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

Abstract

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.

Full text

Functional and molecular adaptations in skeletal
muscle of myoglobin-mutant mice
ROBERT W. GRANGE,
1
* ANNETTE MEESON,
2
* EVA CHIN,
2
KIM S. LAU,
1
JAMES T. STULL,
1
JOHN M. SHELTON,
2
R. SANDERS WILLIAMS,
2,3
AND DANIEL J. GARRY
2,3
Departments of
1
Physiology,
2
Internal Medicine, and
3
Molecular Biology,
University of Texas Southwestern Medical Center, Dallas, Texas 75390
Received 7 November 2000; accepted in final form 20 June 2001
Grange, Robert W., Annette Meeson, Eva Chin, Kim
S. Lau, James T. Stull, John M. Shelton, R. Sanders
Williams, and Daniel J. Garry. Functional and molecular
adaptations in skeletal muscle of myoglobin-mutant mice.
Am J Physiol Cell Physiol 281: C1487–C1494, 2001.—Myoglobin
is a cytoplasmic hemoprotein that is restricted to cardiomyo-
cytes 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 compen-
satory adaptations in the absence of myoglobin. Mice without
myoglobin manifest adaptations in skeletal muscle that in-
clude a fiber type transition (type I to type II in the soleus
muscle), increased expression of the hypoxia-inducible tran-
scription 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 vascu-
lar endothelial growth factor (soleus muscle), as well as in-
creased 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; vasculariza-
tion
SKELETAL MUSCLES of adult mammals include several
specialized subtypes of myofibers that differ with re-
spect to their metabolic capabilities, molecular regula-
tion, contractile physiology, and susceptibility to fa-
tigue (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 cytoplas-
mic hemoprotein that has been proposed to facilitate
oxygen transport in heart and oxidative skeletal myo-
fibers (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 mus-
cle is subject to physiological control. A number of
studies have reported increased myoglobin content in
skeletal muscle of mammals that have adapted to hyp-
oxic 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 skele-
tal muscles to levels equal to those in the heart (29, 37).
Results of clinical studies suggest that myoglobin de-
saturates in proportion to exercise intensity, thus sup-
porting an important role for myoglobin in the facilita-
tion 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 mo-
lecular 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 func-
tional performance of isolated skeletal muscles or in-
tact animals lacking myoglobin.
MATERIALS AND METHODS
Generation of Mb⫺/⫺mice. Homozygous myoglobin-defi-
cient 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 poly-
merase chain reaction of genomic DNA.
Skeletal muscle contractility and cGMP measurements.
Wild-type or Mb⫺/⫺mice were euthanized using pentobar-
bital sodium, and the extensor digitorum longus (EDL) and
the soleus muscles were rapidly excised. Each muscle was
*R. W. Grange and A. Meeson contributed equally to this work.
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
@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.
Am J Physiol Cell Physiol
281: C1487–C1494, 2001.
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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% O
2
-5%
CO
2
) physiological salt solution (pH 7.6, 30°C) containing (in
mM) 120.5 NaCl, 4.8 KCl, 1.2 MgSO
4
, 20.4 NaHCO
3
, 1.6
CaCl
2
, 10 glucose, and 1.0 pyruvate. After a 15-min equili-
bration period, muscles were stimulated via closely flanking
platinum electrodes using square pulses of 0.2-ms duration
at a voltage and a muscle length (L
o
) selected to elicit max-
imal isometric twitch force (model S48, Grass Instruments).
Resting force at L
o
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 N
G
-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. Skel-
etal muscle cGMP content was determined using a radioim-
munoassay as previously described (20, 21).
