Division of Pulmonary/Critical Care Medicine, The Burns and Allen Research Institute, Cedars-Sinai Medical Center, University of California Los Angeles School of Medicine, Los Angeles, California 90048
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ABSTRACT |
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The
impact of a targeted disruption of the Igf1 gene, encoding the
insulin-like growth factor I (IGF-I), on diaphragm (DIA) cellularity
was studied in 2-mo-old homozygous mutant
[IGF-I(/
)] mice and their wild-type
[WT; i.e., IGF-I(+/+)] littermates. DIA fiber types were
classified histochemically. DIA fiber cross-sectional areas (CSA) were
determined from digitized muscle sections, and fiber succinate
dehydrogenase (SDH) activity was determined histochemically using a
microdensitometric procedure. An acidic ATPase reaction was used to
visualize capillaries. Myosin heavy chain (MyHC) isoforms were
identified by SDS-PAGE, and their proportions were determined by
scanning densitometry. The body weight of IGF-I(
/
)
animals was 32% that of WT littermates. DIA fiber type proportions
were unchanged between the groups. The CSAs of types I, IIa, and IIx DIA fibers of IGF-I(
/
) mutants were 63, 68, and 65%,
respectively, those of WT animals (P < 0.001). The DIA
thickness and the number of fibers spanning its entire thickness were
reduced by 36 and 25%, respectively, in IGF-I(
/
) mice
(P < 0.001). SDH activity was significantly increased in all
three types of DIA fibers of IGF-I(
/
) mutants (P < 0.05). The number of capillaries per fiber was reduced ~30% in
IGF-I(
/
) animals, whereas the capillary density was
preserved. The proportions of MyHC isoforms were similar between the
groups. Muscle hypoplasia likely reflects the importance of IGF-I on
cell proliferation, differentiation, and apoptosis (alone or in
combination) during development, although reduced cell size highlights
the importance of IGF-I on rate and/or maintenance of DIA fiber growth
in the postnatal state. Reduced capillarity may result from both direct
and indirect influences on angiogenesis. Improved oxidative capacity
likely reflects DIA compensatory mechanisms in IGF-I(
/
) mutants.
insulin-like growth factor I gene deletion; respiratory muscles; diaphragm fiber size, proportions and number; oxidative capacity; capillarity; myosin heavy chain isoforms
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INTRODUCTION |
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MICE HOMOZYGOUS FOR a targeted disruption of the gene
encoding the insulin-like growth factor I (IGF-I), i.e., Igf1
gene deletion, have recently been described (3, 30, 36). IGF-I is the principal polypeptide growth factor mediating the growth-promoting actions of growth hormone (28). Although the liver is the major source
of circulating IGF-I (i.e., hormonal action), expression of the peptide
within a muscle allows autocrine/paracrine effects locally (28). IGF-I
plays an important role in myogenesis and in the maintenance of
growth/integrity of muscle fibers (17). Indeed, the Igf1 gene
knockout models highlight the importance of IGF-I during embryogenesis,
as significant growth retardation was observed in utero (3, 36). At
birth, the body weights of homozygous mutant
[IGF-I(/
)] mice were ~60% that of
wild-type [WT; i.e., IGF-I(+/+)] littermates (30, 36). Most
of the IGF-I(
/
) pups died at or soon after birth due to
possible "ventilatory failure" (~84-95%, depending on
genetic background; see Refs. 30 and 36). Surviving
IGF-I(
/
) animals continued to exhibit significant
growth retardation such that their body weight at day
60 was only ~30% that of WT adult mice (3). Powell-Braxton and
co-workers (36) observed a reduction in muscle mass. They evaluated
diaphragm, tongue, and heart muscles histologically in neonatal
homozygous IGF-I(
/
) mutants and reported "generalized muscular dystrophy," characterized by vacuolization of muscle fibers
and apparent decreased amounts of myofibrillar material. Subjectively,
muscle fiber cellularity (i.e., number of muscle fibers) did not appear
to be reduced (36). No quantitative measures or analyses were employed,
however, in the analysis of diaphragm or other muscle specimens (30,
36). The diaphragm is the most important inspiratory muscle of
respiration (10). We reasoned therefore that severe derangements in its
cellular composition and/or morphometric indexes of constituent fibers
may explain in part the potential for failure of the respiratory pump
and associated early mortality or reduced life expectancy associated with the IGF-I(
/
) mutation (30, 36).
