Published online before print March 30, 2001, doi: 10.1161/hh0701.089753
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© 2001 American Heart Association, Inc.
Integrative Physiology |
Adaptive Mechanisms That Preserve Cardiac Function in Mice Without Myoglobin
From the Departments of Internal Medicine (A.P.M., N.R., J.M.S., P.P.A.M., Y.K., J.E., R.S.W., D.J.G.), Thoracic and Cardiovascular Surgery (J.M.D., K.H.), and Molecular Biology (R.S.W., D.J.G.), University of Texas Southwestern Medical Center, Dallas.
Correspondence to Daniel J. Garry, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., NB11.200, Dallas, TX 75390-8573. E-mail garry{at}ryburn.swmed.edu
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Abstract |
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Abstract—Mice lacking myoglobin survive to adulthood and meet the circulatory demands of exercise and pregnancy without cardiac decompensation. In the present study, we show that many myoglobin-deficient embryos die in utero at midgestation with signs of cardiac failure. Fetal mice that survive to gestational day 12.5, however, suffer no subsequent excess mortality. Survival in the absence of myoglobin is associated with increased vascularity and the induction of genes encoding the hypoxia-inducible transcription factors 1
and 2, stress
proteins such as heat shock protein 27, and vascular
endothelial growth factor. These adaptations are
evident in late fetal life, persist into adulthood, and are sufficient
to maintain normal myocardial oxygen consumption during stressed
conditions. These data reveal that myoglobin is necessary to support
cardiac function during development, but adaptive responses evoked in
some animals can fully compensate for the defect in cellular oxygen
transport resulting from the loss of myoglobin.
Key Words: myoglobin • transgenic mice • metabolism • hypoxia • vasculature
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myoglobin is a monomeric cytoplasmic hemoprotein that is restricted to cardiomyocytes and oxidative skeletal myofibers.1 2 3 Acute chemical inhibition of oxymyoglobin formation demonstrated that myoglobin is an essential protein in the delivery of oxygen from the erythrocyte to mitochondria during periods of high metabolic demand.1 2 3 4 5 6 7 Surprisingly, we observed that mice with a complete deficiency of myoglobin are viable and have preserved cardiac and skeletal muscle function over a wide range of oxygen levels.8 Furthermore, animals lacking myoglobin are fertile and capable of surviving the hemodynamic stress of pregnancy. We found no evidence of spontaneous deaths or congestive heart failure within a large colony of adult mice (>100 animals) devoid of myoglobin.8
These observations suggested either that previous concepts of myoglobin function in the heart are incorrect or that mice developing in the absence of myoglobin adapt to this deficiency in a manner that maintains respiratory function in the face of an otherwise catastrophic decline in oxygen transport. In the present article, we present new evidence to support the latter viewpoint. In the absence of myoglobin, most embryos succumb to heart failure at midgestation. Those embryos that survive do so by mounting a complex compensatory response that involves extensive reprogramming of cardiac gene expression and serves to maintain myocardial oxygen consumption at normal levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Homozygous myoglobin-deficient (Mb–/–) mice were generated using gene disruption technology as previously described.8 A complete absence of myoglobin mRNA and protein was previously documented.8 Offspring of heterozygote intercrosses (Mb+/– x Mb+/–) from timed pregnancies were harvested at 3 periods during embryogenesis (gestational days 8 to 9 [E8 to E9], E9.5 to E10.5, and E11 to E12.5) and genotyped by Southern blot assays. Viable progeny from the heterozygote intercrosses were genotyped and analyzed as described below.
Histological and
Immunohistochemical Analysis and Vascular Quantitation
All embryos and embryonic tissues were staged,
harvested, fixed in 4% paraformaldehyde overnight,
either paraffin-processed or quick-frozen in liquid nitrogen, and
cryosectioned as previously
described.9 The staging of
embryos was performed by counting the presence of the vaginal plug as
day 0.5 after conception and by the number of somites. Tissue for
genotyping the embryos was obtained from the yolk sac.
