Departments of 1 Physiology, 2 Internal Medicine, and 3 Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
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ABSTRACT |
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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)-1 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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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).
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).
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RESULTS |
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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).
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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.
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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.
|
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.
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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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The authors thank Dennis Belotto for assistance with the electron microscopic analysis that appears in this paper.
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FOOTNOTES |
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* 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.
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