Influences of IGF-I gene disruption on the cellular profile of the diaphragm

Mario Fournier and Michael I. Lewis

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Model and Groups

A CD-1 mouse line was used for the generation of the IGF-I(-/-) mutants by homologous recombination in embryonic stem cells whereby a neomycin resistance gene expression cassette was inserted in a targeting vector at amino acid 15 of the mature protein (exon 3) to interrupt its coding sequence (36). Heterozygous [IGF-I(+/-)] animals were identified using tail DNA and were interbred to generate animals in which both IGF-I alleles were inactivated [i.e., the homozygous IGF-I(-/-) mutants]. Because <= 5% of IGF-I(-/-) animals survived, multiple breeding colonies were developed to generate adult survivors. Young adult male mice from six different litters were studied at 2 mo of age, and IGF-I(-/-) mutants (n = 6) were compared with their WT littermates (n = 6).

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
[NBT-dfz] = <FR><NU>OD</NU><DE><IT>k</IT> × <IT>L</IT></DE></FR>
where OD is the optical density of the muscle fiber measured at 570 nm (the peak absorbance wavelength for NBT-dfz), k is the molar extinction coefficient for NBT-dfz (26,478 mol/cm), and L is the path length (i.e., 6 µm section thickness) for light absorbance. The OD of muscle fibers was determined using a microdensitometric procedure implemented on the computer-based image processing system. The video image was then digitized (8-bit gray level resolution) in a matrix of 1,024 × 1,024 pixels (picture elements). The gray levels of the video scanner were calibrated for photometry (OD units) using a series of neutral density filters (0.004-2.00 OD units; Melles Griot, Irvine, CA). During the SDH reaction, the formation of NBT-dfz in diaphragm fibers increases linearly over a period of at least 7-9 min (6). In reactions where succinate was absent from the reaction medium, there was measurable staining (i.e., reduction of NBT), but the OD did not change significantly across the same time periods. The tissue blank OD also corresponded to the OD measured at time 0 in reactions where succinate was present in the medium. Based on these data, we justified the use of a single end-point measurement of OD at 5 min. From these measurements, a rate of SDH reaction was interpolated. Mean SDH activity of individual diaphragm muscle fibers was determined by averaging the OD of all pixels within outlined muscle fibers. To correct for the nonspecific formation of NBT-dfz, the tissue blank OD for each fiber was subtracted from the OD measured when substrate was added to the incubation medium. From the Beer Lambert equation, the mean SDH activity of each fiber was expressed as millimole fumarate per liter tissue per minute. Approximately 200-300 fibers (i.e., same fibers sampled for the measurement of cross-sectional area) were analyzed for SDH from each specimen.

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 (beta /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 (beta /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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (+/-) and homozygous (-/-) IGF-I mutants. In the latter, there is an absence of any product derived from the WT allele (i.e., using IGF-I primers) but a clear signal using neomycin primers, indicating the disrupted alleles (Fig. 1). Accordingly, serum IGF-I levels were undetectable in IGF-I(-/-) mutants (data not shown). Furthermore, all of the mice used in the present study were descendents from the original colonies developed by Powell-Braxton and colleagues (36) who, in addition, demonstrated in homozygous knockouts complete absence of IGF-I mRNA in the liver and eviscerated carcasses of these animals (see Fig. 1D in Ref. 36).


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 1.   Example of results of PCR analysis genotyping of representative animals depicting wild-type (+/+), heterozygous (+/-), and homozygous (-/-) insulin-like growth factor I (IGF-I) mutant mice generated by intercrossing heterozygous parents. A product generated using IGF-I primers indicates the presence of at least one wild-type allele (WT), and a product using neomycin primers indicates at least one disrupted allele (NEO). Marker at left is a 100-bp DNA ladder confirming size of primers: IGF-I = 366 bp and NEO = 608 bp.

