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© 1996 American Heart Association, Inc.
Articles |
Cardiomyopathy in Transgenic myf5 Mice
From the Department of Microbiology and Immunology (J.G.E., L.A.L.), the Department of Medicine (A.M.), and the Department of Pathology (S.F.), Albert Einstein College of Medicine, Bronx, NY, and the Department of Anatomy (G.E.L., B.K.M.), University of Wisconsin Medical School, Madison.
Correspondence to L.A. Leinwand, Professor and Chair, MCDB CB 347, University of Colorado, Boulder, CO 80309-0347. E-mail leinwand@beagle.colorado.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and Methods Results Discussion References |
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Abstract To explore the compatibility of skeletal and cardiac programs of gene expression, transgenic mice that express a skeletal muscle myogenic regulator, bmyf5, in the heart were analyzed. These mice develop a severe cardiomyopathy and exhibit a significantly shorter life span than do their nontransgenic littermates. The transgene was expressed from day 7.5 post coitum forward, resulting in activation of skeletal muscle genes not normally seen in the myocardium. Cardiac pathology was not apparent at midgestation but was evident by day 2 of postnatal life, and by 42 days, hearts exhibited multifocal interstitial inflammation, fibrosis, cellular hypertrophy, and occasional myocyte degeneration. All four chambers of the heart were enlarged to varying degrees, with the atria demonstrating the most significant hypertrophy (>100% in 42-day-old mice). The transgene and several skeletal muscle–specific genes were expressed only in patchy areas of the heart in heterozygous mice. However, molecular markers of hypertrophy (such as
-skeletal actin and atrial
myosin light chain-1) were expressed with a wider distribution,
suggesting that their induction was secondary to the expression of the
transgene. In older (28-week-old) mice, lung weights were also
significantly increased, consistent with congestive heart
failure. The life span of bmyf5 mice was significantly
shortened, with an average life span of 109 days, compared with at
least a twofold longer life expectancy for nontransgenic littermates.
Expression of the transgene was associated with an increase in
Ca2+-stimulated myofibrillar ATPase in myofibrils obtained
from the left ventricles of 42-day-old bmyf5 mice.
Myocardial bmyf5 expression therefore induces a program of
skeletal muscle gene expression that results in progressive
cardiomyopathy that may be due to incompatibility
of heart and skeletal muscle structural proteins.
Key Words: cardiomyopathy • bmyf5 • transgenic mice • hypertrophy
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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The MDFs, MyoD, myf5, myogenin, and MRF4/herculin/myf6, direct determination and differentiation of skeletal muscle. They are characterized by a highly homologous 70–amino acid segment that contains a bHLH region. This region also shares sequence homology with a number of other transcriptional activators that are involved with cell-fate specification, such as daughterless and twist.1 MDFs are thought to be responsible for the determination and differentiation of myogenic progenitor cells destined to become skeletal muscle cells. The MDF proteins function as transcriptional activators of skeletal muscle–specific genes through distinct cis-acting elements.2 Additionally, they act in collaboration with other transcription factors, such as MEF2.3 MDFs are expressed at high levels during skeletal muscle development and continue to be expressed at low levels in adult skeletal myofibers. Although their role in adult skeletal muscle is unknown, they appear to be influenced by variations in neuronal activity and hormonal levels.4 5 6 The roles for the different MDFs have been examined by determining the temporal and spatial patterns of expression7 and the effects of targeted disruption of each of these genes.8 In all cases of inactivation of one or more MDFs, mice were born with beating hearts, clearly indicating that targeted disruption of the MDFs had no apparent effect on cardiac formation or development.