The aim of this study was therefore to assess, in a quantitative
fashion, morphometric and biochemical indexes of individual diaphragm
muscle fibers in both adult male mice homozygous for a targeted
deletion of the Igf1 gene and WT littermates. We
postulated that both fewer and smaller-size diaphragm muscle
fibers would be noted in the adult IGF-I(/
) mutants,
based on the proliferative, differentiating, and growth-promoting
actions of IGF-I in muscle cell cultures (17).
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METHODS |
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Animal Model and Groups
A CD-1 mouse line was used for the generation of the IGF-I(Genotyping
The genotypes of the mice were determined by analyses of genomic DNA extracted from tail clips obtained before weaning. DNA isolation was performed according to specifications of the manufacturer (Puregene DNA Isolation Kit; Gentra Systems). Samples of DNA were amplified with PCR as follows: 35 cycles in 100-µl reactions in PCR buffer containing 1 mM of each dNTP and 40 U/ml of Taq DNA polymerase and oligonucleotides primers. For detection of the WT allele, primers were derived from sequences upstream of exon 3 (I1 = 5'-GACCAGTAGCAAAGGACTTACCAC and 3' of the neomycin insertion in the exon; I2 = 5'-AAGTAAAAGCCCCTCGGTCCACAC), resulting in a 366-bp product. For detection of neomycin, primers (N1 = 5'-TGACTGGGCACAACAGACAATCGG and antisense N2 = 5'-GTAGCCAACGCTATGTCCTGATAG) producing a 608-bp fragment were used. To give bands of an equal intensity in the heterozygotes, a ratio of 4:1 IGF-I-neomycin primer was used in each reaction (see Fig. 1).Histochemical Procedures
Muscle preparation and fiber morphology . Under deep general anesthesia [ketamine (100 mg/kg ip) and xylazine (7.5 mg/kg ip)], two adjacent strips of the midcostal diaphragm were dissected. The first segment of diaphragm was frozen in liquid nitrogen and was used for biochemical analysis. Excised length was determined upon dissection of the second diaphragm segment. The second strip of diaphragm was mounted on cork at this resting excised length between liver segments (to facilitate sectioning of the extremely thin delicate diaphragm muscle) and then rapidly frozen in isopentane, which had been cooled to its melting point by liquid nitrogen. Serial cross sections (5 µm thickness) were cut using a cryostat (Reichert-Jung; model 2800E), rapidly fixed in cold acetone, and processed for hematoxylin and eosin (H&E).
Fiber type proportions. Muscle fibers were classified based on myofibrillar ATPase (mATPase) after alkaline (pH = 9.6) and acid (pH = 4.0, 4.25, and 4.45) preincubations from serial sections of 10 µm thickness (7, 12). One serial section was fixed in 2% paraformaldehyde at pH = 7.4 for 2 min at room temperature and then was preincubated at pH = 10.4 [modification (21) of the method by Guth and Samaha (25)]. These staining procedures allow the classification of fibers into types I, IIa, IIb, IIx, and IIc (21, see also Ref. 24). In each muscle, at least 700 fibers were analyzed. Previously, we verified concordance between immunohistochemical [i.e., identification of myosin heavy chain (MyHC) phenotype] and histochemical classification of fiber types, with 95% or more correspondence between the mATPase-based and the major isoform of MyHC expressed in single muscle fibers (21, 24, see also below).
