Functional and molecular adaptations in skeletal muscle of myoglobin-mutant mice

Robert W. Grange1,*, Annette Meeson2,*, Eva Chin2, Kim S. Lau1, James T. Stull1, John M. Shelton2, R. Sanders Williams2,3, and Daniel J. Garry2,3

Departments of 1 Physiology, 2 Internal Medicine, and 3 Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myoglobin is a cytoplasmic hemoprotein that is restricted to cardiomyocytes and oxidative skeletal myofibers and facilitates oxygen delivery during periods of high metabolic demand. Myoglobin content in skeletal muscle increases in response to hypoxic conditions. However, we previously reported that myoglobin-null mice are viable and fertile. In the present study, we define important functional, cellular, and molecular compensatory adaptations in the absence of myoglobin. Mice without myoglobin manifest adaptations in skeletal muscle that include a fiber type transition (type I to type II in the soleus muscle), increased expression of the hypoxia-inducible transcription factors hypoxia-inducible factor (HIF)-1alpha and HIF-2 (endothelial PAS domain protein), stress proteins such as heat shock protein 27, and the angiogenic growth factor vascular endothelial growth factor (soleus muscle), as well as increased nitric oxide metabolism (extensor digitorum longus). The resulting changes in angiogenesis, nitric oxide metabolism, and vasomotor regulation are likely to account for preserved exercise capacity of animals lacking myoglobin. These results demonstrate that mammalian organisms are capable of a broad spectrum of adaptive responses that can compensate for a potentially serious defect in cellular oxygen transport.

transgenic mice; oxygen metabolism; hypoxia; vascularization


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLES of adult mammals include several specialized subtypes of myofibers that differ with respect to their metabolic capabilities, molecular regulation, contractile physiology, and susceptibility to fatigue (10, 29, 37). This diversity of skeletal myofibers enables the muscle to fulfill a variety of functional demands. Skeletal myofibers are capable of responding to altered functional demands or metabolic state by reprogramming gene expression so as to alter their specialized phenotypic characteristics (29, 37).

Myoglobin is an evolutionarily conserved cytoplasmic hemoprotein that has been proposed to facilitate oxygen transport in heart and oxidative skeletal myofibers (10, 28, 29, 37, 38). This cytoplasmic protein was the first protein to be subjected to definitive structural analysis and has been the subject of ongoing interest to biologists (29). Myoglobin expression in skeletal muscle is subject to physiological control. A number of studies have reported increased myoglobin content in skeletal muscle of mammals that have adapted to hypoxic environments such as those living at high altitude or engaging in prolonged underwater diving (4, 16, 22, 26). Furthermore, chronic electrical stimulation of the motor nerve augments myoglobin expression in skeletal muscles to levels equal to those in the heart (29, 37). Results of clinical studies suggest that myoglobin desaturates in proportion to exercise intensity, thus supporting an important role for myoglobin in the facilitation of oxygen transport during periods of high metabolic demand (8, 25, 31, 32).

Garry et al. (11) recently pursued a gene disruption strategy to generate mice with a complete absence of myoglobin. Surprisingly, such animals are viable and have preserved cardiac and skeletal muscle function over a wide range of oxygen levels. We hypothesized that preserved function results from cellular and molecular adaptations that are evoked as a response to myoglobin deficiency. In the present study, we describe important cellular and molecular adaptations that are likely to account for the surprisingly preserved functional performance of isolated skeletal muscles or intact animals lacking myoglobin.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of Mb-/- mice. Homozygous myoglobin-deficient mice (Mb-/-) were generated using gene disruption technology as previously described (11). Age- and gender-matched wild-type and heterozygous myoglobin mice were bred and genotyped using Southern blot analysis and polymerase chain reaction of genomic DNA.

Skeletal muscle contractility and cGMP measurements. Wild-type or Mb-/- mice were euthanized using pentobarbital sodium, and the extensor digitorum longus (EDL) and the soleus muscles were rapidly excised. Each muscle was suspended between a fixed clamp at the base of a jacketed organ bath and a Grass FTO3C isometric force transducer (11). Muscles were maintained in an oxygenated (95% O2-5% CO2) physiological salt solution (pH 7.6, 30°C) containing (in mM) 120.5 NaCl, 4.8 KCl, 1.2 MgSO4, 20.4 NaHCO3, 1.6 CaCl2, 10 glucose, and 1.0 pyruvate. After a 15-min equilibration period, muscles were stimulated via closely flanking platinum electrodes using square pulses of 0.2-ms duration at a voltage and a muscle length (Lo) selected to elicit maximal isometric twitch force (model S48, Grass Instruments). Resting force at Lo was typically ~1 g (19, 20, 35).