The myocardium originates in a region of rostral mesoderm called the cardiac crescent. The primitive heart appears as a simple tube between 7.5 and 8 days PC.9 10 Although a number of transcription factors have been isolated that are expressed in the heart,11 12 none of these has been shown to be cardiac specific. In Drosophila, tinman and cripto are expressed before heart formation and lie in a critical pathway for heart development.13 14 One potential cardiac determination factor has been reported by Litvin et al,15 with homology to the second helix of MyoD. An antibody directed against this helix supershifts a complex consisting of chick heart extract and the E-box of the muscle creatine kinase gene. This implicates a bHLH protein in myocardial gene expression. This finding might be expected, since several cardiac-specific genes contain the E-box motif within their promoters.16 17
The ability of MDFs to activate a skeletal program of gene
expression has been demonstrated in a variety of cell
types.18 19 In most cases, expression results in
conversion of the host cell to that of a myogenic cell. Ectopic
expression of MyoD in the hearts of transgenic mice activated
some muscle-specific skeletal sarcomeric genes and resulted in
embryonic lethality with severe cardiac abnormalities in fetuses
heterozygous for the transgene.20 Cardiac and skeletal
muscle share some common structural features, and during development,
many isoforms of sarcomeric proteins are coexpressed in both types of
muscle, such as -skeletal actin and ß-MHC. However, many of
the sarcomeric components of skeletal and cardiac muscle are distinct
and show restricted patterns of expression. In an attempt to further
our understanding of the compatibility of cardiac and skeletal muscle
genetic programs, we have studied the effects of bmyf5
expression in the murine myocardium. Heterozygous mice
survive to adulthood but have severe pathology and develop heart
failure. The results demonstrate that ectopic expression of
bmyf5 activates a skeletal muscle gene program that
is incompatible with normal cardiac function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Transgenic Mice
Transgenic animals were generously provided by R. Santerre and C. Smith (Eli Lilly Laboratories) and have been previously described.21 Briefly, the coding region and 3' untranslated regions of the bovine myf5 cDNA were cloned into the unique EcoRI site of pEMSVscribe, which contains the Moloney LTR.22 The line used in the present study (line 25-37) contained more than 30 copies of the transgene. All animals studied were heterozygous and were bred against an FVB/NJ background. Animals were maintained in the animal care facility in accordance with the Guide for Care and Use of Laboratory Animals (Department of Health and Human Services Publication NIH 86-23). To determine presence of the transgene, a portion of the tail was removed from young animals. From this sample, genomic DNA was prepared, and the presence of the transgene was determined as described by Santerre et al.21
Histology and Immunocytochemistry
Hearts from 15.5-day
embryos and 2- and 42-day-old mice were
removed and placed in 4% formaldehyde/PBS overnight at 4°C. They
were infiltrated with paraffin and sectioned on a microtome (MicroM).
Sections were stained using hematoxylin and eosin or Masson's
trichrome. For immunocytochemical analysis, sections were fixed
in 3.7% formaldehyde/PBS for 5 minutes at room temperature before
being permeabilized with 0.5% Triton X-100/PBS for 5
minutes. Nonspecific binding was blocked by incubation with 5% normal
goat serum overnight at 4°C. The primary antibody was then added, and
the sections were incubated overnight at 4°C. The tissues were washed
three times in 0.05% Tween 20/PBS for 10 minutes. A secondary antibody
conjugated to horseradish peroxidase was added, and the sections were
incubated for 60 minutes at 37°C, followed by washing as described
above, before being developed with diaminobenzidine and counterstained
with hematoxylin.
RNA Analysis
For Northern analysis, RNA was prepared from
heart or
skeletal muscle by the method of Chomczynski and Sacchi.23
Total RNA (10 µg) was separated on a 1% agarose/formaldehyde gel.
The quality and relative amounts of RNA were determined from ethidium
bromide staining. RNA was transferred to a nylon membrane (BioTrans,
ICN Biochemicals) by capillary action. Probes used were isolated from
unique cDNA coding regions of embyronic MHC,24
-skeletal actin,25 ANF,26
-tubulin,27 and cardiac LC128 and were
labeled by random priming with [32P]dCTP.
Single-stranded oligonucleotides were also used for
-MHC (5'-GAGGGTCTGCTGGAGAGG-3') and ß-MHC
(5'-TGTTGCAAAGGCTCCAGGTCTGAGGGCTTC-3').29 Blots were
hybridized in 6x SSC, 1% SDS, and 100 µg/mL sonicated salmon DNA.
Blots probed with cDNA were prehybridized and hybridized at 65°C;
blots probed with oligonucleotides were hybridized at
55°C. Washes (2x SSC/2% SDS, 1x SSC/2% SDS, and 0.5x
SSC/2%
SDS) were performed at 65°C or the calculated hybridization
temperature. Quantification of hybridized bands was carried out using a
phosphorimager (Molecular Dynamics).