Animal Body Weights

At 2 mo of age, the body weight of IGF-I(-/-) male animals (11.7 ± 2.8 g) was only ~32% that of WT littermates (36.5 ± 2.4 g; P < 0.01). Despite marked difference in body weights, the IGF-I(-/-) mutants appeared to exhibit a "proportional" reduction in body dimensions.

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).


View larger version (120K):
[in this window]
[in a new window]
 
Fig. 2.   Photomicrographs of standard hematoxylin and eosin stain of fresh frozen diaphragm sections from WT (A) and IGF-I(-/-) (B) mice. Note that there was an absence of histopathological features but smaller cell sizes in muscle fibers of IGF-I(-/-) mutants compared with WT mice. Both diaphragm sections in A and B were viewed with an objective of ×63 magnification. Calibration bar = 50 µm.

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.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Diaphragm fiber type proportions (A), mean cross-sectional areas (CSA; B), and relative contribution of fiber types to total diaphragm costal area (C). Data based on 6 animals/group, which also represent 6 different litters. Note that the CSAs of type I, IIa, and IIx fibers were reduced 32-37% in IGF-I(-/-) mice compared with WT littermates. * Significantly different from WT mice (P < 0.001). Values are means ± SD.

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Diaphragm thickness and relative number of fibers in IGF-I (-/-) and 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).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Succinate dehydrogenase (SDH) activity in diaphragm fibers of IGF-I(-/-) and WT mice. Data based on 6 animals/group, which also represent 6 different litters. Note that SDH activity was significantly higher in all 3 major types of diaphragm fibers of IGF-I(-/-) mutants. * Significantly different from WT mice (P < 0.05). Values are means ± SD.

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(-/-) animals. [There was, however, a trend for an increase in the proportion of MyHC 2X isoform within the IGF-I(-/-) mutants (P = 0.06; Fig. 5B).] Furthermore, the absence of bands characteristic of developmental MyHC isoforms (i.e., embryonic and neonatal) confirm the above observation (see Fiber proportions) that only the normal mature forms of this contractile protein are expressed in adult IGF-I(-/-) mice.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5.   A: SDS-PAGE of myosin heavy chain (MyHC) isoforms extracted from diaphragm of WT and IGF-I(-/-) mutants depicting the presence of adult isoforms in their migration pattern. B: proportions of MyHC isoforms in the diaphragms of IGF-I(-/-) and WT mice. Data based on 6 animals/group, which also represent 6 different litters. Note that, although no differences were observed between the groups, there was a strong tendency for the proportions of MyHC 2X to be increased in the IGF-I(-/-) mutants (P = 0.06). Values are means ± SD.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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(-/-) mouse model, transcripts of the myogenic factors MyoD, myogenin, MRF-4, and myf-5 were reported qualitatively similar to those of WT animals (30). The above comments refer to those myoblasts allowed to proceed to terminal differentiation and does not preclude impaired differentiation affecting at least some myoblasts during development (as discussed above).

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(-/-) diaphragm were not significantly altered, although there was a trend for the proportion of MyHC 2X to be increased. In view of the preservation of diaphragm fiber proportions and estimated relative contribution of each fiber type to total diaphragm costal area in the IGF-I(-/-) animals, the distinct trends noted in the expression of MyHC 2X isoform would suggest enhanced coexpression of isoforms within individual fibers. The functional sequelae of such changes are unclear but may well reflect adaptive changes aimed at preserving the force-generating properties of the diaphragm.

