate
challenges in mild hypercapnia (inspired CO2
fraction = 0.03). First- and second-filial generations and two
backcross progeny were also studied to assess response distributions of
HVR phenotypes relative to the parental strains. Although the
minute ventilation (
E) during hypoxia
was comparable between the parental strains, breathing frequency
(f) and tidal volume were significantly different; C3 mice
demonstrated a slow, deep HVR relative to a rapid, shallow phenotype of
B6 mice. The HVR profile in B6C3F1/J mice suggested that
this offspring class represented a third phenotype, distinguishable from the parental strains. The distribution of HVR among
backcross and intercross offspring suggested that the inheritance
patterns for f and E during
mild hypercapnic hypoxia are consistent with models that
incorporate two genetic determinants. These results further suggest
that the quantitative genetic expression of alleles derived from C3 and
B6 parental strains interact to significantly attenuate individual HVR
in the first- and second-filial generations. In conclusion, the genetic
control of HVR in this model was shown to exhibit a relatively simple
genetic basis in terms of respiratory timing characteristics.
C3H/HeJ; C57BL/6J; hypoventilation; hypercapnic ventilation; segregation analysis
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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DISTRIBUTIONS OF HYPOXIC VENTILATORY responses (HVR) in healthy human subjects have been described by Weil and colleagues (10, 18, 31). This investigative group and others (6, 29) have provided strong evidence for the existence of high- and low-responsive human subpopulations. A six- to sevenfold difference appears to exist between individuals occupying the extremes of HVR distributions (10). Furthermore, familial background has been shown to be important in determining different phenotypic profiles of hypoxic and hypercapnic ventilation among healthy individuals (4, 19, 30). A bimodal distribution of high- and low-responsive subgroups provides strong evidence that a small number of major genetic determinants in humans emerge to influence differential hypoxic ventilatory phenotypes (10).
Although the strongest evidence that genetic determinants influence HVR in humans originates from twin studies, the results are inconsistent (2, 11, 13, 20, 29). For example, Arkinstall et al. (2) suggested that tidal volume (VT), but not breathing frequency (f), was influenced by genetic factors during hypercapnic challenge. In contrast, Kawakami et al. (11) were unable to detect any significant genetic influence on breathing pattern in response to either hypercapnia or hypoxia. More recently, Kobayashi et al. (13) suggested that genetic factors influenced hypoxic ventilation the most in the presence of hypercapnia. Because it is often difficult to dissect variability among human volunteers into genetic and environmental components, interpretations of human studies are often uncertain. As an alternative approach, the present study examines HVR variation between two standard inbred mouse strains to study heritable ventilatory traits unique to hypoxia, which serve to define genetic components separate from variation within strains.
Our laboratory has assessed within-strain (environmental component) and between-strain (genetic component) variability among many different inbred mouse strains and demonstrated a wide spectrum of HVR characteristics (25). These studies led us to focus on two strains, C3H/HeJ (C3) and C57BL/6J (B6), which occupy the extremes of numerous strain distribution patterns including baseline (i.e., room air) hypoxic and hypercapnic ventilatory traits. Using quantitative genetic approaches, we exploited the phenotypic variation derived from segregant and nonsegregant offspring classes of C3 and B6 parental strains. The overall objectives of this study can be summarized in two ways: 1) to dissect differential ventilatory traits into discernible and meaningful phenotypes to describe acute hypoxic ventilatory mechanisms (14), and 2) to enumerate the genes that have major effects in determining different ventilatory phenotypes (22). Because a substantial homology exists between human and mouse genomes (21), achieving a significant understanding of the variation in HVR phenotypes between inbred parental strains and their progeny can contribute to the discovery of novel genetic determinants in humans. The results from the present study are consistent with genetic hypotheses that incorporate two genes; their interaction determines different phenotypic profiles of hypoxic ventilation.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Inbred mouse strains are the product of 20 or more generations of brother-sister matings, making individuals within a strain genetically identical and homozygous at every locus (9). Inbred strains offer unique advantages to understanding the genetic control of breathing because 1) genetic background can be certified, 2) environmental factors can be strictly controlled in a laboratory setting, 3) empirical variation between strains can be partitioned into genetic and environmental components, and 4) libraries of phenotypic and genotypic markers are available for complementary association and linkage studies.