cGMP formation was examined in EDL and soleus muscles under each of two conditions. In the first condition, one EDL and one soleus muscle from each Mb+/+ or Mb-/- mouse was treated for 30 min with 1 mM NG-nitro-L-arginine (NLA), a nitric oxide synthase (NOS) inhibitor, solubilized in 1 N HCl. During the final 30 s of this treatment, the muscles were stimulated at 30 Hz (20, 21). We previously reported (21) a significant increase in cGMP content in EDL but not soleus muscles at this frequency and duration of stimulation. In the second condition, the contralateral EDL and soleus muscles from each mouse were treated for 30 min with vehicle (1 N HCl) and stimulated the final 30 s at 30 Hz. At the conclusion of electrical stimulation for both conditions, muscles were rapidly frozen in liquid nitrogen and stored at -80°C. Skeletal muscle cGMP content was determined using a radioimmunoassay as previously described (20, 21).

To examine the role of myoglobin availability in inducing low-frequency fatigue, we measured force recovery after fatigue of the soleus and EDL muscles. Muscles were fatigued using a previously defined protocol involving 350-ms, 100-Hz tetani repeated every 4 s for 2 min, then every 3 s for 2 min, etc., until force reached 30% of initial levels (1). On average, EDL muscles fatigued in 3-4 min whereas soleus muscles fatigued in 9-10 min. Muscle force output was measured at a range of frequencies (1, 40, 80, 120 Hz) both before and 60 min after fatigue. To assess low-frequency fatigue, the 60-min recovery data were plotted as a percentage of the initial force at each frequency. Data are presented as means ± SE (n = 6 muscles in each group).

RNA isolation and RT-PCR. Total RNA was isolated from skeletal muscle of adult 3-mo-old male Mb+/+, Mb+/-, and Mb-/- mice using the Tripure isolation kit (Boehringer Mannheim). Four micrograms of total RNA were used in each reverse transcription reaction (Retro-script, Ambion). Complementary DNA (2 µl) was then used as a template for the PCR reaction in a 20-µl reaction volume including 100 ng of each primer, 2 mM MgCl2, Taq buffer, and 1 U of Taq polymerase (GIBCO-BRL). Eighteen microliters of each PCR reaction were loaded on a 2% agarose gel as previously described (11, 12). Semiquantitative RT-PCR was performed as described previously (12) under conditions in which the abundance of each amplified cDNA varied linearly with input RNA. PCR primer pairs (F, forward; R, reverse) used for this study included myoglobin (F, 5'-ACCATGGGGCTCAGTGATGGGGAG-3'; R, 5'-CAGGTACTTGACCGGGATCTTGTGC-3'), heat shock protein 27 (F, 5'-TTCACCCGGAAATACACGCT-3'; R, 5'-GCTCCAGACTGTTCAGACTT-3'), cytochrome oxidase subunit VIaH (COX VIaH) (F, 5'-GACAATGGCTCTGCCTCTAAAGG-3'; R, 5'-CATCAAGGGTGCTCATAACCGGT-3'), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F, 5'-GTGGCAAAGTGGAGATTGTTGCC-3', R, 5'-GATGATGACCCGTTTGGCTCC-3'), vascular endothelial growth factor (VEGF) (F, 5'-GGATCCATGAACTTTCTGCTGTCT-3'; R, 5'-GCATTCACATCGGCTGTGCTGTAG-3'), hypoxia-inducible factor (HIF)-1 (F, 5'-GATGAGTTCTGAACGTCGAAAAGAAAAGT-3'; R, 5'-GAAGTTTTCTCACACGTAAATAACTGATGGTG-3'), HIF-2 (ePAS) (F, 5'-GGAGCAGCTCAGAGCTGAGGAAGGAG-3'; R, 5'-GGACAGGAGCTTATGTGTCCGAAGGAAG-3'), myosin heavy chain (MHC) I (F,5'-AAGGAGCAGGACACCAGCGCCCA-3'; R, 5'-GATCTACTCTTCATTCAGGC-3'), MHCIIX (F, 5'-AAGGAGCAGGACACCAGCGCCCA-3'; R, 5'-ATCTCTTTGGTCACTTTCCTGCT-3'), and troponin I (TnIs) (F, 5'-TGCTGAAGAGCCTGATGCTA-3'; R, 5'-GAACATCTTCTTGCGACCTTC-3').

Analysis of fiber type in muscles from intact animals. Skeletal muscles from adult myoglobin-null and wild-type male mice were harvested as previously described (10, 11). Sections of soleus muscle from Mb+/+ and Mb-/- (n = 3 in each group) were stained using a metachromatic staining protocol as previously described (6, 10, 27). The proportion of fast and slow fibers was quantified by three observers who were blinded to the genotype of the animals. Fibers expressing fast myosin were identified in 8-µm serial cryosections of the same muscles and immunostained using a monoclonal antibody that recognizes MHC type I isoform (NOQ7.5.4D, 1:16,000; Sigma, St. Louis, MO), MHC type IIa (SC-71, 1:1,200; generously provided by Dr. S. Schiaffino, Padva, Italy), and a monoclonal antibody that recognizes all myosin fast isoforms (mouse monoclonal antiserum; MY-32, 1:500; Sigma) and detected using either a FITC-conjugated goat anti-mouse IgG (Jackson Immunochemicals, West Grove, PA) or peroxidase-conjugated secondary antisera and DAB. For immunohistochemistry, sections were incubated overnight at 4°C with the primary antibody, which was detected with a fluorophor-conjugated secondary antiserum as previously described (10, 11).