The onset of transgene expression was determined by RT-PCR. Male mice positive for the transgene were used for breeding with control female mice. The presence of a vaginal plug was designated 0.5 day PC. Pregnant females were killed at select times after conception, and the embryos were removed. The embryos were divided and used for preparation of genomic DNA and total RNA, except for the 4.5-day and 7.5-day PC embryos. Because of limiting amounts of tissue, simultaneous preparation of DNA and RNA was achieved by use of TRI reagent (Iowa Biotech Inc). To determine the presence of the transgene, PCR analysis, as indicated above, was performed on genomic DNA. For determination of expression of the transgene, an RT/thermocycling (RT-PCR) reaction was performed. An antisense primer common to the first exon of the bovine and murine myf5 genes was incubated with 1 µg total RNA and AMV-RT (Promega) for 60 minutes at 42°C. After this, species-specific primers unique to the 5' untranslated regions were used in a thermocycling reaction to amplify the single-strand cDNA. A portion of each reaction was run on a 4% 1x TAE (mmol/L: Tris-Cl 10 [pH 7.4], sodium acetate 10, and EDTA 1) agarose gel and visualized by ethidium bromide staining.
In Situ Hybridization
Embryos were fixed in 4% PBS,
dehydrated, and infiltrated with
paraffin. The paraffin blocks were cut to give sections of 5 to 7 µm.
The hybridization and posthybridization procedures are detailed in
Lyons et al.30 The myogenin and MyoD1 probes were used as
described by Sassoon et al.31 The murine myf5
probe was used as described by Ott et al.32 The embryonic
MHC probe was used as described by Lyons et al.30 The
-skeletal actin probe was used as described by Sassoon et
al.25 The ALC1 probe was used as described by Lyons et
al.28 The MEF2C probe was used as described by Edmondson
et al.33 The MEF2B probe was used as described by Lyons et
al.34
Cardiac Contractile Proteins
Cardiac myofibrils were purified
using established
methods.35 Ca2+-activated ATPase
activity of cardiac myofibrils was measured at 30°C in the presence
of (mmol/L) Tris-Cl 50 (pH 7.6), CaCl2 10, KCl 300, and ATP
5 using 0.1 to 0.3 mg of protein.36 Myofibrillar ATPase
activities were measured in the presence of 3 mmol/L Mg2+
and either with 2.5 mmol/L EDTA or across a range of free
Ca2+ concentrations. ATPase data are expressed as
micromoles Pi per minute per milligram protein. Myosin
isoenzymes were analyzed by nondenaturing electrophoresis as
described by D'Albis et al,37 and the relative amounts
were estimated by integration of densitometric scans.
Electron Microscopy
Hearts were perfused with a PBS solution
before being
infused with fixation solution (4% paraformaldehyde,
2.5% glutaraldehyde, and 100 mmol/L calcium
cacodylate). They were then fixed in Trump's EM fixative for 5 days
and postfixed in 2% osmium for 1 hour. Hearts were then embedded in
acetonitrile/araldite/epoxy resin overnight, flat-embedded in
araldite/epoxy resin, and allowed to polymerize for 2 days. Thin
sections were stained with uranyl acetate and then by lead citrate.
Thin sections were examined using a Siemens No. 102 microscope.
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Developmental Profile of Transgene Expression
Transgenic mice were produced to express the bovine myf5 gene under the control of the MSV LTR in their brains and hearts. Mice heterozygous for the transgene exhibited abnormal morphology in both tissues.21 In the brain, this abnormality took the form of striated myofibers, and in the heart, the manifestation was an increase in size. To further characterize these mice and to define their cardiovascular pathology, hearts were analyzed histologically and characterized for their expression of the transgene and for activation of skeletal muscle genes. To determine when, during development, the transgene was expressed, RT-PCR was performed. Embryos were collected and used to determine the presence of the transgene and its expression pattern. In order to understand the pathogenesis, it was important to determine the onset of transgene expression to the developmental events of cardiac morphogenesis. It was observed that the transgene was not expressed in 4.5-day PC embryos but was expressed in 7.5-day PC embryos and thereafter (data not shown). These data indicate that the transgene was expressed before the formation of the heart, although since whole embryos were assayed, it cannot be determined whether this expression occurred in tissues that were destined to become the myocardium.