Reproductively mature male and female inbred mice of the B6 and C3 strains and the B6C3F1/J [i.e., female B6 × male C3 (F1)] progeny were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in the animal facilities at Johns Hopkins University. Backcross [i.e., female B6 × male F1 (B6BX) and female C3 × male F1 (C3BX)] and intercross [i.e., B6C3F2 (F2)] progeny were generated, and animals were weaned within 4-5 wk. From the breeding colonies, randomly selected male backcross and intercross progeny were placed in cages containing 4-6 animals and housed for an additional 6-12 wk before testing. All experiments were performed between 0900 and 1800; only male mice were used for experiments. The environments before and during the experiments, as well as during animal handling, were highly standardized. Water and chow (Agway Pro-Lab RMH 1000) were provided ad libitum. All animal protocols were reviewed and approved by the Animal Care and Use Committee of the Johns Hopkins School of Public Health.Whole body plethysmography. Ventilatory function was assessed by whole body plethysmography (5, 8, 25, 26) under unanesthetized and unrestrained conditions. Each animal was permitted to acclimate in the chamber at least 30 min before ventilation measurements were obtained. Chamber temperature was maintained within the thermoneutral zone for mice (i.e., 26-28°C) and was recorded with each ventilatory measurement using a type T thermocouple device. Compressed air was humidified (90% relative humidity) and directed through the chamber at a flow rate of ~300 ml/min. At constant chamber volume, changes in pressure due to inspiratory and expiratory temperature fluctuations were measured using a differential pressure transducer (model 8510B-2, Endevco).
In addition to intermittently evaluating ventilation at baseline [inspired O2 fraction (FIO2) = 0.21 and inspired CO2 fraction (FICO2) = 0.00 in N2], we also assessed ventilatory responses during a sequence of acute (3-5 min) inspirate challenges: 1) mild hypercapnic hypoxia (MHX; FICO2 = 0.03 and FIO2 = 0.10 in N2) and 2) mild hypercapnic normoxia (MNX; FICO2 = 0.03 and FIO2 = 0.21 in N2) admixtures. The inspired air was analyzed for O2 and CO2 by a mass spectrometer (model 1100, Perkin Elmer) before and after each ventilatory measurement and was maintained within 1% of target conditions. In the MHX condition, mild hypercapnia was added to the hypoxic inspirate challenge to oppose the loss of CO2 during hypoxic ventilation; hence, the MNX condition represents a complementary reference, on par with the baseline condition, to evaluate ventilatory responses that are unique to acute hypoxia. Although blood-gas determinations have been recently accomplished in mice (e.g., see Refs. 12, 15), the large number of animals required to assess inheritance patterns among different offspring classes prohibited the routine use of blood-gas determinations in the present study. Each animal was weighed after the challenge protocol and subsequently used in other laboratory studies.Data acquisition. The analog signal generated from the pressure transducer was recorded as a digital input using a data acquisition system (Keithley Instruments) and a dedicated computer. Data were acquired at an input frequency of 100 Hz, and peak inspiration and expiration were determined from ~15 consecutive tidal breaths. On rare occasions, data were not secured by computer, and f, VT, and inspiratory time (TI) were estimated from four tidal breaths within a 6-s strip-chart recording, as described elsewhere (25). Least-squares regression analysis was used to compare the two methods of ventilatory data acquisition, and suitable reproducibility (r2 = 0.99) was established between the two ventilatory measurements. Pressure-transducer calibrations were performed daily with the use of a 50-µl gas-tight syringe while chamber temperature was maintained at a level similar to that in experimental ambient conditions.