Vascular density in skeletal muscles. Adult Mb+/+ and Mb-/- soleus muscle or EDL were immersion fixed overnight in methyl-Carnoy's fixative, paraffin embedded, sectioned, and stained using the biotinylated lectin Bandiera simplicifolia lectin B4 (BSLB4, 10 µg/ml; Vector Labs) (7). Substrate was developed with 3,3'-diaminobenzidine, and slides were either coverslipped or lightly counterstained with hematoxylin and analyzed microscopically. For each animal, capillary density was examined at three separate levels and quantified by two blinded investigators.

Image analysis. Stained sections were examined with either a BioRad MRC 1024 confocal microscope equipped with a krypton/argon laser (BioRad Life Science Group) or a Leica Laborlux-S microscope equipped with bright- and darkfield optics and an Optronics VI-470 CCD camera. Image processing was completed with Adobe Photoshop 5.0 and printed with a Kodak XLS 8600 PS printer.

Statistical analysis. Statistical analysis utilized a multi-way ANOVA for repeated measures with Duncan's post hoc analysis (BMDP software; Sepulveda, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Disruption of the myoglobin gene. The targeting strategy was designed to delete exon 2 of the myoglobin gene, which encodes the heme binding domain. Details of the targeting procedure and genotypic analysis have been described previously (11). As shown with in situ hybridization techniques (10, 34), myoglobin expression is restricted to the heart (not shown) and oxidative skeletal myofibers in Mb+/+ animals but absent in the cardiac and skeletal muscles of myoglobin-mutant animals (Fig. 1). Burkholder et al. (3) previously reported that the adult mouse soleus muscle consists of slow oxidative (58%) and fast oxidative glycolytic (42%) myofibers whereas the EDL consists of fast oxidative glycolytic (51%) and fast glycolytic (49%) myofibers (3).


View larger version (70K):
[in this window]
[in a new window]
 
Fig. 1.   In situ hybridization for myoglobin in adult hindlimb skeletal muscle. A: darkfield illumination of a transverse section of Mb+/+ adult skeletal muscle. Silver grains, representing myoglobin expression, are heterogeneously deposited over oxidative skeletal myofibers including the oxidative slow-twitch soleus muscle. B: darkfield illumination of a transverse section of Mb-/- adult skeletal muscle. Note absence of signal in mutant skeletal muscle. Bar = 500 µm. S, soleus muscle; RG, red gastrocnemius muscle; WG, white gastrocnemius muscle; Pl, plantaris muscle.

Myoglobin-deficient mice were fertile and had no apparent functional limitations under ambient conditions. Light microscopic and ultrastructural analysis revealed no significant structural abnormalities or qualitative differences in mitochondria of skeletal myocytes that lack myoglobin (Fig. 2).


View larger version (190K):
[in this window]
[in a new window]
 
Fig. 2.   Morphological assessment of Mb+/+ and Mb-/- skeletal muscle. Structural integrity and mitochondrial density is preserved in the presence (A) and absence (B) of myoglobin in adult soleus muscle shown by a histochemical staining technique for succinate dehydrogenase. Bar = 50 µm. There are no qualitative differences in mitochondrial content or structural abnormalities between the Mb+/+ (C) and Mb-/- (D) soleus muscle. Bar = 0.5 µm.

Fiber type transformation in absence of myoglobin. We previously reported (10, 28) that myoglobin is expressed selectively in oxidative type I (slow) and type IIa (fast oxidative) skeletal muscle fibers. To determine whether there are fiber type differences in the absence of myoglobin, we assessed the proportion of slow versus fast fibers within the soleus muscles of adult male Mb+/+ and Mb-/- animals. Using a metachromatic staining assay, we observed a significant fiber type transition in the Mb-/- soleus muscle (Fig. 3, A-C). In the absence of myoglobin, the percentage of slow (type I) myofibers in the soleus muscles was decreased to 41.6 ± 2.9% compared with 52.6 ± 2.5% in wild-type animals (P < 0.05). Similarly, there was a significant increase (P < 0.05) in the type IIa fibers in the Mb-/- soleus muscle (55.5 ± 2.9%) compared with the Mb+/+ soleus muscle (45.7 ± 2.4%). These results were confirmed with an alternative histochemical staining technique for myosin ATPase activity and specific immunohistochemical staining of slow and fast MHC isoforms. Furthermore, these results were corroborated by the observation of a transition involving MHC and TnI isoforms of gene expression in adult Mb-/- soleus muscle using RT-PCR analysis (Fig. 3, D and E). These results reveal cellular adaptations likely to enhance functional performance of skeletal muscle in the absence of myoglobin.