Immunohistochemistry was performed as previously
described.2 9 The
whole-mount immunohistochemical staining protocol included overnight
fixation of the embryonic hearts in 4%
paraformaldehyde and overnight incubation with both the
primary (rabbit anti-CD31 or platelet-endothelial
cell adhesion molecule 1 [PECAM-1]; 1:50, Pharmingen) and secondary
antiserum (horseradish peroxidase–conjugated goat anti-rat serum;
1:100, Vector Labs). The embryonic hearts were then incubated with
dimethylaminoazobenzene/NiCl2 solution,
postfixed overnight in 4% paraformaldehyde/0.1%
glutaraldehyde, and photographed using an
Olympus SZH stereomicroscope.
In addition to whole-mount immunohistochemical staining with PECAM-1, we used a second technique to identify the endothelial cells of blood vessels in the developing and adult wild-type and mutant hearts. Embryonic (E12.5 to E16.5) or adult male Mb+/+ and Mb–/– hearts were isolated, immersion-fixed overnight in methyl-Carnoy’s fixative, embedded in paraffin, sectioned, and stained using the biotinylated lectin Bandiera simplicifolia lectin B4 (10 µg/mL; Vector labs).10 Substrate was developed with dimethylaminoazobenzene, and slides were either cover-slipped or lightly counterstained with hematoxylin and analyzed microscopically (magnification of 20x to 100x). For each embryo (n=3 for each genotype), the entire section was analyzed at 3 separate levels. For each adult animal, 30 fields were examined at 3 separate levels (n=3 for each genotype). The vasculature was quantified by 2 blinded investigators. Statistical analysis was performed with a Student’s paired t test.
RNA Isolation and Reverse
Transcription–Polymerase Chain Reaction
Total RNA was isolated from individually dissected
embryonic or adult hearts or whole embryos using the Tripure isolation
kit (Boehringer Mannheim). Four micrograms of total RNA were
used in each reverse transcription (RT) reaction (Retro-script,
Ambion). Complementary DNA (2 µL) was then used as a template for the
polymerase chain reaction (PCR) in a 20-µL reaction volume including
100 ng of each primer, 2 mmol/L MgCl2, Taq
buffer, and 1 U of Taq polymerase (GIBCO/BRL). Fifteen microliters of
each PCR reaction were loaded on a 2% agarose gel, as previously
described.9 For myoglobin, a
single forward primer was positioned to include exon 1, and
amplification by PCR (25 cycles) was done using reverse primers
complementary to sequences from exon 2. Semiquantitative RT-PCR using
RNA isolated from individually dissected embryonic (E12.5) or adult
(3-month-old) hearts was performed as previously
described.9 For each primer
pair, PCR conditions were used to establish linearity of the output
signal relative to the input template
DNA.9 PCR primer pairs used
for this study are included in the online data supplement (located at
http://www.circresaha.org).
Image Analysis
Stained sections were examined with a Leitz
Laborlux-S microscope equipped with an Optronics VI-470 CCD camera and
Scion Image 1.62 analysis software. Image processing was
completed using Adobe Photoshop 5.0 and printed using a Kodak XLS 8600
PS printer.
Hemodynamic Studies
(Working Heart Preparation)
Mice (25 to 30 g) were euthanized, and their
thoracic contents were excised and placed in a 4°C perfusate.
The heart and aorta were dissected free and perfused in the Langendorff
mode at 37.5°C with a (retrograde) perfusion pressure of 100 cm
H2O. The preparation was then converted to a
working mode with a left atrial pressure of 15 cm
H2O and an anterograde perfusion
pressure of 80 cm
H2O11 12
(online data supplement).
13C
Metabolic Studies
Hearts were perfused in the working mode as described
above. After stabilization with a standard Krebs-Henseleit buffer, the
buffer was changed to the following mixture of
physiological substrates:
3-13C lactate (1.2 mmol/L),
1,3-13C acetoacetic acid (0.17 mmol/L),
3-13C pyruvate (0.12 mmol/L), BSA
(0.75%), U-13C labeled fatty acids
(0.5 mmol/L; consisting of 50% palmitic acid, 23% oleic acid,
15% linoleic acid, 11% palmitoleic acid, and 1% stearic acid), and
unlabeled glucose (8
mmol/L).13 After 30 minutes
of perfusion, hearts were freeze-clamped and extracted in perchloric
acid, neutralized with KOH, reconstituted in
D2O, and analyzed by nuclear magnetic
resonance (NMR)
spectroscopy.14
Proton-decoupled 13C NMR spectra were
obtained at 150.87 MHz using a Varian Inova spectrometer with a
broadband probe, a 45-degree observe pulse, an interpulse delay of
1.5 s, 43 488 data points, and 4000 scans.