In computing VT, the amplitudes of the inspiratory and expiratory limbs of each tidal breath were averaged and the body temperature of each animal was assumed to be constant at 37°C. Exploratory studies were performed to investigate the role of hypoxia-induced hypothermia in modifying our ventilatory measurements. We used a radiotelemetry system (Data Sciences, International) to detect changes in deep-body temperature, and hypoxia-induced strain differences between C3 and B6 mice were not observed during a 3- to 5-min sampling period. Variations of 1-2°C in deep-body temperature account for <5% error in computing VT. The data from each animal at baseline and during MNX and MHX were used to make the following computations. Minute ventilation (E) was calculated as the product of f
and VT. Expiratory time (TE) was determined
from total respiratory time (Ttot) minus TI, mean inspiratory flow was calculated as the ratio of VT to
TI, and the inspiratory fraction was computed as the ratio
of TI to Ttot. In exploratory studies, the
respiratory timing was measured by positioning a strain gauge on the
lateral aspect of the chest wall in anesthetized mice and assessing the
minimum and maximum excursions of the analog signal generated by the
strain gauge. These experiments demonstrated that the moments of end
inspiration and end expiration during baseline and challenged breathing
coincided with the maximum and minimum excursions of the chest wall,
respectively. In terms of VT,
E, and VT/TI,
we normalized individual results for body weight.
Data analysis.
The statistical procedures were initiated by examining each of
seven traits among the C3 and B6 progenitors (n = 20 mice per strain) and the F1 progeny (n = 14 mice) by two-way
repeated-measures ANOVA to examine the strain and inspirate challenge
effects (i.e., treatments). Post hoc mean comparisons were performed
using a Duncan's multiple range test and were considered significant
at the 
-level of 0.01.
2 distribution (with degrees of
freedom equal to the difference in the number of independent parameters
being estimated between the hypothesis and the unrestricted model).
Although the use of a 2 distribution is not always
exact, it serves as an approximation for determining which
trait-treatment combinations best fit (i.e., large P values) a
simple genetic basis (7, 22).
When two loci are involved, the most general model allows for nine
different genotypic means, one for each of the nine genotypes possible
when there are two alleles at each of two loci. Within each locus,
there may be no dominance; that is, the average for heterozygotes is
halfway between the averages of the two homozygotes. There may also be
no epistasis; that is, genotypic effects attributable to one locus are
additive to effects due to a second locus. Furthermore, allelic effects
at two loci may be equal in magnitude. If each one of these three
conditions is met, the two-locus model exhibits equal and additive
allele effects. The two loci may be linked or unlinked, the latter with
equal and additive effects providing the most parsimonious two-locus
model. Between this alternative and the general model, incorporating
further restrictions offers additional possibilities, including
1) zero-average dominance (i.e., the dominance effects of the
two loci are equal but are opposite) and 2) certain symmetry
restrictions for two-locus models, such as symmetry A (i.e., the mean
difference between two recombinant genotypes in one backcross is
similar to that in the other when expressed relative to the mean
difference between their respective nonrecombinants) as described
elsewhere (22).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Variation in ventilatory responses among C3, B6, and F1 mice.
In Fig. 1, representative traces of C3, B6,
and F1 mice at baseline and during acute hypoxic challenge are
depicted. At baseline and during hypoxic challenge, C3 mice demonstrate
a slow, deep breathing pattern relative to the rapid, shallow pattern
of B6 and F1 mice. Spirograms are also illustrated in Fig. 1, which highlight the composite changes in breathing pattern from baseline to
hypoxic ventilation. The relative irregularity associated with the
pressure trace of F1 mice on hypoxia is noteworthy.
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E
responses are depicted for C3, B6, and F1 mice at baseline and during
MNX and MHX challenges. As previously reported (25, 27), the
E responses at baseline were not different among the three groups of mice. With MNX or MHX challenge, increases in E from baseline were
observed in each animal. The E
response during MNX was significantly (P < 0.01) greater in the B6 progenitor compared with both C3 and F1 mice. During MHX, the
E response was significantly (P < 0.01) different between B6 and F1 mice.
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E responses were dissected into
f and VT components, results for C3 mice were shown
to be significantly (P < 0.01) different from those for B6
and F1 mice with respect to both f and VT responses (Fig.
3, A and B). The strain
variation in breathing pattern occurred at baseline and during each
inspirate challenge. Although the f responses were similar between B6
and F1 mice across treatments, VT responses during MNX and
MHX were significantly (P < 0.01) diminished in F1 compared
with B6 mice.
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E responses can also be dissected
into different components that incorporate
VT/TI and TI/Ttot (Fig.