Life Span of Transgenic Animals
All of the animals studied
were heterozygous. Although the
transgenic females were fertile, they exhibited a decreased incidence
of conception, and most pregnant females went into apparent heart
failure, as evidenced by labored breathing and
cardiomyopathy. Additionally, transgenic animals
were frequently found dead during routine visits. Autopsies were not
routinely done, although in animals examined, heart enlargement was in
evidence. The survival rates of the bmyf5 transgenic mice
were compared with a group of randomly chosen littermate control mice
(Fig 1
). For the bmyf5 transgenic mice, the
mean life age at the time of death was 109±19 days, with no
significant gender differences. At the end point of the study (220
days), none of the wild-type littermates had died. Not included
were animals that were euthanized because they appeared to be suffering
from labored breathing, presumably due to heart failure. Therefore,
this estimate of life expectancy may be an underestimate.
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Cardiac Hypertrophy in bmyf5
Mice
Heart chamber weights were determined in animals at 6 to 7 weeks
and 6 months of age. In animals that were 6 to 7 weeks of age, body
weights were not significantly different between the groups (control,
21.4±0.6 g; bmyf5, 21.3±0.9 g), but all four chambers
exhibited increases in mass. Both atria were increased >100%, whereas
both ventricles were increased >10% (Table). Tissue
weights were also obtained from those surviving to 6 months. Greater
increases in the ventricles and left atria were observed in the
6-month-old animals compared with the 6-week-old animals
(Table). Additionally, in the 6-month-old mice, there was a
significant increase in the ratio of lung to body weight, suggesting
the presence of congestive heart failure. These data may
represent an underestimate of the extent of disease, since not
all animals maintained in the colony survived for 6 months. However,
the data clearly demonstrate that cardiac enlargement exists in the
transgenic animals and that it appears to progress with age.
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Histopathology in bmyf5 Transgenic Hearts
A previous
report21 suggested fibrosis and some
myocyte degeneration in bmyf5 mice. We extended these
studies to include a time course as well as electron microscopic
analysis. Since bmyf5 expression produced
hypertrophy of all four cardiac chambers, evidence of
cellular pathology seemed likely. Hearts from 15.5-day embryos
demonstrated no apparent pathology (data not shown). However, in both
2-day-old and 6-week-old bmyf5 animals, there were
clear indications of pathology (Fig 2). In the
2-day-old animals, myocyte degeneration was observed as well as an
increased number of interstitial cells. Multifocal
interstitial cellular infiltration and edema were also
observed. The presence of these cells in immunologically naive neonates
suggests that they do not represent a cellular immune response.
In the 6-week-old animals, multifocal interstitial
inflammation and fibrosis were observed along with myocyte
hypertrophy. Additionally, myocyte degeneration and
necrosis were also observed. The left ventricular free wall
and base, papillary muscle, and left ventricular
subendocardial wall and midwall all exhibited these pathological
features. In an attempt to identify the interstitial cells,
sections were stained with an anti-desmin antibody, a
muscle-specific marker. Whereas the ventricular
myocytes showed a characteristic striated pattern, the
interstitial cells were desmin negative, suggesting that
there were not any cells of myogenic lineage (Fig 3C).
There was no obvious evidence of multinucleated skeletal muscle cells
in the hearts of these animals, suggesting that expression of
bmyf5 did not convert cells in the myocardium to
morphologically distinct skeletal muscle cells.
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Staining with
Masson's trichrome defines type 1 collagen associated
with fibrosis present within the heart. Within control hearts, some
collagen (which stains blue) is present (Fig 3A
). In the hearts
from bmyf5 mice, there were varying degrees of fibrosis
present, with some areas displaying remarkable fibrosis
interspersed among areas of relatively normal morphology (Fig
3B).
Although the degree of fibrosis varied among animals and across a
single section, there was clear evidence of significant damage within
the bmyf5 hearts.