View larger version (86K):
[in this window]
[in a new window]
 
Fig. 3.   Fiber composition of adult male Mb+/+ and Mb-/- soleus muscles. A: representative section from metachromatic dye-ATPase-stained Mb+/+ adult male soleus muscle. B: representative section from metachromatic dye-ATPase-stained Mb-/- male soleus muscle. I, type I fibers; IIa, type IIa fibers; IIb, type IIb fibers. C: quantitative results of metachromatic histochemical analysis reveals an 11% decrease in type I (slow) fibers in Mb-/- soleus muscle (41.6 ± 2.9%) compared with the wild-type (+/+) control (52.6 + 2.5%) (n = 3 animals for each group). Bars represent means ± SE. *Statistically significant difference (P < 0.05). D: semiquantitative RT-PCR analysis of RNA isolated from adult male wild-type, heterozygote (+/-), or myoglobin-null soleus muscle. Note decreased expression of myosin heavy chain (MHC) I and troponin I slow (TnIs) and an increased expression of MHCIIX in the myoglobin-mutant soleus muscle compared with the wild-type control (performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. E: quantitative results of selected transcripts relative to wild-type expression (n = 3 animals for each group). *Statistically significant difference (P < 0.05).

Muscle performance in Mb-/- mice. We reported previously (11) that the exercise capacity of Mb-/- mice is indistinguishable from that of Mb+/+ mice measured with a standard exercise treadmill protocol. We hypothesized, however, that twitch properties may be influenced by the fiber type transition or metabolic disturbances (i.e., hypoxia) that occur in the Mb-/- skeletal muscle. Low-frequency fatigue is defined by a selective reduction in force at low (i.e., 30-50 Hz) but not high (i.e., 100 Hz) stimulation frequencies (1, 5, 24) after a series of fatiguing contractions. Because muscles are still capable of achieving maximum force output, it is unlikely that there is impairment of the contractile proteins. Previous studies showed that low-frequency fatigue occurs in muscles with predominantly fast-twitch fibers (i.e., EDL) but not those with predominantly slow-twitch fibers (i.e., soleus) (1, 5, 24). Using an isolated muscle preparation, we observed a difference in the extent of low-frequency fatigue between Mb+/+ and Mb-/- mice (Fig. 4). Low-frequency fatigue was observed in EDL muscles irrespective of genotype. For example, after the fatigue protocol, force responses at 40 Hz were 48.4 ± 3.5% and 55.4 ± 9.0% of initial force in Mb+/+ and Mb-/- EDL muscles, respectively. In soleus muscles, however, low-frequency fatigue was observed only in Mb-/- mice (88.0 ± 3.7% of initial force at 40 Hz; P < 0.05) and not in Mb+/+ mice (102.0 ± 4.0% of initial force at 40 Hz). There were no significant differences in force at the higher frequencies (80 and 120 Hz) between Mb+/+ and Mb-/- EDL or soleus muscles. The presence of low-frequency fatigue in the Mb-/- soleus muscle may reflect the fiber type transition (see Fig. 3) and/or it may occur as a consequence of hypoxia.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Low-frequency fatigue in Mb-/- soleus (Sol) muscles. Recovery of force was determined 60 min after a well-established fatigue protocol and was plotted as a percentage of the initial force at each frequency (1). Low-frequency fatigue was observed in Mb+/+ and Mb-/- extensor digitorum longus (EDL) muscles (Mb+/+ 48.4 ± 3.5%, Mb-/- 55.4 ± 9.0% of initial force at 40 Hz). Low-frequency fatigue was observed in Mb-/- soleus muscles (88.0 ± 3.7% at 40 Hz) but was absent in Mb+/+ soleus muscles (102.0 ± 4.0% at 40 Hz). Data are presented as means ± SE (n = 6 muscles). *Statistically significant difference (P < 0.05).

Cellular adaptations in Mb-/- skeletal muscle. Oxygen availability is influenced by a number of cellular mechanisms (17, 30, 33, 36). We have observed that the myoglobin-mutant heart has a significant increase in vascularization (e.g., capillary density; Ref. 15 and Meeson and Garry, unpublished data), and we hypothesized that a similar mechanism could facilitate oxygen delivery in myoglobin-mutant skeletal muscle. We observed a 21% increase in capillary density involving the slow-twitch oxidative soleus muscle of myoglobin-mutant mice (1,252 ± 9 capillaries/mm2) compared with wild-type controls (1,031 ± 15 capillaries/mm2) (Fig. 5). This vascular adaptation was not shown in the Mb-/- fast-twitch EDL muscle because no differences were observed in capillary density between Mb+/+ (935 ± 4 capillaries/mm2) and Mb-/- (992 ± 11 capillaries/mm2) genotypes. In the absence of any changes in vascularization associated with the fast-twitch EDL, we hypothesized that oxygen delivery, in the absence of myoglobin, may be facilitated by a nitric oxide (NO)-mediated mechanism.