Fiber cross-sectional areas. Muscle fiber cross-sectional area was determined from digitized muscle sections, using a computer-based image processing system composed of a Leitz Laborlux S (Leica) microscope, CCD video camera system (model VI-470; Optronics Engineering, Goleta, CA), high-resolution Trinitron color video monitor (model PVM-1343MD; Sony), 486 DX-50 MHz PC with a Targa+ imaging board (Truevision,), and Mocha image analysis software (version 1.20; Jandel, San Rafael, CA). A stage micrometer was used to calibrate the imaging system. The cross-sectional area of 200-300 individual fibers (i.e., sampled from those used in the analysis of fiber proportions) was determined from the number of pixels within manually outlined fiber boundaries.
Relative number of fibers. Costal diaphragm thickness was determined from multiple measurements (8 per sample) along the midcostal region. The number of fibers spanning the diaphragm thickness (i.e., from abdominal to thoracic surface) was determined at each measurement site to determine a relative index of muscle fiber number.
Fiber succinate dehydrogenase activity.
Fiber oxidative capacity was determined by quantifying the activity of
succinate dehydrogenase (SDH; a key enzyme in the tricarboxylic acid
cycle) in individual muscle fibers as previously described (5, 6, 12,
44). Briefly, in the histochemical reaction for SDH, the progressive
reduction of nitroblue tetrazolium (NBT) to an insoluble colored
compound [a diformazan (dfz)] is used as a reaction
indicator. The reduction of NBT is mediated by H+ released
in the conversion of succinate to fumarate. Sections (6 µm) were
incubated with and without (tissue blanks) succinate (60 mM) as
substrate. The concentration of NBT-dfz deposited within a muscle fiber
was calculated using the Beer Lambert equation
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Fiber capillarization. An acidic ATPase reaction was used to visualize capillaries surrounding individual muscle fibers from 10-µm-thick serial muscle cross sections (12, 45). This technique has been validated by our group against other commonly used methods [i.e., amylase-periodic acid-Schiff (44) and alkaline phosphatase (unpublished results; also see Ref. 45)]. This ATPase method identifies only the arterial end of the capillary bed by staining the capillary endothelium. Furthermore, the ATPase technique used in this study does not distinguish between perfused and nonperfused vessels. The indexes of capillarity determined were 1) capillary-to-fiber ratio, i.e., the total number of capillaries divided by the total number of fibers within the muscle section and 2) capillary density, i.e., the number of capillaries per unit cross-sectional area.
Immunohistochemical Identification of MyHC Isoforms
Anti-MyHC monoclonal antibodies (41) were used for the indirect immunoperoxidase identification of MyHC isoforms. Serial cryosections (10 µm thickness) matching the ATPase stains were exposed to one of the following antibodies (diluted in PBS): BA-D5 (1:10) reacting with MyHC 1; SC-71 (1:10) reacting with MyHC 2A; BF-F3 (1:10) reacting with MyHC 2B; BF-35 (1:20) reacting with all MyHCs except 2X; and BF-B6 (1:10) reacting with embryonic and neonatal MyHCs. These mouse antibodies (IgG1) were generously provided by Regeneron Pharmaceuticals (Tarrytown, NY). Sections were then rinsed and exposed to peroxidase-conjugated secondary antibody (horse anti-mouse IgG; 1:200) while control sections were exposed only to secondary antibodies. Visualization was obtained after diaminobenzidine reaction and nickel amplification.Electrophoretic Identification of MyHC Isoforms
For the myofibril extraction, 10-mg muscle samples were homogenized by hand on ice in 20 vol of cold buffer (46). The homogenate was centrifuged at 4°C, the supernatant was discarded, and the pellet was resuspended. The final pellet was resuspended in 10 vol of the buffer. Myofibril content was determined using a micro-bicinchoninic acid (BCA) protein assay kit (Pierce) and was quantified using an ELISA reader at 550 nm. Samples of purified myofibrils were diluted 1:8 in a denaturing sample buffer. Final myofibrillar protein concentration was ~0.125 µg/µl. Proteins were denatured in the same buffer by boiling samples for 2 min before loading. The determination of the MyHC composition was performed using an SDS-PAGE technique with 8% separating gels (48). Each well was loaded with 0.7-0.9 µg of protein extract. Electrophoresis was performed using a Bio-Rad Mini-Protean II system for a duration of 25 h at constant 80 V with running buffers kept at 4-7°C.The gels were stained with silver nitrate (Bio-Rad Silver Stain Plus