To examine the impact of this pathology on
the ultrastructure of the
myocyte and the sarcomere, electron microscopy was carried out. Shown
in Fig 4 are representative electron
micrographs from the heart of a 6-week-old bmyf5 animal.
The photographs indicate that within the same heart, it was evident
that adjacent fibers may display differing degrees of organization.
There were many regions in which active growth was evident, as
indicated by the presence of large numbers of ribosomes and newly
formed sarcomeres (Fig 4A). Other cells in the process of
forming
sarcomeres had disorganized Z lines (Fig 4B). Many regions
contained
well-organized sarcomeres, and there were no indications of a
significant change in their morphology. Also observed were many
fibrotic cells and large deposits of collagen (data not shown). These
data suggest that the myocardium from 6-week-old
bmyf5 mice appeared to be compensating for the extracellular
damage by actively forming new sarcomeres, consistent with
cellular hypertrophy.
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Induction of Skeletal Muscle Gene Expression
It is clear from
the above analysis that expression
of the bmyf5 transgene is disruptive to the
myocardium. The simplest hypothesis is that expression of
bmyf5 in the heart has activated a skeletal muscle
program of gene expression that is incompatible in some way with that
normally found in the heart. Given the considerable homology between
many of the structural proteins in cardiac and skeletal muscle, it
might be expected that the programs would be compatible. To determine
whether transgene expression induced the downstream expression of
skeletal muscle genes, analysis of left ventricular
RNA was performed by RT-PCR and Northern analysis. Several
skeletal muscle genes, including myogenin, herculin, and embryonic MHC,
were induced in 6-week-old hearts, as assayed by RT-PCR (data not
shown). Northern analysis indicated that
ventricular myosin LC1, -MHC, and -tubulin were
not significantly altered by expression of the bmyf5
transgene (Fig 5). Embryonic MHC was undetected by
Northern analysis. Given the observed hypertrophy
in 6-week-old animals, RNA was also analyzed for the
induction of a number of markers classically associated with
compensatory hypertrophy, including ß-MHC,
-skeletal actin, ANF, and ALC1a. Expression of these genes was
increased in the left ventricles of bmyf5 mice compared with
the left ventricles of littermate control mice (Fig 5). These
data
clearly show that there is an increase in the expression of gene
products associated with compensated hypertrophy.
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Localization of Skeletal Muscle Transcripts
The dispersed
pathology across the hearts of transgenic animals
suggested that bmyf5 expression may not be uniform. To
determine the distribution of bmyf5 and other skeletal
muscle transcripts, sections were cut from the hearts of 2-day-old
animals and hybridized to riboprobes for several different skeletal
muscle gene products (Fig 6). Two distinct patterns
of expression are evident from the sections shown: patchy expression
and relatively uniform expression. Expression of bmyf5 was
not evenly distributed throughout the heart. This patchy distribution
of expression was also observed for the myogenic regulator myogenin
(Fig 6B) and for the muscle transcription factors MEF2B (Fig
6C) and
MEF2C (data not shown) as well as embryonic skeletal MHC (Fig
6D).
Counterstaining the slides with eosin visualized cell boundaries. It
was determined that the patchy expression patterns of the MDFs and
embryonic MHC were associated with cardiomyocytes and not
the fibrotic or desmin-negative regions (data not shown). A second
group of genes showed a very different, broader distribution pattern
including several structural muscle gene products, such as
-skeletal actin (Fig 6F) and ALC1a (Fig
6E). Additionally, it
was observed that -skeletal actin was expressed in the atria of
the transgenic animals but not control animals. The genes whose
expression patterns showed a broader distribution have previously been
shown to be associated with compensatory hypertrophy. This
suggests that they are not directly induced by bmyf5
expression but are likely to represent a secondary response.
This hypothesis is also supported by the observation that the
expression patterns of ANF and ß-MHC were not induced by
bmyf5 expression in the hearts of 2-day-old animals but
were induced by 6 weeks of age (Fig 5). In the hearts of
2-day-old
animals, expression of the bmyf5 transgene did not
noticeably alter the expression of several structural proteins and
transcription factors, including perinatal MHC, ALC2A, muscle creatine
kinase, smooth muscle -actin, dystrophin, MyoD, sno, MRF4,
GATA-4, MEF2D, and Nkx-2.5 (data not shown). These data indicate that
the expression of some skeletal muscle gene products was directly
dependent upon expression of the transgene, whereas the response
patterns of others suggest a compensatory response.