View larger version (86K):
[in this window]
[in a new window]
 
Fig. 5.   Analysis of vascularization in Mb+/+ and Mb-/- skeletal muscle. Representative micrographs for Mb+/+ and Mb-/- soleus (A and B) and EDL (C and D) stained with a vascular tissue-specific lectin are shown in A-D. Note a 21% increase in capillary density associated with the Mb-/- soleus muscle (B) compared with the wild-type control (A). There are no differences noted in the vasculature of the mutant (D) and the wild-type (C) EDL. Bar = 20 µm. E: quantitation of the capillaries reveals a significant increase in the Mb-/- soleus muscle (1,252 ± 9 capillaries/mm2) compared with the Mb+/+ soleus muscle (1,031 ± 15 capillaries/mm2). No significant changes are evident in the EDL (n = 3 animals for each group). Bars represent means ± SE. *Statistically significant difference (P < 0.05).

Previous studies reported that the relative content and activity of neuronal NOS (nNOS) are increased in fast-twitch compared with slow-twitch skeletal muscles (2, 19, 21). Electrical stimulation of the fast-twitch EDL but not the slow-twitch soleus muscles results in increased NO and an increased cGMP content (21). These studies suggest that contraction of fast-twitch muscles activates nNOS to produce NO, which may in turn diffuse to smooth muscles of adjacent blood vessels (i.e., arterioles) and activate soluble guanylyl cyclase (sGC), resulting in increased cGMP and dilation of the vasculature to facilitate oxygen delivery (20). To determine whether biochemical effects attributable to NO generation are altered in muscles of myoglobin-null mice, we measured cGMP content after a 30-Hz electrical stimulation in Mb+/+ and Mb-/- soleus and EDL muscles. On electrical stimulation, cGMP content in the EDL from Mb+/+ mice increased significantly from 3.09 ± 0.62 to 6.17 ± 0.58 fmol cGMP/mg muscle wet wt (n = 8 muscles; P < 0.05). In the absence of myoglobin, we observed a further increase (4-fold; P < 0.05) in cGMP content (3.34 ± 0.75 to 13.21 ± 1.78 fmol cGMP/mg wet wt; n = 8 muscles) that was significantly greater compared with the stimulated Mb+/+ EDL (Fig. 6). This increase was attenuated with exposure to a NOS inhibitor, NLA, further supporting the presence of an enhanced NO-mediated mechanism that stimulates cGMP formation in fast-twitch muscles of Mb-/- mice, presumably to promote oxygen delivery [cGMP content after exposure to NLA was 3.12 ± 0.56 (n = 4) and 5.39 ± 1.13 (n = 5) fmol cGMP/mg wet wt in Mb+/+ and Mb-/- EDL, respectively]. Resting levels of cGMP in soleus muscles from Mb+/+ and Mb-/- mice [4.49 ± 1.34 (n = 6) and 5.3 ± 1.87 (n = 6) fmol cGMP/mg wet wt, respectively] were not significantly changed after electrical stimulation [5.93 ± 1.02 (n = 8) and 6.1 ± 0.88 (n = 9) fmol cGMP/mg wet wt for Mb+/+ and Mb-/- mice, respectively] (Fig. 6).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Increased cGMP formation in electrically stimulated Mb-/- EDL muscle compared with control. EDL and soleus muscles were isolated from Mb+/+ and Mb-/- adult male mice and electrically stimulated (30 Hz for 15 s; Stim) in the presence or absence of NG-nitro-L-arginine (NLA). cGMP content was measured in muscle extracts by RIA. A: 2.1-fold increase in cGMP was observed in Mb-/- EDL compared with Mb+/+ EDL after electrical stimulation and was attenuated by the nitric oxide synthase inhibitor NLA. B: no significant increase in cGMP was observed in stimulated Mb+/+ or Mb-/- soleus muscles. Bars represent means ± SE. *Statistically significant difference (P < 0.05).