To examine the role of myoglobin availability in inducing
low-frequency fatigue, we measured force recovery after fa-
tigue 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). Com-
plementary DNA (2 ␮l) was then used as a template for the
PCR reaction in a 20-␮l reaction volume including 100 ng of
each primer, 2 mM MgCl
2
,Taq buffer, and1UofTaq
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⬘-ACCATGGGGCTCAGTGAT-
GGGGAG-3⬘;R,5⬘-CAGGTACTTGACCGGGATCTTGTGC-3⬘),
heat shock protein 27 (F, 5⬘-TTCACCCGGAAATACACGCT-
3⬘;R,5⬘-GCTCCAGACTGTTCAGACTT-3⬘), cytochrome oxi-
dase subunit VIaH (COX VIaH) (F, 5⬘-GACAATGGCTCTGC-
CTCTAAAGG-3⬘;R,5⬘-CATCAAGGGTGCTCATAACCGGT-
3⬘), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F,
5⬘-GTGGCAAAGTGGAGATTGTTGCC-3⬘,R,5⬘-GATGATG-
ACCCGTTTGGCTCC-3⬘), vascular endothelial growth factor
(VEGF) (F, 5⬘-GGATCCATGAACTTTCTGCTGTCT-3⬘;R,5⬘-
GCATTCACATCGGCTGTGCTGTAG-3⬘), hypoxia-inducible
factor (HIF)-1 (F, 5⬘-GATGAGTTCTGAACGTCGAAAAGAAA-
AGT-3⬘;R,5⬘-GAAGTTTTCTCACACGTAAATAACTGATGGTG-
3⬘), HIF-2 (ePAS) (F, 5⬘-GGAGCAGCTCAGAGCTGAGGAAG-
GAG-3⬘;R,5⬘-GGACAGGAGCTTATGTGTCCGAAGGAAG-3⬘),
myosin heavy chain (MHC) I (F,5⬘-AAGGAGCAGGACAC-
CAGCGCCCA-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⫽3in
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 express-
ing fast myosin were identified in 8-␮m 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 over-
night at 4°C with the primary antibody, which was detected
with a fluorophor-conjugated secondary antiserum as previ-
ously described (10, 11).
Vascular density in skeletal muscles. Adult Mb⫹/⫹and
Mb⫺/⫺soleus muscle or EDL were immersion fixed over-
night in methyl-Carnoy’s fixative, paraffin embedded, sec-
tioned, and stained using the biotinylated lectin Bandiera
simplicifolia lectin B4 (BSLB4, 10 ␮g/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 ei-
ther 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 process-
ing 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
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 expres-
sion 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 an-
imals (Fig. 1). Burkholder et al. (3) previously reported
that the adult mouse soleus muscle consists of slow
oxidative (58%) and fast oxidative glycolytic (42%)
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myofibers whereas the EDL consists of fast oxidative
glycolytic (51%) and fast glycolytic (49%) myofibers (3).
Myoglobin-deficient mice were fertile and had no
apparent functional limitations under ambient condi-
tions. Light microscopic and ultrastructural analysis
revealed no significant structural abnormalities or
qualitative differences in mitochondria of skeletal myo-
cytes that lack myoglobin (Fig. 2).
Fiber type transformation in absence of myoglobin.
We previously reported (10, 28) that myoglobin is ex-
pressed 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 con-
firmed with an alternative histochemical staining tech-
nique for myosin ATPase activity and specific immu-
nohistochemical staining of slow and fast MHC
isoforms. Furthermore, these results were corrobo-
rated 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, Dand E).
These results reveal cellular adaptations likely to en-
hance functional performance of skeletal muscle in the
absence of myoglobin.
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 proto-
col. We hypothesized, however, that twitch proper-
ties 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 con-
tractions. Because muscles are still capable of
achieving maximum force output, it is unlikely that
there is impairment of the contractile proteins. Pre-
vious studies showed that low-frequency fatigue oc-
curs in muscles with predominantly fast-twitch fi-
bers (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 differ-
ence 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, how-
ever, 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
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, repre-
senting myoglobin expression, are heteroge-
neously 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 ␮m. S, soleus
muscle; RG, red gastrocnemius muscle; WG,
white gastrocnemius muscle; Pl, plantaris
muscle.
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 ␮m. There are no qualitative differences in
mitochondrial content or structural abnormalities between the
Mb⫹/⫹(C) and Mb⫺/⫺(D) soleus muscle. Bar ⫽0.5 ␮m.