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-beta ] 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(-/-) mice so that the normal number of motoneurons could also be maintained. In a separate study in IGF-I(-/-) mice of the same age, the number of motoneurons in lumbar spinal cord were preserved (4), which implies similar data for phrenic motoneurons at the cervical level. It has been suggested that the number of motoneurons ultimately surviving the period of normal or programmed cell death is coupled to the number of primary myotubes (e.g., see Ref. 31). Therefore, the late embryonic period may be more dependent on the presence of IGF-I, and the smaller number of fibers present in the diaphragm of IGF-I(-/-) mutants would then be the result of a lower rate of secondary myotube formation. This has implications for the ultimate size of motor units (i.e., the number of muscle fibers innervated by a single phrenic motoneuron) in the diaphragm of IGF-I(-/-) mice. We postulate that the diaphragm motor unit size would be reduced because of fewer target fibers available for innervation. The consequence of this would be 1) reduced force production of each motor unit, which would also be compounded by the reduced size of individual diaphragm fibers, and 2) the need to increase diaphragm motor unit recruitment to achieve forces required during resting ventilation or increased ventilatory demand. This suggests that the total force-generating capacity of the diaphragm (the most important respiratory muscle) would be reduced, and thus its functional force reserve would be reduced also, particularly under conditions of increased load. The increased SDH activity, as noted above, likely reflects the enhanced recruitment profile of the diaphragm.

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adesanya, OO, Zhou J, Samathanan C, Powell-Braxton L, and Bondy CA. Insulin-like growth factor 1 is required for G2 progression in the estradiol-induced mitotic cycle. Proc Natl Acad Sci USA 96: 3287-3291, 1999[Abstract/Free Full Text].

2.   Arthur, WT, Vernon RB, Sage EH, and Reed MJ. Growth factors reverse the impaired sprouting of microvessels from aged mice. Microvasc Res 55: 260-270, 1998[ISI][Medline].

3.   Baker, J, Liu J-P, Robertson EJ, and Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75: 73-82, 1993[ISI][Medline].

4.   Beck, KD, Powell-Braxton L, Widmer H-R, Valverde J, and Hefti F. Igf1 gene disruption results in reduced brain size, CNS hypomyelination, and loss of hyppocampal granule and striatal parvalbumin-containing neurons. Neuron 14: 717-730, 1995[ISI][Medline].

5.   Blanco, CE, Fournier M, and Sieck GC. Metabolic variability within individual fibers of the cat tibialis anterior and diaphragm muscles. Histochem J 23: 366-374, 1991[ISI][Medline].

6.   Blanco, CE, Sieck GC, and Edgerton VR. Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibers. Histochem J 20: 230-243, 1988[ISI][Medline].

7.   Brooke, MH, and Kaiser KK. Muscle fiber types: how many and what kind? Arch Neurol 23: 369-379, 1970[ISI][Medline].

8.   Coleman, ME, Demayo F, Yin KC, Lee HM, Geske R, Montgomery C, and Schwartz RJ. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J Biol Chem 270: 12109-12116, 1995[Abstract/Free Full Text].

9.   Daughaday, WH, and Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 10: 68-91, 1989[Abstract].

10.   De Troyer, A, and Estenne M. Functional anatomy of the respiratory muscles. Clin Chest Med 9: 175-193, 1988[ISI][Medline].

11.   Dwyer, CM, Madgwick A, Ward SS, and Stickland NC. The effects of maternal undernutrition in early gestation on the development of fetal myofibres in the guinea pig. Reprod Fertil Dev 7: 1285-1292, 1995[ISI][Medline].

12.   Enad, JG, Fournier M, and Sieck GC. Oxidative capacity and capillary density of diaphragm motor units. J Appl Physiol 67: 620-627, 1989[Abstract/Free Full Text].

13.   Engert, JC, Berglund EB, and Rosenthal N. Proliferation precedes differentiation in IGF-I-stimulated myogenesis. J Cell Biol 135: 431-440, 1996[Abstract].

14.   Ewton, DZ, Falen SL, and Florini JR. The type II IGF receptor has low affinity for IGF-I analogs: pleiotypic actions of IGFs on myoblasts are apparently mediated by the type I receptor. Endocrinology 120: 115-124, 1987[Abstract].