3, E and F). Although there were no strain differences
with respect to VT/TI at baseline, B6 mice
demonstrated a significantly (P < 0.01) greater response compared with C3 and F1 mice during MNX. The
VT/TI response during MHX was also
significantly (P < 0.01) different between B6 and F1 mice.
With respect to TI/Ttot, F1 mice devoted a
significantly (P 
0.01) greater fraction of time to inspiration
compared with B6 mice at baseline and during both MNX and MHX.
Segregation analysis of offspring derived from C3 and B6
progenitors.
In the present study, single-locus, polygenic, and mixed models were
rejected for each ventilatory trait at baseline and during each
inspirate challenge. As shown in Table 1, a
best-fit genetic hypothesis was established for 15 of 21 possible
trait-treatment combinations. For many of the combinations, a
two-unlinked general model or a two-unlinked equal and additive locus
model was determined to be the most parsimonious model. In 5 of the 15 cases in which a two-locus model fitted the data, approximate P
values (i.e., using 2 distributions) exceeded 0.25, that
is, for TI and TE at baseline, VT/TI during MNX, and TI and
TI/Ttot during MHX. In an additional 5 of 15 cases, approximate P values were between 0.15 and 0.25, that
is, for f and TI/Ttot at baseline,
VT during MNX, and E and f
during MHX. For the remaining cases, a weaker fit was demonstrated by
approximate P values <0.10. In summary, hypotheses that are supported by approximate P values in excess of 0.25 can be
critically evaluated as having a relatively simple genetic basis.
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E, f, TI, and TI/Ttot during MHX. The range of
E responses among C3BX offspring was
similar to the C3 progenitor, whereas the ranges of B6BX and F2
responses more broadly incorporated responses that exceeded the
progenitors (Fig. 4A). With respect to f during MHX, the
breathing pattern difference between C3 and B6 mice was associated with differences among backcross and intercross progeny (Fig. 4B). The responses of C3BX mice favored the C3 progenitor, whereas the
majority of B6BX and F2 responses favored the B6 and F1 progenitors. In
many ways, TI during MHX reflected and accentuated the
inheritance pattern of f (Fig. 4C). The range of TI
responses in the C3BX progeny was similar to that in the C3 progenitor,
whereas the majority of B6BX and F2 responses resembled those of the B6
and F1 progenitors. The two-unlinked additive locus model for
TI/Ttot during MHX (Table 1) differed from
other traits, and the segregation plot exhibited a different
inheritance pattern (Fig. 4D). The range of
TI/Ttot responses was similar among the two
backcross and the intercross progeny and encompassed a range delineated by the two progenitors and F1 mice.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study demonstrates three different phenotypic profiles of
hypoxic ventilation among C3 and B6 parental strains and their F1
offspring. The strain variation in baseline breathing pattern between
C3 and B6 progenitors appears to be preserved during acute hypoxic
challenge. However, hypoxic VT responses of F1 mice are
significantly attenuated relative to the parental strains (Fig.
3B), which results in a reduced hypoxic
E response in this offspring class (Fig.
2). Considering the results from previous studies, hypoxic
E responses of F1 mice are shown to be
uniquely blunted relative to numerous inbred strains of mice, including
many standard strains as well as the BXH recombinant inbred strains
derived from F1 progenitors (23, 25). These observations suggest that a
combination of alleles derived from C3 and B6 parental strains
interacts to express hypoventilation as a phenotype of acute
hypoxic exposure. Therefore, the hypoventilatory response of F1 is
likely a multifactorial phenotype controlled by a suite of genetic determinants.
Consistent with the first objective of this study,
E responses at baseline and during MNX
and MHX challenges are dissected into meaningful components to further
delineate the differences between ventilatory phenotypes. If
E responses are examined in terms of f
and VT components (Fig. 3, A and B), f
responses at baseline and during each inspirate challenge are
remarkably similar between B6 and F1 mice. In contrast, distinctions
between B6 and F1 mice are defined by VT and TI
differences during challenge. Alternatively, if
E responses are evaluated in terms of
VT/TI and TI/Ttot
components, the F1 phenotype is distinguishable by a protracted
TI/Ttot that occurs at baseline and during both
MNX and MHX challenges. As shown in Fig. 3F, an increase in
TI/Ttot appears to represent a ventilatory
compensation that augments the inspiratory phase and optimizes the
volume of each tidal breath. During MHX, however, F1 mice fail to
achieve adequate VT, even when accompanied by a protracted
TI/Ttot. The protracted
TI/Ttot at baseline of F1 mice appears to
utilize reserves normally exploited during ventilatory adjustments to
acute hypercapnic or hypoxic challenge.