Isoproterenol-Induced Cardiac Hypertrophic
Challenge
Ventricular hypertrophy was
induced in age- and sex-matched adult Mb+/+
and Mb–/– mice by continuous
administration of isoproterenol (0.028 g/mL at a rate of 1.0 µL/h),
as described
previously.15 16
After 7 days of continuous infusion, the mice were weighed and
euthanized. The hearts were excised, weighed, and either fixed in 4%
paraformaldehyde or frozen for isolation of total
RNA.2 Cardiac
hypertrophy was determined by the comparison of heart
weight to body weight, histological analysis,
and gene expression.
Ischemic
Injury/Cardiomyopathic Challenge
After continuous anesthesia with 2.0%
isoflurane, age- and sex-matched adult Mb+/+
and Mb–/– mice underwent a left
thoracotomy/pericardiotomy to expose the left ventricle, and the left
anterior descending coronary artery was ligated with 8-0
prolene sutures. The residual pneumothorax was evacuated to restore
negative pressure, and buprenorphine (0.05 mg/kg) was administered for
postoperative pain control. Two months after ischemic injury,
transthoracic echocardiography was
performed under isoflurane anesthesia. The mice were
weighed and euthanized, and their hearts were excised, weighed, fixed
in 4% paraformaldehyde, and processed for
histological analysis.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Morphogical Phenotype of the Myoglobin Mutant Embryos
Analysis of a large group of embryos from 16 male and 16 female heterozygote intercrosses at sequential stages of embryonic development demonstrated a Mendelian distribution of genotypes (1:2:1 ratio) only in embryos at E9.0 days or younger (online data supplement). Between days E9.5 and E10.5, the majority of embryos homozygous for the mutated myoglobin allele (Mb–/–) exhibited a number of severe defects. These included congestive heart failure manifested by prominent pericardial effusion and vascular insufficiency manifested by diffuse hemorrhages, a generalized developmental delay, and reduced size (Figure 1
). Similar defects were observed more rarely in
animals bearing a single copy of the myoglobin-null allele
(Mb+/–). Of the myoglobin-null embryos that
developed beyond this stage (E11.0 days or older), there was no
evidence of further fetal loss or structural abnormalities (online data
supplement). These findings were noted in 2 strains of mice (C57BL/6
and Sv 129).
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To further define the morphological defects associated with
reduced expression of myoglobin, histological sections
of Mb+/+, Mb+/–
(data not shown), and Mb–/– embryos were
compared. Histological analysis of runted
mutant embryos revealed evidence of myocardial thinning, congestive
heart failure, and vascular insufficiency with peripheral
hemorrhage
(Figure 1
). Other than a generalized developmental delay,
morphological defects in Mb–/– embryos
were limited to the cardiovascular system, and other
major developmental events were not noted until the time of death.
Among the 12 failing Mb–/– embryos at E9.5
to E10.5, 4 had a severe pericardial effusion consistent with
congestive heart failure and 8 had extensive hemorrhages.
Ultrastructural analysis of phenotypically abnormal but viable
Mb–/– hearts at E10.5 revealed evidence of
edema in cells of both the endocardial and myocardial layers, increased
cytoplasmic vacuoles, and a less well-developed sarcomeric myosin actin
contractile apparatus compared with normal wild-type
littermates (data not shown). The TdT-mediated dUTP nick-end labeling
assay showed no evidence of an apoptotic process associated
with the hearts of failing Mb–/– embryos
(data not shown), which is consistent with an oncotic process
associated with the demise of the mutant embryos.
Increased Vascularity in Surviving
Mb–/– Embryos and Adult Mice
Hearts were dissected free from
Mb–/– and Mb+/+
embryos that survived to E12.5. Immunostaining to
detect PECAM-1, a marker for endothelial cells,
demonstrated hypervascularity in the viable
Mb–/– heart (n=3) compared with wild-type
(Mb+/+) hearts (n=3)
(Figure 2). These results were confirmed using a specific
lectin that identifies the endothelial cells. Further,
we observed that the formation of myocardial vascularization was
established earlier during cardiac development in the absence of
myoglobin
(Figure 2). There was a 48% increase in capillary density in
the ventricles of Mb–/– mice at E13.5
(Figure 2).