Reprogramming of skeletal muscle gene expression in the absence of myoglobin. Pharmacological studies using chemical inhibitors demonstrated an important role for myoglobin in oxygen transport within striated muscles (2, 10, 19, 29). We hypothesized that tissue hypoxia resulting from myoglobin deficiency would stimulate cellular defense mechanisms that are based on hypoxia-inducible gene expression. Using semiquantitative RT-PCR analysis, we observed enhanced expression in Mb-/- adult soleus muscle of a number of genes that are known to be induced in response to hypoxic conditions. HIF-1, HIF-2 (ePAS), stress proteins, and VEGF are all markedly induced in the myoglobin-mutant soleus muscle (Fig. 7). Similar molecular profiles (excluding VEGF) were observed in the EDL (data not shown). These changes in gene expression plausibly drive cellular adaptations such as increased vascularization and NO-mediated vasorelaxation that preserve skeletal muscle function when myoglobin is absent.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7.   Semi-quantitative RT-PCR analysis of RNA isolated from 3-mo-old male wild-type (+/+), heterozygote (+/-), or myoglobin-null (-/-) littermates. Note increased expression of hypoxia-inducible factor (HIF)-1, HIF-2 [endothelial PAS domain protein (ePAS)], vascular endothelial growth factor (VEGF), stress proteins [heat shock protein (hsp) 27] and cytochrome oxidase subunit VIaH (COX) in myoglobin-mutant soleus muscle compared with Mb+/+ control soleus muscle (performed in triplicate). Similar molecular profiles of gene expression were observed in the EDL (data not shown). B: quantitative results of selected transcripts relative to wild-type expression (n = 3 muscles for each group). *Statistically significant difference (P < 0.05). Mb, myoglobin.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using a gene targeting strategy, we previously reported (11) that myoglobin-mutant mice tolerate the hemodynamic challenges associated with pregnancy and have a normal exercise capacity. We proposed that knockout mice surviving in the absence of myoglobin do so by developing powerful cellular and molecular adaptations that maintain oxygen transport.

In the present study, we observed no apparent changes in mitochondria or sarcomeric ultrastructure of skeletal myocytes that lack myoglobin. Changes in fiber type, however, were observed in myoglobin-knockout mice. Slow (type I) myofibers were decreased, whereas fast (type II) myofibers were increased in the soleus of myoglobin-mutant mice. Fiber type alterations assessed by a metachromatic staining assay were confirmed by changes in the expression of specific isoforms of MHC and TnI and were paralleled by changes in physiological variables (i.e., response to a fatigue protocol such as low-frequency fatigue).

Mammalian skeletal muscle is capable of responding to altered functional demands and metabolic challenges such as hypoxia by changing gene expression to promote morphological, biochemical, and functional adaptations. Myoglobin-mutant mice manifest a variety of such adaptive responses in a manner sufficiently robust to compensate almost fully for a defect in oxygen transport that otherwise would result from myoglobin deficiency. Our current findings indicate that preserved exercise capacity in the absence of myoglobin is attributable, at least in part, to reprogramming of gene expression within skeletal muscle and compensatory cellular responses that include increased vasculogenesis in soleus muscles and enhanced NO metabolism in EDL muscles. Intracellular hypoxia is likely to be an inciting stimulus for these adaptations, through mechanisms that include induction of HIF-1, a basic-helix-loop-helix-PAS protein (17, 30, 33, 36). HIF-1 is a transcriptional activator of downstream target genes that increase oxygen delivery (e.g., angiogenic growth factors and NOS) or that facilitate ATP production in the absence of oxygen (e.g., glucose transporters and glycolytic enzymes) (30, 33, 36).

The increase in vascular density of the soleus muscle is a fundamentally important compensatory adaptation when myoglobin is absent. An increased number of capillaries would be expected to reduce the mean diffusion distance for oxygen in the adult soleus muscle and promote oxygen delivery. An additional adaptation in Mb-/- fast-twitch muscle groups such as the EDL involves NO metabolism. nNOS, a Ca2+/calmodulin-dependent enzyme, is present in skeletal muscle, although the relative content and activity of nNOS appear to be much greater in fast-twitch compared with slow-twitch muscle (2, 19, 21). NOS converts L-arginine and molecular oxygen to NO and L-citrulline. NO binds to the heme-containing proteins, and its intracellular effects are primarily mediated by sGC. This protein is present in smooth muscle cells and activates an important relaxation cascade when activated by NO (23). Electrical stimulation of the EDL but not the soleus muscle results in increased cGMP content (21). We have proposed that stimulation of fast-twitch muscle activates nNOS to produce NO that can diffuse to smooth muscles of adjacent blood vessels (i.e., arterioles) and activate sGC, resulting in increased cGMP (20). In the present study, we observed cGMP to be significantly increased after electrical stimulation of the Mb-/- EDL, which was attenuated by the addition of a NOS inhibitor. These results are consistent with the hypothesis that in the absence of myoglobin, oxygen delivery is maintained by a NO-mediated vasodilation mechanism in fast-twitch muscles such as the EDL.

Our present data illustrate a spectrum of responses to impaired oxygen transport that are likely to account for the remarkably complete compensatory adaptation of skeletal muscle to myoglobin deficiency. Further examination of the cellular and molecular mechanisms of this powerful adaptive response ultimately could lead to advances in therapy of patients with myopathy or vascular disease.