One hypothesis, which considers inadequate VT responses accompanied by relatively prolonged TI, suggests that lung volume and compliance are different among the strains. A recent study demonstrates C3 and B6 strain variations in lung pressure-volume characteristics (28), including a 50% greater lung volume in C3 mice compared with age- and weight-matched B6 and F1 mice. Although C3 and B6 strain differences in baseline breathing pattern are not solely dependent on covariation in lung volume or compliance, the role that lung mechanics plays during acute hypoxic or hypercapnic ventilation may be quite different. As demonstrated by a recent study in rats (3), lung mechanics and hypoxic ventilation interact through an increase in end-expiratory lung volume. If this mechanism is applied to our genetic mouse model, then strain differences in hypoxic ventilatory phenotypes may be influenced by lung structural variation. For example, strain differences in elastic recoil properties likely emerge to influence TE responses during challenge (Fig. 3D). Although it is reasonable to suggest that interactions between lung mechanics and hypoxic ventilation explain C3 and B6 variations, phenotypic differences between B6 and F1 mice are not likely attributable to lung structure because lung volume and compliance are similar in these mice. Hence, alternative ventilatory control mechanisms, such as chemosensitivity or central integration, appear to be modified by a combination of C3 and B6 alleles inherited by F1 mice.
Variation in mechanisms of chemosensitivity and integration is a
hypothesis supported by C3 and B6 strain differences in
E responses to mild hypercapnia (25, 26).
The results of the present study advance this hypothesis by suggesting
that a reduced hypercapnic E
response, a phenotype of C3 mice, is inherited by the F1 offspring
(Fig. 2). Therefore, our model of the hypoxic hypoventilation
characteristic of F1 mice incorporates a weak response to inspirate
changes in CO2. Although attenuated
E responses of C3 mice differ from B6
mice in mild hypercapnia, the parental responses are not
distinguishable when the same inspirate challenge includes hypoxia. The
E responses of F1 mice, however, remain
significantly lower than those of the B6 progenitors. This attribute of
F1 mice is accompanied by a markedly attenuated
VT/TI during MNX and MHX (Fig. 3E),
which may indicate a reduction in the neural "drive" associated
with inspiration during chemical challenges. In summary, although
E responses at baseline do not differ
among the three groups, C3 and F1 mice demonstrate a lower
CO2 ventilatory sensitivity relative to B6 mice.
One unifying hypothesis considers a simplified model in which C3 and B6 mice differ in two major ways. First, advantages derived from greater lung volume and compliance incrementally dictate the magnitude and pattern of breathing during MNX and MHX challenges in C3 mice (28). On the other hand, the ventilatory response to chemical challenge in B6 mice is progressively governed by a robust ventilatory chemosensitivity relative to C3 mice (25). Therefore, it seems logical to postulate that the hypoventilatory phenotype of F1 mice can be anticipated if interactive alleles for small lung volume and blunted chemosensitivity are dominant in heterozygous genotypes. In the most parsimonious model, these prior expectations would suggest that at least two major genetic determinants influence C3 and B6 strain differences in HVR.
Segregation analyses. Although heritability of MNX and MHX ventilatory traits can be assessed with the use of response distributions of C3, B6, and F1 mice, specific genetic hypotheses, including single-gene, two-gene, and polygenic models, can be tested by incorporating responses obtained from backcross and intercross progeny. When considering these hypotheses in the present study, the objective was to evaluate the response variation among backcross and intercross progeny to determine whether these distributions fit patterns of inheritance ascribable to C3, B6, and F1 mice. For each ventilatory trait at baseline and during challenge, specific inheritance patterns emerge to enumerate major gene effects and to advance potential gene interactions. By evaluating each trait-treatment combination in this way, critical mechanisms of ventilatory control are underscored as models having relatively simple genetic bases. These results also serve to support future linkage studies that explore genotype-phenotype associations.