kit). Dried stained gels with duplicate samples were scanned two times
using a UVP Image Store 5000 system, and densitometric measurements
were performed with its Gel Documentation and Software System. After
background subtraction, the relative contribution of each band within a
gel was determined by the ratio of the total gray level within the area
of a specific band to that of the cumulative gray level of all the
bands present in a sample. The specificity of each band has been
demonstrated based on immunoblotting identification after
electrophoretic transfer (41). This method allows the separation of
MyHC 1 (/slow), 2A, 2B, 2X, embryonic, and neonatal isoforms from
denatured myofibrils of skeletal muscle. In adult muscle, the fastest
migrating band corresponds to MyHC 1 (
/slow), followed in order by
MyHC 2B, MyHC 2X, and MyHC 2A. The densitometric analysis of each
identified MyHC isoform was performed in duplicate on two samples for
each muscle, and the average relative content of each MyHC isoform was estimated.
Statistical Analysis
The distribution of all data was tested for normality before parametric analyses. Statistical analysis of group means was then performed (GraphPad Instat, GraphPad Software, version 2.04) using an unpaired Student's t-test. An alpha level of 0.05 was used to compare differences in independent groups and to determine overall significance. All data are represented as means ± SD. ![]() |
RESULTS |
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Animal Genotyping
PCR analysis was used to confirm the genotype of all animals. Figure 1 depicts an example of such analysis for genotyping of representative animals performed just before weaning, showing WT (+/+) mice together with heterozygous (+/
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Animal Body Weights
At 2 mo of age, the body weight of IGF-I(Histochemical Studies
Fiber morphology.
Based on the H&E staining of muscle sections, there was no evidence of
histopathology in diaphragm fibers of IGF-I(/
) animals (Fig. 2). No obvious structural differences
in the appearance of the morphological features of the diaphragm fibers
between IGF-I(
/
) mutants and WT littermates were observed
other than the average smaller size of individual fibers in the
IGF-I(
/
) mice (Fig. 2; see also below).
|
Fiber proportions.
The mATPase stain of individual diaphragm fibers of
IGF-I(/
) animals was also similar to that of WT mice (not
shown). No differences were observed in the proportions of diaphragm
muscle fibers between IGF-I(
/
) and WT animals (Fig.
3A). Because a small
proportion of fibers classified as type IIc were present in diaphragm
of IGF-I(
/
) mice only, there was a question as to the
presence of immature types of muscle fibers. Serial muscle sections
were therefore analyzed for the MyHC phenotype, thus allowing a direct
comparison in the same individual muscle fibers between histochemical
and immunohistochemical techniques. The type IIc fibers from these
serial sections of IGF-I(
/
) mutants were found to
coexpress MyHC 1 and 2A isoforms only. There was no expression of
developmental (i.e., embryonic and neonatal) MyHC isoforms within any
of the diaphragm fibers. Overall, the diaphragm MyHC profile
corresponded well with the mATPase staining.
|
Fiber cross-sectional areas.
The cross-sectional areas of all diaphragm fibers were
significantly reduced in the IGF-I(/
) group compared with
WT animals (P < 0.001; Fig. 3B). In
IGF-I(
/
) animals, the cross-sectional areas of types I,
IIa, and IIx fibers were 63, 68 and 65%, respectively, those observed
in WT littermates (Fig. 3B). Taking into account fiber
proportions and cross-sectional areas, the estimated relative contribution of the different fiber types to total costal diaphragm area was preserved in the IGF-I(
/
) group (Fig.