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Adult Mb–/– mice also
exhibited an increased vascular supply in the atria (46% increase) and
ventricles (28% increase) compared with wild-type controls
(Figure 3). These findings are in agreement with those of
Godecke et al,17 who used
ultrastructural morphometric techniques and reported a 31% increase in
capillaries in the myoglobin mutant ventricle. There were no apparent
differences in the ultrastructural morphology or the histochemical
staining of succinate dehydrogenase activity, a marker of mitochondrial
content (online data
supplement).1 18
We also observed no apparent differences in the profile of serum
electrolytes (data not shown), hemoglobin concentration, or hematocrit
between age- and sex-matched adult Mb+/+ or
Mb–/– mice (online data
supplement).
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Reprogramming of Cardiac Gene Expression in the
Absence of Myoglobin
Using northern analysis and semiquantitative
RT-PCR analysis, we observed enhanced expression in
Mb–/– (and
Mb+/–) embryonic and adult ventricles of a
number of genes that are known to be induced in response to hypoxic
conditions. Hypoxia-inducible factor-1 (HIF-1), HIF-2
(ePAS), stress proteins (heat shock proteins), and vascular
endothelial growth factor are all markedly induced in
the myoglobin-deficient ventricles of both embryos
(Figure 4) and adults
(Figure 5 and online data supplement). These changes in gene
expression plausibly drive cellular adaptations, such as increased
vascularization, that preserve cardiac function when myoglobin is
absent.
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Hemodynamic Analysis of
Mb+/+ Versus
Mb–/– Hearts
Using the working heart preparation, we previously
reported no significant differences in cardiac performance
between wild-type and myoglobin mutant hearts, even under hypoxic
conditions.8 Analysis
of hemodynamics in working hearts perfused with a
physiological mixture of substrates under normoxic
conditions revealed no significant differences in coronary
flow, cardiac output, or oxygen consumption in
Mb–/– and Mb+/+
hearts (online data supplement and the
Table
above). The results obtained in the present study agree
closely with the hemodynamic measurements obtained by
Grupp et al12 using the
working heart preparation in wild-type mice.
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Myocardial oxygen consumption has a number of determinants,
including heart rate, contractility, systolic
wall tension, and substrate
utilization.1 13 14
Under physiological conditions, the adult heart
generates energy or ATP primarily from the oxidative
phosphorylation of fatty acids as the preferred
substrate. To define the preferred myocardial substrate for oxidative
phosphorylation in the absence of myoglobin, working
heart preparations were perfused with
13C-labeled substrates and then
analyzed using NMR
spectroscopy.13 14
We found that fatty acids were the preferred substrate in both
Mb–/– and wild-type hearts. The relative
use of fatty acids and acetoacetate for the production of
acetyl-coenzyme A units feeding into the Krebs cycle was similar in the
2 groups, but there was a modestly greater use of
13C-labeled lactate in the hearts from
Mb–/– animals
(Table).
The use of these 13C-labeled substrates in
the present study agrees with previously published results using
this technique in the rat
heart.14
Working heart preparations were then challenged by the
administration of the ß-adrenergic agonist isoproterenol. Despite a
20% increase in heart rate, no differences were observed between
Mb+/+ and Mb–/–
hearts with respect to myocardial oxygen consumption in response to
isoproterenol stimulation
(Figure 6). These results support the conclusion that cardiac
function in response to short-term adrenergic stimulation is preserved
in the absence of myoglobin.
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O2; A) or heart rate (B) under baseline conditions (Pre) or after stimulation with isoproterenol (1x10–6 mol/L; Post) between Mb+/+ (n=5) and Mb–/– (n=6) hearts. Histograms depict mean±SEM. Following a 7-day continuous infusion of isoproterenol in adult Mb+/+ (E) and Mb–/– (F) mice, the hearts were excised and noted to be increased in size compared to the wild-type (C) and knockout (D) controls. No significant histological differences were noted between chronically stimulated Mb+/+ (G) and Mb–/– (H) hearts using a Masson trichrome stain. I, Northern blot analysis was undertaken using total RNA isolated from the ventricles of Mb+/+ and Mb–/– mice in the absence (-) of or following a 7-day continuous infusion of isoproterenol (+). There was a similar induction of atrial natriuretic factor (ANF) expression in wild-type and Mb–/– ventricles after isoproterenol stimulation. Mb indicates myoglobin; GAPDH, glyceraldehyde phosphate dehydrogenase. Bar=2.5 mm in C, D, E and F; bar=50 µm in G and H.