    ACKNOWLEDGEMENTS

The authors thank Dennis Belotto for assistance with the electron microscopic analysis that appears in this paper.


    FOOTNOTES

* R. W. Grange and A. Meeson contributed equally to this work.

This work was supported by grants from the Muscular Dystrophy Association, the American Heart Association, Texas Affiliate, the National Institutes of Health (AR-40849, HL-54794, and HL-06296), and the D. W. Reynolds Foundation.

Present address of R. W. Grange: Dept. of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

Address for reprint requests and other correspondence: D. J. Garry, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., NB11.200, Dallas, TX 75390-8573 (E-mail: daniel.garry{at}utsouthwestern.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 November 2000; accepted in final form 20 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allen, DG, Lee JA, and Westerblad H. Intracellular calcium and tension during fatigue in isolated single muscle fibers from Xenopus laevis. J Physiol (Lond) 415: 433-458, 1989[Abstract].

2.   Balon, TW, and Nadler JL. Nitric oxide release is present from incubated skeletal muscle preparations. J Appl Physiol 77: 2519-1521, 1994[Abstract/Free Full Text].

3.   Burkholder, TJ, Fingado B, Baron S, and Lieber RL. Relationship between muscle fiber types and sizes and muscle architectural properties in the mouse hindlimb. J Morphol 221: 177-190, 1994[ISI][Medline].

4.   Butler, PJ, and Jones DR. The comparative physiology of diving in vertebrates. Adv Comp Physiol Biochem 8: 179-364, 1982[ISI][Medline].

5.   Chin, ER, Balnave CD, and Allen DG. Role of intracellular calcium and metabolites in low-frequency fatigue of mouse skeletal muscle. Am J Physiol Cell Physiol 272: C550-C559, 1997[Abstract/Free Full Text].

6.   Chin, E, Olson EA, Richardson JA, Yang Q, Humphries C, Shelton J, Wu H, Zhu W, Bassel-Duby R, and Williams RS. A calcineurin-dependent pathway controls skeletal muscle fiber type. Genes Dev 12: 2499-2509, 1998[Abstract/Free Full Text].

7.   Coffin, JD, Harrison J, Schwartz S, and Heimark R. Angioblast differentiation and morphogenesis of the vascular endothelium in the mouse embryo. Dev Biol 148: 51-62, 1991[ISI][Medline].

8.   Conley, K, and Jones C. Myoglobin content and oxygen diffusion: model analysis of horse and steer muscle. Am J Physiol Cell Physiol 271: C2027-C2036, 1996[Abstract/Free Full Text].

9.   Connett, R, Gayeski T, and Honig C. Lactate accumulation in fully aerobic, working, dog gracilis muscle. Am J Physiol Heart Circ Physiol 246: H120-H128, 1984[Abstract/Free Full Text].

10.   Garry, DJ, Bassel-Duby R, Richardson JA, Grayson J, Neufer PD, and Williams RS. Postnatal development and plasticity of specialized muscle fiber characteristics in the hindlimb. Dev Genet 19: 146-156, 1996[ISI][Medline].

11.   Garry, DJ, Ordway GA, Lorenz JN, Radford NB, Chin ER, Grange RW, Bassel-Duby R, and Williams RS. Mice without myoglobin. Nature 395: 905-908, 1998[ISI][Medline].

12.   Garry, DJ, Yang Q, Bassel-Duby R, and Williams RS. Persistent expression of MNF identifies myogenic stem cells in postnatal muscles. Dev Biol 188: 280-294, 1997[ISI][Medline].

13.   Gayeski, T, Connett R, and Honig C. Minimum intracellular PO2 for maximum cytochrome turnover in red muscle in situ. Am J Physiol Heart Circ Physiol 252: H906-H915, 1987[Abstract/Free Full Text].

14.   Gayeski, T, and Honig C. Intracellular PO2 in long axis of individual fibers in working dog gracilis muscle. Am J Physiol Heart Circ Physiol 254: H1179-H1186, 1988[Abstract/Free Full Text].

15.   Godecke, A, Flogel U, Zanger K, Ding Z, Hirchenhain J, Decking U, and Schrader J. Disruption of myoglobin in mice induces multiple compensatory mechanisms. Proc Natl Acad Sci USA 96: 10495-10500, 1999[Abstract/Free Full Text].

16.   Guyton, GP, Stanek K, Schneider RC, Hochachka P, Hurford W, Zapol D, Liggins G, and Zapol W. Myoglobin saturation in free-diving Weddell seals. J Appl Physiol 79: 1148-1155, 1995[Abstract/Free Full Text].

17.   Hochachka, PW. The metabolic implications of intracellular circulation. Proc Natl Acad Sci USA 96: 12233-12239, 1999[Abstract/Free Full Text].