3C).
Relative number of fibers.
The thickness of the diaphragm in IGF-I(/
)
mutants was reduced by 36% compared with WT siblings (P < 0.001; Table 1). The number of fibers
spanning the entire thickness of the diaphragm from thoracic to
abdominal surfaces was significantly reduced in IGF-I(
/
)
animals compared with WT littermates (P < 0.001; Table 1).
Thus the relative number of fibers in the diaphragm of
IGF-I(
/
) animals was reduced by 25%, suggesting marked
muscle hypoplasia compared with WT mice.
|
Fiber oxidative capacity.
Mean SDH activity was found to be significantly higher in all
types of fibers, i.e., types I (17%), IIa (14%), and IIx (23%), within the diaphragm of IGF-I(/
) mice compared with WT
animals (P < 0.05; Fig. 4).
|
Fiber capillarity.
The average number of capillaries per fiber (i.e.,
capillary-to-fiber ratio) was reduced by 30% in the
IGF-I(/
) mutants compared with WT littermates (1.44 ± 0.15 vs. 2.05 ± 0.29; P < 0.05). The number of capillaries
per unit area (i.e., capillary density) was not significantly different
between IGF-I(
/
) mutants and WT mice (3.28 ± 0.04/µm2 × 10
3 vs. 2.57 ± 0.73/µm2 × 10
3).
MyHC Isoforms
The MyHC phenotype of the diaphragm of adult mice was evaluated by SDS-PAGE and revealed the presence of four bands. The fastest migrating band within the gel corresponds to MyHC 1, followed in order by MyHC 2B, MyHC 2X, and MyHC 2A isoforms (Fig. 5A). The proportions of MyHC isoforms were not significantly altered in IGF-I(
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DISCUSSION |
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Mice homozygous for a targeted disruption of the Igf1 gene
exhibited marked growth retardation, diaphragm muscle hypoplasia, reduced diaphragm fiber size, reduced diaphragm fiber capillarity, preserved capillary density, and increased oxidative capacity of muscle
fibers. No evidence of muscle fiber dystrophy or loss of fiber
structural integrity was noted in the IGF-I(/
) mice.
Diaphragm Fiber Number
Mitogenic responses to IGF-I have been well characterized in vitro using a variety of different cell types, including skeletal muscle cells (27, 33; also see Ref. 17 for review). It appears from cell culture experiments that proliferative responses to IGF-I precede myogenic differentiation and that, although proliferative responses ensue, myogenic differentiation is inhibited through a process independent of its proliferative effects (13, 15, 40). Thus the reduced number of diaphragm muscle fibers (i.e., hypoplasia) noted in the present study may reflect impairments in the proliferative phase, the differentiation process, programmed cell death, or varied combinations of these developmental processes.With regard to impairment in proliferation, Igf1, Igf2,
and Igf1 receptor (IGF-IR) gene deletion models suggest that
muscle hypoplasia may be the result of fewer proliferative events
arising from an altered cell cycle rate (3). For example, a
prolongation of cell cycle time has been reported in fibroblasts
isolated from IGF-IR(/
) embryos (42). In addition, a
recent study on in vivo cell cycle dynamics in uterine cells of
IGF-I(
/
) mice demonstrated a significantly prolonged
transit through the G2 phase of mitosis (1). IGF-I
activates the D-type cyclins as an early event in the proliferation of
cultured myoblasts (13). Because mice lacking the cyclin D1 gene are
also characterized by a phenotype of reduced growth (43), lack of IGF-I
may thus exert indirect effects with regard to cell size and number.