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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. Oxy-
gen 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 Mee-
son 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/mm
2
) compared with wild-type
controls (1,031 ⫾15 capillaries/mm
2
) (Fig. 5). This vas-
cular adaptation was not shown in the Mb⫺/⫺fast-
twitch EDL muscle because no differences were observed
in capillary density between Mb⫹/⫹(935 ⫾4 capillaries/
mm
2
) and Mb⫺/⫺(992 ⫾11 capillaries/mm
2
) genotypes.
In the absence of any changes in vascularization associ-
ated with the fast-twitch EDL, we hypothesized that
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 metachro-
matic 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).
Fig. 4. Low-frequency fatigue in Mb⫺/⫺soleus (Sol) muscles. Recov-
ery 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). *Statisti-
cally significant difference (P⬍0.05).
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oxygen delivery, in the absence of myoglobin, may be
facilitated by a nitric oxide (NO)-mediated mechanism.
Previous studies reported that the relative content
and activity of neuronal NOS (nNOS) are increased in
fast-twitch compared with slow-twitch skeletal mus-
cles (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 ves-
sels (i.e., arterioles) and activate soluble guanylyl cy-
clase (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 signif-
icantly 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 signif-
icantly greater compared with the stimulated Mb⫹/⫹
EDL (Fig. 6). This increase was attenuated with expo-
sure 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 deliv-
ery [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 signif-
icantly 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).
Reprogramming of skeletal muscle gene expression in
the absence of myoglobin. Pharmacological studies us-
ing chemical inhibitors demonstrated an important
role for myoglobin in oxygen transport within striated
Fig. 5. Analysis of vascularization in
Mb⫹/⫹and Mb⫺/⫺skeletal muscle.
Representative micrographs for Mb⫹/⫹
and Mb⫺/⫺soleus (Aand B) and EDL
(Cand D) stained with a vascular tis-
sue-specific lectin are shown in A–D.
Note a 21% increase in capillary den-
sity associated with the Mb⫺/⫺soleus
muscle (B) compared with the wild-
type control (A). There are no differ-
ences noted in the vasculature of the
mutant (D) and the wild-type (C) EDL.
Bar ⫽20 ␮m. E: quantitation of the
capillaries reveals a significant in-
crease in the Mb⫺/⫺soleus muscle
(1,252 ⫾9 capillaries/mm
2
) compared
with the Mb⫹/⫹soleus muscle
(1,031 ⫾15 capillaries/mm
2
). No signif-
icant changes are evident in the EDL
(n⫽3 animals for each group). Bars
represent means ⫾SE. *Statistically
significant difference (P⬍0.05).
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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 semi-
quantitative 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 pro-
teins, and VEGF are all markedly induced in the myo-
globin-mutant soleus muscle (Fig. 7). Similar molecu-
lar profiles (excluding VEGF) were observed in the
EDL (data not shown). These changes in gene expres-
sion plausibly drive cellular adaptations such as in-
creased vascularization and NO-mediated vasorelax-
ation that preserve skeletal muscle function when
myoglobin is absent.
DISCUSSION
Using a gene targeting strategy, we previously re-
ported (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
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 N
G
-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).
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.
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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 alter-
ations 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 chal-
lenges such as hypoxia by changing gene expression to
promote morphological, biochemical, and functional
adaptations. Myoglobin-mutant mice manifest a vari-
ety 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 pre-
served 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 vasculogen-
esis in soleus muscles and enhanced NO metabolism in
EDL muscles. Intracellular hypoxia is likely to be an
inciting stimulus for these adaptations, through mech-
anisms 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 adapta-
tion when myoglobin is absent. An increased number of
capillaries would be expected to reduce the mean dif-
fusion 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 Ca
2⫹
/calmodulin-
dependent enzyme, is present in skeletal muscle, al-
though the relative content and activity of nNOS ap-
pear 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 impor-
tant 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., arte-
rioles) 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, oxy-
gen delivery is maintained by a NO-mediated vasodi-
lation 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.
The authors thank Dennis Belotto for assistance with the electron
microscopic analysis that appears in this paper.
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 Uni-
versity, Blacksburg, VA 24061.
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