Recent studies have applied a similar approach to C3 and B6 differences in baseline breathing pattern (27). As reiterated in Table 1, response variation in f and TI at baseline is shown to be consistent with a two-gene model. In addition, strain differences in TE and TI/Ttot at baseline are traits inherited in a pattern consistent with two-gene models. The two-unlinked equal and additive locus model for TE at baseline ranks among the strongest evidence of a genetically determined ventilatory trait in this study. In general, the inheritance patterns associated with C3 and B6 differences in ventilation at baseline appear to emphasize strain variation in respiratory timing mechanisms, and, in particular, the TI and TE results warrant further consideration. In this regard, a recent study showed significant linkage results, suggesting that a quantitative trait locus, assigned the symbol Itbq1, on mouse chromosome 3 determines a substantial proportion of strain variation in TI at baseline between C3 and B6 mice (24). With respect to hypoxic ventilatory traits, results from the present study suggest that strain variation inE during MHX is determined by a
relatively small number of genetic determinants (Table 1). As shown in
Fig. 4A, the attenuated hypoxic E
response of F1 mice is conferred to a subgroup of C3BX and F2 progeny. These results support the hypothesis that alleles derived from the C3
progenitor contribute to the hypoxic hypoventilatory phenotype determined by a blunted sensitivity to chemical challenge. Strain differences in f responses during hypoxic challenge are also
consistent with a two-unlinked equal and additive locus model. These
results appear to be reflected in the TI responses during
MHX. In Fig. 4C, a majority of TI responses of B6BX
and F2 mice resemble B6 and F1 responses. In contrast, a small number
of F2 and many of the C3BX responses mimic C3 mice. Like the
inheritance patterns associated with ventilatory responses at baseline,
strain variations in HVR highlight respiratory timing mechanisms. The
two-unlinked locus models for TI and
TI/Ttot are associated with relatively high
probabilities (Table 1), which suggest that these inheritance patterns
have relatively simple genetic bases and warrant further consideration.
Whether there are common genetic determinants regulating respiratory
timing mechanisms during both baseline and MHX treatments is unclear.
However, when this question was addressed with BXH recombinant inbred
strains, the results suggested that the genetic regulation of hypoxic
TI responses does not cosegregate with TI responses at baseline (23). It is clear that hypoxic
TI/Ttot responses provided a relatively robust
indication that this trait is determined by a small number of genes. As
shown in Fig. 4D, TI/Ttot responses
above 50% occur in both backcross and intercross progeny. Therefore,
the combination of alleles that regulates the atypical
TI/Ttot response of F1 mice is inherited by
backcross and intercross progeny.
Segregation analysis also supports the conclusion that C3 and B6 strain
differences in VT responses during mild hypercapnia are
determined by two genes (Table 1). Because only a moderate increase in
VT from MNX to MHX was observed in C3 and F1 mice, the
significance of the VT responses during both MNX and MHX
may indicate that the genetic mechanisms regulating differential
VT responses are common between the two inspirate
challenges (Fig. 3B). As shown in Fig. 5A, the range of
responses in the backcross and intercross offspring classes
incorporates individuals that resemble responses of F1 mice with
respect to reductions in VT during MNX. The two-unlinked
equal and additive locus model associated with
VT/TI responses during MNX represents the best
fit relative to all other trait-treatment combinations. The attenuated
hypercapnic VT/TI response characteristic of C3
and F1 mice is largely inherited by C3BX and F2 progeny. Therefore, if
blunted CO2 response is a phenotype of the C3 progenitor
and VT or VT/TI represents affected variables, as few as two genes determine this outcome. Furthermore, the
moderate VT response from MNX to MHX in F1 mice (Fig.
3B) supports the position that F1 mice respond to MNX and MHX
in a manner similar to B6 mice in terms of the f phenotype but that the
VT phenotype of F1 mice is unique.