With regard to the differentiation process, Florini and Ewton (16) reported that the IGFs are potent stimulators of terminal myoblast differentiation in vitro and that IGF-I induces expression of myogenin, a transcription factor belonging to the MyoD myogenesis gene family (18). Transgenic mice overexpressing IGF-I in muscle have demonstrated accelerated myogenic differentiation (8). We therefore cannot discount that absence of IGF-I results in impaired differentiation as described in muscle cell cultures in which IGF-I antisense oligonucleotides were introduced (19). It is of interest to note that reduced numbers of limb and diaphragm muscle fibers were reported in models of prenatal undernutrition, a condition associated with reduced IGF-I levels (11, 37, 50). The reduction in fiber numbers was reported to be related to impaired secondary myotube formation (11, 50).
With regard to programmed cell death, or apoptosis, there are data to
suggest that IGF-I may prevent premature cell death in a variety of
cell types, perhaps best characterized in hematopoietic cells (49).
IGF-I, IGF-II, and insulin have also been shown to inhibit
differentiating myoblast cell death in cultured C2 muscle cell
lines (47). Thus we cannot preclude that the absence of IGF-I may have
contributed to accelerated apoptosis in IGF-I(/
) mutants,
resulting in fewer myoblasts proceeding to terminal differentiation.
In addition, it is unclear if the presence of intact IGF-II and IGF-IR
function during embryogenesis compensated to some extent to minimize
the degree of hypoplasia noted in IGF-I(/
) mice (14).
This possibility is suggested from the more severe phenotypes exhibited
in IGF-IR(
/
) mice and in double mutants after targeted disruption of both Igf1 and Igf2 genes compared with
either single knockouts (30).
Diaphragm Fiber Size and Integrity
The IGFs are potent stimulators of terminal myoblast differentiation in vitro (16), and IGF-I induces expression of myogenin, a transcription factor belonging to the MyoD myogenesis gene family (18). The present study suggests that myogenic differentiation occurred, as evidenced by the absence of dystrophic changes in individual muscle fibers, normal fiber type proportions, the presence of only adult MyHC isoforms, and similar amounts of total MyHC and myofibrils per gram of diaphragm noted in WT littermates. This suggests that intact IGF-II and IGF-IR function, was together with other myogenic factors during embryogenesis, was sufficient to promote terminal myoblast differentiation in the absence of IGF-I (17, 26). However, in the IGF-IR(We postulate therefore that reduced diaphragm fiber size reflects
influences after terminal differentiation. IGFs pre- and postnatally
promote anabolism and growth due to enhanced amino acid uptake and
protein synthesis and likely reduced protein degradation (28). During
embryogenesis, both IGF-I and IGF-II play a key growth-promoting role;
however, in postnatal life, serum levels and physiological roles of
IGF-II are considerably diminished (9). The significant growth
retardation after birth [body weight ~60% at birth to ~30%
of normal at 2 mo of age (3, and present study)] supports the
importance of IGF-I in the maintenance of growth. The significant
reduction in the cross-sectional areas of all diaphragm fiber types in
the present study is thus compatible with the reduced rate and/or
maintenance of muscle fiber growth. Although the reduction in diaphragm
fiber size appears less than for body weights in IGF-I(/
)
survivors, failure to account for all factors making up cell volume
(e.g., reduced muscle fiber length) may underestimate muscle effects.
Alternatively, we cannot discount differing tissue specificity to the
lack of IGF-I and/or the modulating influences of other recently
identified anabolic or repressive mediators on diaphragm muscle fibers
[e.g., interleukin-15 (39), glial growth factor 2 (20),
growth/differentiation factor 8 (32)].
MyHC, SDH, and Capillarity
The proportions of MyHC isoforms in the IGF-I(The increment in oxidative capacity in diaphragm fibers in
IGF-I(/
) mutants suggests an adaptive response, possibly
due to increased recruitment and/or frequency coding of diaphragm motor
units compared with WT animals. The source of this adaptation is
unknown but could result from increased diaphragm work to distend a
structurally abnormal lung and/or to compensate for a weaker diaphragm
muscle secondary to hypoplasia. In addition, we have previously
evaluated the effects of IGF-I administration on the SDH activity of
rat diaphragm fiber types and have observed no effects (29). Increased
oxidative capacity of skeletal muscle has also been reported to occur
after exercise training under conditions such as malnutrition, which is
known to reduce serum IGF-I levels (22). These data strengthen our
postulate that the increased SDH activity noted in the present study
primarily reflects a response to enhanced recruitment, as opposed to
direct or indirect influences of Igf1 deletion.