15.   Ewton, DZ, Roof SL, Magri KA, McWade FJ, and Florini JR. IGF-II is more active than IGF-I in stimulating L6A1 myogenesis: greater mitogenic actions of IGF-I delay differentiation. J Cell Physiol 161: 277-284, 1994[ISI][Medline].

16.   Florini, JR, and Ewton DZ. Insulin acts as a somatomedin analog in stimulating myoblast growth in serum free medium. In Vitro 17: 763-768, 1981[ISI][Medline].

17.   Florini, JR, Ewton DZ, and Coolican SA. Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17: 481-517, 1996[Abstract].

18.   Florini, JR, Ewton DZ, and Roof SL. IGF-1 stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mol Endocrinol 5: 718-724, 1991[Abstract].

19.   Florini, JR, Magri KA, Ewton DZ, James PL, Grindstaff K, and Rotwein PS. "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II. J. Biol Chem 266: 15917-15923, 1991[Abstract/Free Full Text].

20.   Florini, JR, Samuel DS, Ewton DZ, Kirk C, and Sklar RM. Stimulation of myogenic differentiation by a neuregulin, glial growth factor 2. J Biol Chem 271: 12699-12702, 1996[Abstract/Free Full Text].

21.   Fournier, M, and Lewis MI. Muscle fiber type composition in the hamster diaphragm (Abstract). Am J Respir Crit Care Med 151: A806, 1995.

22.   Fuge, KW, Crews EL, Pattengale PK, Holloszy JO, and Shank RE. Effects of protein deficiency on certain adaptative responses to exercise. Am J Physiol 215: 660-663, 1968[ISI][Medline].

23.   Goad, DL, Rubin J, Wang H, Tashjian AH, Jr, and Patterson C. Enhanced expression of vascular endothelial growth factor in human SaOS-2 osteoblast-like cells and murine osteoblasts induced by insulin-like growth factor I. Endocrinology 137: 2262-2268, 1996[Abstract].

24.   Gorza, L. Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined histochemical myosin ATPase and anti-myosin monoclonal antibodies. J Histochem Cytochem 38: 257-265, 1990[Abstract].

25.   Guth, L, and Samaha FJ. Procedure for the histochemical determination of actomyosin ATPase. Exp Neurol 28: 365-367, 1970[ISI][Medline].

26.   Han, VKM, and Hill DJ. The involvement of insulin-like growth factors in embryonic and fetal development. In: The Insulin-Like Growth Factors Structure and Biological Functions, edited by Schofield P. N.. New York: Oxford, 1992, p. 178-220.

27.   Johnson, SE, and Allen RE. The effects of bFGF, IGF-I and TGF-beta on RMo skeletal muscle cell proliferation and differentiation. Exp Cell Res 187: 250-254, 1990[ISI][Medline].

28.   Jones, JI, and Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16: 3-34, 1995[ISI][Medline].

29.   Lewis, MI, LoRusso TJ, and Fournier M. Anabolic influences of insulin-like growth factor I and/or growth hormone on the diaphragm of young rats. J Appl Physiol 82: 1972-1978, 1997[Abstract/Free Full Text].

30.   Liu, J-P, Baker J, Perkins AS, Robertson EJ, and Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Ifg1r). Cell 75: 59-72, 1993[ISI][Medline].

31.   McLennan, IS. Quantitative relationships between motoneuron and muscle development in Xenopus laevis: implications for motoneuron cell death and motor unit formation. J Comp Neurol 271: 19-29, 1988[ISI][Medline].

32.   McPherron, AC, Lawler AM, and Lee S-J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387: 83-90, 1997[ISI][Medline].

33.   McWade, FJ, Ewton DZ, and Florini JR. Mitogenic competence factor, a serum component that is necessary for IGFs to stimulate myoblast proliferation and suppresses their ability to stimulate differentiation (Abstract). In: Prog 77th Ann Meet Endocr Soc Washington, DC: Endocr Soc, 1995, p. 520.