Isoproterenol-Induced Cardiac
Hypertrophy
To assess the response to long-term adrenergic
stimulation in this knockout model, ventricular
hypertrophy was induced by a 7-day continuous
administration of
isoproterenol.15 16
Using this well-characterized model of cardiac hypertrophy,
we determined the functional and structural adaptations in response to
adrenergic stimulation in mice that lacked myoglobin. No significant
differences in heart weight were noted between age- and sex-matched
wild-type and myoglobin mutant mice. Knockout and wild-type mice that
were subjected to long-term isoproterenol infusion had a 34%
(P<0.005) and 22%
(P<0.05) increase,
respectively, in the heart weight to body weight ratio (online data
supplement) and an induction of atrial natriuretic factor
expression in the ventricle compared with their respective controls
(Figure 6). There was no evidence of congestive heart failure
in either Mb+/+ or
Mb–/– mice in response to this stimulus.
Histological analysis
(Figure 6) after long-term isoproterenol infusion revealed
the presence of focal regions of myocardial fibrosis, but the frequency
of such abnormalities did not seem to differ between
Mb+/+ and Mb–/–
animals.
Ischemic Injury and
Cardiomyopathy
As a further challenge, ischemic injury was
induced surgically in wild-type and myoglobin mutant mice. We observed
no differences in survival during the perioperative
period or during a 2-month survival period between wild-type or
knockout mice. Furthermore, 2 months after the ischemic insult,
wild-type and Mb–/– mice had comparable
increases in heart size and decreases in left ventricular
function
(Figure 7). Using transthoracic
echocardiography, fractional shortening was
depressed in wild-type (wild-type control [n=5], 0.60±0.02;
wild-type with coronary ligation [n=3], 0.34±0.05;
P<0.001) and myoglobin
mutant (Mb–/– control [n=5], 0.59±0.03;
Mb–/– with coronary ligation
[n=3], 0.34±0.05; P<0.001]
mice after coronary artery ligation. Although
myoglobin-deficient mice survived this ischemic challenge,
these data do not exclude the possibility that myoglobin may function
in oxygen metabolism during the early phase(s) of
ischemic injury.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed that a majority of embryos lacking myoglobin die, apparently of congestive heart failure and vascular insufficiency, within the brief period (E9.5 to E10.5) of gestation that corresponds to the initial expression of myoglobin in wild-type embryos. Increased lethality was also observed in embryos (E9.5 to E10.5) who were haploinsufficient for myoglobin, suggesting that myoglobin content was important for viability during this critical period of cardiovascular development. However, a proportion of embryos without myoglobin survive past this critical developmental stage, and surviving animals exhibit grossly normal cardiovascular function as adults.8 17
These new data establish that myoglobin is important during cardiac development at a stage (E9.5 to 10.5 days) associated with the onset of rhythmic cardiac contractions and increasing metabolic demands that precedes the establishment of a functional coronary vasculature.19 20 21 22 It is logical to surmise that the rapid growth of the heart that occurs during this period of development, particularly the expansion of the ventricular wall, increases the distance oxygen must diffuse from the ventricular cavity to reach cells closer to the epicardial surface of the heart.22 The establishment of the coronary vasculature is ultimately required to supply oxygen to the expanding myocardium, and even subtle differences in the timing of vasculogenesis and cardiac growth may be sufficient to increase the dependence of working myocytes on myoglobin to facilitate oxygen delivery. Remarkably, the requirement for myoglobin at this stage of development is not absolute, and some animals mount an adaptive response that sustains myocardial function.
Our current findings suggest that the ability to survive without myoglobin is based on increased vasculogenesis, driven by intracellular hypoxia, and ultimately establishes a new steady-state in which oxygen transfer is preserved in the absence of myoglobin. This conclusion is supported by the increased expression of hypoxia-responsive genes, including those encoding angiogenic growth factors, and morphological evidence of hypervascularity in Mb–/– embryos that survive past E12.5 and into adult life.