The possible mechanisms accounting for fewer capillaries per fiber in
the IGF-I(/
) mutants most likely reflect the absence of
IGF-I, which has been reported to have both direct angiogenic properties (e.g., 2, 34) and indirect angiogenic effects mediated by
enhancing the gene expression of vascular endothelial growth factor
(23, 38). By contrast, there is no evidence that other conventional
angiogenic factors [basic fibroblast growth factor (FGF), acidic
FGF, platelet-derived growth factor, and transforming growth
factor-
] and newly described factors (e.g., angiopoietins, ephrins, leptin, and chemokines) are directly influenced by IGF-I. From
the current data, it is not possible to weigh the relative importance
of direct vs. indirect effects of the absence of IGF-I on diaphragm
capillarity. It should be noted, however, that, despite the decreased
capillary-to-fiber ratio, the preserved number of capillaries per unit
area suggests adequate diffusion of oxygen and other substrates. Thus,
although we postulate that changes in SDH activity reflect altered
activation history of the diaphragm in IGF-I(
/
) mutants,
the preserved capillary density negates the need for further adaptation
and therefore any need to increase the local microcirculation through
non-IGF-I-dependent pathways.
Functional Implications and Significance
The impact of targeted disruption of the Igf1 gene on growth (i.e., body weight) starts to take place on embryonic day 13.5 (3). At this time, primary myotube formation is complete, and there is a 2-day hiatus before the appearance of the second generation of myotubes (35). It is likely that the normal number of primary myotubes was preserved in the IGF-I(The IGF-I knockout model also demonstrates the importance of IGF-I in the maintenance of body growth and muscle fiber size and may be of value in evaluating the importance of IGF-I in several conditions associated with very low IGF-I levels (e.g., severe chronic malnutrition/wasting syndromes due to a variety of causes). The impact of low IGF-I on the diaphragm is of major importance as the diaphragm is rhythmically active throughout life and necessary for effective alveolar ventilation. The present model suggests a reduced total force-generating capacity and force reserve of the diaphragm. This is perhaps best evidenced by the low survival rate (5%) of IGF-I knockout mice, with nonsurvivors thought to succumb from respiratory failure.
In summary, the major effects of Igf1 gene deletion on the
diaphragm are 1) reduced muscle fiber number (hypoplasia) and
capillarity and 2) reduced anabolic effects, most notably in
the postnatal state, reflecting a reduced rate and/or maintenance of
growth of muscle fibers as evidenced by reduced cell size. Reduced
diaphragm fiber number may reflect influences of Igf1 gene
deletion on cell proliferation, differentiation, and programmed cell
death either alone or in combination. Reduced diaphragm capillarity may
result from impaired angiogenesis in the IGF-I(/
) mice.
By contrast, biochemical alterations in oxidative capacity of
individual diaphragm muscle fibers likely reflect increased activation
of the diaphragm in surviving adult IGF-I(
/
) mutants.
These findings likely have wide applicability to a variety of other
skeletal muscles.
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ACKNOWLEDGEMENTS |
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We acknowledge the advice, encouragement, and support of Dr. Lyn Powell-Braxton (Genentech) and Dr. Ken Dorshkind for help in developing our breeding colonies. We also thank Dr. Philip Barnett for critical review of the manuscript, Xiayou Da and Dr. Nancy Wu for technical assistance, and Debbie Craig for secretarial assistance.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grant HL-47537.
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.
Address for reprint requests and other correspondence: M. Fournier, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Rm. 6732, Los Angeles, CA 90048 (E-mail: mario.fournier{at}cshs.org).
Received 26 October 1999; accepted in final form 29 October 1999.
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