34.   Nicosia, RF, Nicosia SV, and Smith M. Vascular endothelial growth factor, platelet-derived growth factor, and insulin-like growth factor-1 promote rat aortic angiogenesis in vitro. Am J Pathol 145: 1023-1029, 1994[Abstract].

35.   Ontell, M, and Kozeka K. Organogenesis of the mouse extensor digitorum longus muscle: a quantitative study. Am J Anat 171: 149-161, 1984[ISI][Medline].

36.   Powell-Braxton, L, Hollingshead P, Warburton C, Dowd M, Pitts-Meek S, Dalton D, Gillet N, and Stewart TA. IGF-1 is required for normal embryonic growth in mice. Genes Dev 7: 2609-2617, 1993[Abstract].

37.   Prakash, YS, Fournier M, and Sieck GC. Effects of prenatal undernutrition on developing rat diaphragm. J Appl Physiol 75: 1044-1052, 1993[Abstract].

38.   Punglia, RS, Lu M, Hsu J, Kuroki M, Tolentoni MJ, Keough K, Levy AP, Levy NS, Goldberg MA, D'Amato RJ, and Adamis AP. Regulation of vascular endothelial growth factor expression by insulin-like growth factor I. Diabetes 46: 1619-1626, 1997[Abstract].

39.   Quinn, L, Haugk K, and Grabstein K. Interleukin-15: a novel anabolic cytokine for skeletal muscle. Endocriology 136: 3669-3672, 1995[Abstract].

40.   Rosenthal, SM, and Cheng Z-Q. Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts. Proc Natl Acad Sci USA 92: 10307-10311, 1995[Abstract].

41.   Schiaffino, S, Gorza L, Sartore S, Saggin L, Ausoni S, Vianello M, Gundersen K, and Lomo T. Three myosin heavy chain isoforms in type 2 skeletal muscle fibers. J Muscle Res Cell Motil 10: 197-205, 1989[ISI][Medline].

42.   Sell, C, Dumenil G, Deveaud C, Miura M, Coppola D, DeAngelis T, Rubin R, Efstratiadis A, and Baserga R. Effect of null mutation of the insulin-like growth factor I receptor on growth and transformation of mouse embryo fibroblasts. Mol Cell Biol 14: 3604-3612, 1994[Abstract].

43.   Sicinski, P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner H, Haslam SZ, Bronson RT, Elledge SJ, and Weinberg RA. Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82: 621-630, 1995[ISI][Medline].

44.   Sieck, GC, Cheung TS, and Blanco CE. Diaphragm capillarity and oxidative capacity during postnatal development. J Appl Physiol 70: 103-111, 1991[Abstract/Free Full Text].

45.   Sillau, AH, and Banchero N. Visualization of capillaries in skeletal muscle by the ATPase reaction. Pfluegers Arch 369: 269-271, 1977[ISI][Medline].

46.   Solaro, RJ, Pang DC, and Briggs FN. The purification of cardiac myofibrils with Triton X-100. Biochem Biophys Acta 245: 259-262, 1971[ISI][Medline].

47.   Stewart, CEH, and Rotwein P. Insulin-like growth factor-II is an autocrine survival factor for differentiating myoblasts. J Biol Chem 271: 11330-11338, 1996[Abstract/Free Full Text].

48.   Talmadge, RJ, and Roy RR. Electrophoretic separation of rat skeletal muscle myosin heavy-chain isoforms. J Appl Physiol 75: 2337-2340, 1993[Abstract].

49.   Williams, GT, Smith CA, Spooncer E, Dexter TM, and Taylor DR. Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 343: 76-79, 1990[ISI][Medline].

50.   Wilson, SJ, Ross JJ, and Harris AJ. A critical period for formation of secondary myotubes defined by prenatal undernutrition. Development 102: 815-821, 1988[Abstract].


Am J Physiol Endocrinol Metab 278(4):E707-E715
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society