Perspectives. In our inbred mouse model, ventilatory responses at baseline and during acute inspirate challenges are shown to be complex traits largely influenced by a discrete number of genetic determinants. Although different combinations of f and VT can be implemented to achieve a given ventilatory outcome, adjustments in the respiratory cycle from baseline to hypoxic ventilation generally involve increased inspiratory drive, decreased expiratory time, and an abbreviated inspiratory "off switching" (16). These ventilatory control mechanisms appear to be operating differently among C3, B6, and F1 mice. In particular, variation in inspiratory drive is genetically determined in this model and emerges as a critical trait predominantly affecting inspirate changes in CO2. Although acute hypoxia also induces a decreased expiratory time in C3, B6, and F1 mice, variation in this trait may prove to be most influenced by lung volume and compliance differences between strains. In this model, other mechanisms, independent of pulmonary mechanics, dictate differences in HVR between B6 and F1 mice. Finally, hypoxia appears to differentially influence the inspiratory off-switching mechanism in C3, B6, and F1 mice. The relative decrease in TI from baseline to hypoxia is greater in C3 mice compared with B6 and F1 mice (Figs. 1 and 3C). In addition, the inspiratory fraction of F1 mice is unique relative to the parental strains (Fig. 3F). As the inspiratory fraction rises and approaches 50% in response to hypoxia, the respiratory cycle approximates a sinusoid, which implies inspiratory and expiratory flows are equal. Any additional increases in ventilation must occur by incremental increases in VT or concomitant decreases in both TI and TE. Together, these observations suggest that mechanisms regulating the transition from inspiration to expiration may be genetically different among C3, B6, and F1 mice.
Limitations. Although the genomic location of alleles that regulate differential ventilatory responses among C3, B6, and F1 mice remains to be elucidated, our exploratory studies lead us to believe that the genetic control of ventilation is limited to the 19 autosomes. In relatively small samples (data not shown), there are no gender differences in either of the parental strains. The absence of gender dimorphisms suggests that the sex chromosomes are not likely to be the origin of major genetic effects. In addition, the ventilatory responses of the reciprocal F1 (i.e., female C3 × male B6, C3B6F1) are comparable to the F1 (i.e., B6C3F1) results reported in this study. This precludes an effort to investigate the reciprocal intercross and excludes the role of genetic imprinting or DNA variation originating from mitochondrial genes. Finally, our experiments were conducted during the period of a circadian cycle in which mice are likely to sleep. Because sleep/wake state was not measured during our studies, we are uncertain of the extent to which our results reflect state dependence. Although baseline measurements may incorporate state-dependent effects, the ventilatory responses during MNX and MHX are less likely affected because animals are routinely alerted by acute environmental changes such as modifying the inspirate gas composition.
In this study, dissecting HVR illustrates the complex nature inherent to the genetic basis of ventilatory control mechanisms. The equivocal results in human studies appear more plausible after similar hypotheses were tested in our simple genetic model. Although the variation of HVR among human volunteers spans a six- to sevenfold difference between low and high responders (10), the results from the present study appear to incorporate a fivefold difference in hypoxicE between low and high responders
(i.e., F2 progeny in Fig. 4A). This species comparison is
not meant to suggest that individual variation in hypoxic response in
humans follows a relatively simple genetic basis such as shown with
results in mice. In contrast, the simple genetic models described in
the present study could be quite different if other standard inbred
strains of mice are used as progenitors. In summary, in the two strains
studied, differential ventilatory traits during acute hypoxia are
determined by a relatively small number of genetic determinants that
predominantly influence the control of respiratory timing mechanisms.
This conclusion differs from the genetic control of traits associated
with hypercapnia. Furthermore, alleles derived from the C3 and B6
parental strains interact to confer hypoxic hypoventilatory phenotypes
in the first- and second-filial generations.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. Machiko Shirahata for critical review of this manuscript.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-53700 and National Institute of General Medical Sciences Grant GM-28356. Results from the segregation analysis reported in this manuscript were obtained using the SAGE program, which is supported by a Public Health Service Resource Grant (1-P41-RR-03655) of the National Center for Research Resources.
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. §1734 solely to indicate this fact.
Original submission in response to a special call for papers on "Hypoxia Influence on Gene Expression."
Address for reprint requests and other correspondence: C. G. Tankersley, Division of Physiology, School of Hygiene and Public Health, The Johns Hopkins Univ., 615 N. Wolfe St., Baltimore, MD 21205.
Received 9 February 2000; accepted in final form 21 March 2000.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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P < 0.01 F1 vs. B6.