18.   Honig, C, Gayeski T, Federspiel A, Clark A, and Clark P. Muscle O2 gradients from hemoglobin to cytochrome: new concepts, new complexities. Adv Exp Med Biol 169: 23-38, 1984[ISI][Medline].

19.   Kobzik, L, Beid MB, Bredt DS, and Stamler JS. Nitric oxide in skeletal muscle. Nature 372: 546-548, 1994[ISI][Medline].

20.   Lau, KS, Grange RW, Chang W-J, Kamm KE, Sarelius I, and Stull J. Skeletal muscle contractions stimulate cGMP formation and attenuate vascular smooth muscle myosin phosphorylation via nitric oxide. FEBS Lett 431: 71-74, 1998[ISI][Medline].

21.   Lau, KS, Grange RW, Isotani E, Sarelius IH, Kamm KE, Huang PL, and Stull J. nNOS and eNOS modulate cGMP formation and vascular response in contracting fast-twitch skeletal muscle. Physiol Genomics 2: 21-27, 2000[Abstract/Free Full Text].

22.   Lenfant, C. High altitude adaptation in mammals. Am Zool 13: 447-456, 1973[ISI].

23.   Lincoln, TM, Cornwell TL, Komalavilas P, Macmillan-Crow LA, and Boerth N. The nitric oxide-cyclic GMP signaling system. In: Biochemistry of Smooth Muscle Contraction, edited by Barany M.. New York: Academic, 1996, p. 257-268.

24.   Lindinger, MI, Heigenhauser G, and Spriet L. Effects of intense swimming and tetanic electrical stimulation on skeletal muscle ions and metabolites. J Appl Physiol 63: 2331-2339, 1987[Abstract/Free Full Text].

25.   Mole, P, Chung Y, Tran T, Sailusuta N, Hurd R, and Jue T. Myoglobin desaturation with exercise intensity in human gastrocnemius muscle. Am J Physiol Regulatory Integrative Comp Physiol 277: R173-R180, 1999[Abstract/Free Full Text].

26.   Nevo, E. Adaptive convergence and divergence of subterranean mammals. Annu Rev Ecol Syst 10: 269-308, 1979[ISI].

27.   Ogilvie, RW, and Feeback DL. A metachromatic dye-ATPase method for the simultaneous identification of skeletal muscle fiber types I, IIA, IIB and IIC. Stain Technol 65: 231-241, 1990[ISI][Medline].

28.   Parsons, WJ, Richardson JA, Graves KH, Williams RS, and Moreadith RW. Gradients of transgene expression directed by the human myoglobin promoter in the developing mouse heart. Proc Natl Acad Sci USA 90: 1726-1730, 1993[Abstract].

29.   Pette, D, and Staron RS. Mammalian skeletal muscle fiber type transitions. Int Rev Cytol 17: 143-222, 1997.

30.   Ratcliffe, PJ, O'Rourke JF, Maxwell PH, and Pugh CW. Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression. J Exp Biol 201: 1153-1162, 1998[Abstract/Free Full Text].

31.   Richardson, R, Leigh J, Wagner P, and Noyszewski E. Cellular PO2 as a determinant of maximal mitochondrial O2 consumption in trained human skeletal muscle. J Appl Physiol 87: 325-331, 1999[Abstract/Free Full Text].

32.   Richardson, R, Noyszewski E, Kendrick K, Leigh J, and Wagner P. Myoglobin O2 desaturation during exercise: evidence of limited O2 transport. J Clin Invest 96: 1916-1926, 1995[ISI][Medline].

33.   Semenza, GL. Perspectives on oxygen sensing. Cell 98: 281-284, 1999[ISI][Medline].

34.   Shelton, JM, Lee MH, Richardson JA, and Patel SB. Microsomal triglyceride transfer protein during mouse development. J Lipid Res 41 (4): 532-537, 2000[Abstract/Free Full Text].

35.   Thomas, GD, Sander M, Lau KS, Huang P, Stull J, and Victor R. Impaired metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle. Proc Natl Acad Sci USA 95: 15090-15095, 1998[Abstract/Free Full Text].

36.   Wenger, RH. Mammalian oxygen sensing, signaling and gene regulation. J Exp Biol 203: 1253-1263, 2000[Abstract/Free Full Text].

37.   Williams, RS, and Neufer PD. Regulation of gene expression in skeletal muscle by contractile activity. In: Handbook of Physiology. Regulation and Integration of Multiple Systems. Bethesda, MD: Am Physiol Soc, 1996, sect. 12, chapt. 25, p. 1124-1150.

38.   Wittenberg, BA, and Wittenberg JB. Transport of oxygen in muscle. Annu Rev Physiol 51: 857-878, 1989[ISI][Medline].


Am J Physiol Cell Physiol 281(5):C1487-C1494
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society