The observation that embryos surviving beyond E12.5 suffer little or no subsequent mortality and maintain relatively normal circulatory function as adults is consistent with either of two conclusions with respect to the importance of myoglobin in the adult heart. Fetal death at E9.5 to E10.5 may result from a developmental bottleneck at this stage that is based on a critical requirement for myoglobin unique to the period that precedes the development of a coronary circulation,19 20 21 22 and myoglobin may have lesser physiological importance at later stages. Alternatively, the ability to survive the fetal stage may select for those animals that establish and maintain powerful adaptive mechanisms that remain active as the heart matures in postnatal life. Our data argue in favor of the latter viewpoint.
In adult mice, we showed that cardiac function is preserved under baseline conditions and in response to adrenergic stimulation in mice without myoglobin. Furthermore, myoglobin-deficient mice survived ischemic injury and suffered left ventricular dysfunction comparable to that of wild-type animals exposed to the same coronary ligation protocol. We observed no abnormalities with respect to serum electrolytes, hemoglobin concentration, or hematocrit in myoglobin-mutant mice. A comprehensive hemodynamic analysis showed normal cardiac output, stroke volume, heart rate, coronary flow, and myocardial oxygen consumption in Mb–/– hearts. Substrate utilization was relatively preserved in the absence of myoglobin. Although fatty acids remained the preferred metabolic substrate, there was a modest increase in lactate utilization in the myoglobin mutant heart. Future studies will further examine substrate utilization in the myoglobin-deficient heart in response to fatiguing exercise and environmentally challenging conditions.
In contrast to knockout mice that lack either desmin23 or creatine phosphokinase,24 25 we observed no pathological changes in mitochondria or the sarcomeric ultrastructure of cardiomyocytes from mice that lack myoglobin. Furthermore, myoglobin mutant mice are capable of developing cardiac hypertrophy, induced by long-term ß-adrenergic stimulation, without apparent hemodynamic compromise.
Our current findings also indicate that survival in the absence of myoglobin is attributable to reprogramming gene expression within the myocardium and compensatory cellular responses that include increased vasculogenesis. Intracellular hypoxia is likely to be an inciting stimulus to these adaptations, through mechanisms that include the induction of HIF-1, a basic-helix-loop-helix-PAS protein.26 Target genes induced by HIF-1 increase oxygen delivery (eg, angiogenic growth factors and nitric oxide synthase) or facilitate ATP production in the absence of oxygen (eg, glucose transporters and glycolytic enzymes).26 27 Disruption of the HIF-1 gene results in embryonic lethality at midgestation in association with developmental arrest and cardiovascular malformations.28 29 Even haploinsufficiency at the HIF-1 locus impairs cardiovascular responses to long-term hypoxia.30 These results collectively support an essential role for HIF-1 as a master regulator of oxygen homeostasis.
Other results of the present study are largely in agreement with those of Godecke et al,17 who confirmed our initial observation of preserved cardiac function in mice without myoglobin. They observed increased vascular density in the hearts of Mb–/– mice. Our present report provides the first description of fetal death in embryos without myoglobin and is the first to define specific changes in gene expression that are likely to drive the adaptive changes in vascular supply.
In summary, we demonstrate that myoglobin plays an important role during cardiac development. In the absence of myoglobin, heart failure and circulatory insufficiency lead to death in the majority of embryos. This requirement for myoglobin is not absolute, and a minority of animals mount an adaptive response that reprograms gene expression and increases myocardial vascularity; this adaption is sufficient to maintain oxygen transfer to cardiomyocytes and to preserve circulatory function. A more complete definition of the cellular, physiological, and molecular mechanisms of this powerful adaptive response could conceivably lead to advances in therapy for patients with ischemic heart disease.
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Acknowledgments |
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This work was supported by grants from the Texas Affiliate of the American Heart Association, the National Institutes of Health (AR40849, HL54794, and HL06296), and the D.W. Reynolds Foundation.
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Footnotes |
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Original received March 13, 2000; resubmission received January 16, 2001; revised resubmission received March 7, 2001; accepted March 7, 2001.
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