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Functional Consequences of bmyf5
Expression
It is unclear how transgene expression and the resulting
pathology
affect cardiovascular function, but our results suggest
compensatory hypertrophy. We have previously observed that
the fractional shortening found in 6-month-old bmyf5
mice was slightly depressed compared with that found in littermate
control mice.38 These data indicate that there was a
functional change in the pumping capacity of the bmyf5
hearts. Several pathological and physiological
models have been shown to alter the contractile protein profile of the
heart and its functional properties. BALB/c mice that overexpress the
-skeletal actin isoform in their hearts have been shown to have
an increase in contractility.39 Pregnant
female mice have been shown to have an increase in the ß-MHC isoform
expression concomitant with a decrease in
Ca2+-activated ATPase activity.40
To
determine whether ectopic bmyf5 expression caused a shift
in the biochemical profile of cardiac myofibrils,
Ca2+-activated ATPase activity was determined.
Myofibrils from the left ventricle were partially purified, and
Ca2+-activated myofibrillar ATPase activity was
measured as a function of Ca2+ concentration. Myofibrils
from hearts of bmyf5 mice compared with littermate control
mice had a significant increase in the
Ca2+-activated ATPase activity, which is a measure
of the physiologically relevant
actin-activated ATPase activity (Fig 7,
top). In the presence of high salts and absence of
Mg2+, actin and myosin are dissociated; thus, the measured
activity reflects inherent myosin ATPase
activity.41 42 It
was observed that Mg2+-free
Ca2+-activated ATPase activity was not
significantly different between the hearts of control and
bmyf5 mice (Fig 7, bottom). Additionally,
electrophoresis
under nondenaturing conditions of the same myofibrillar preparations
did not indicate a shift in the isoform distribution of the MHCs nor
the stoichiometry of the major structural proteins (data not shown).
These data suggest that there was not a detectable shift in MHC
content. However, the discrete areas of bmyf5 expression may
not be sufficient to produce enough proteins to detect a shift in the
MHC isoform when myofibrils from the total left ventricle are
analyzed. Nevertheless, changes in
Ca2+-activated myofibrillar ATPase activity
indicate that a functional change at the biochemical level has resulted
from transgene expression.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Expression of the bovine myf5 transgene promotes a pattern of skeletal muscle gene expression in the myocardium with ensuing myocardial pathology. The distribution of activated gene expression assumed two forms. One pattern of expression correlated with the expression of bmyf5. A second pattern of skeletal muscle gene expression that included
-skeletal actin and ALC1 extended beyond the
region of bmyf5 expression. The activation of these
molecular markers of hypertrophy suggests a pattern of
primary cellular pathology followed by compensatory
hypertrophy. From one point of view, these results are
somewhat surprising, because of the high degree of sequence identity
among many heart and skeletal contractile proteins. Additionally, since
the sarcomeric structure of heart and skeletal muscle are very similar,
protein integration ought to be functionally compatible. However, the
extraordinarily small changes that appear to be critical for the
etiology of familial hypertrophic cardiomyopathy
suggest the very fine control under which the sarcomere is constructed
and maintained.43 None of the bHLH MDF factors affiliated with skeletal muscle formation have been detected in the developing normal myocardium. Thus, this group of bHLH proteins does not play a role in cardiomyogenesis. However, several cardiac-specific genes contain the E-box motif in their regulatory regions, suggesting that they may be influenced by bHLH proteins and that there may be cardiac equivalents for the MDFs. Although several homeobox proteins have been implicated, to date, no bHLH proteins have been reported that direct the formation of the myocardium.11 12 One promising candidate is the anti-H2 activity described by Litvin et al.15
There was no indication of a morphological transformation of cells in the myocardium into skeletal muscle fibers. This suggests that although the myocardium may be permissive for the MDFs, in terms of muscle-specific gene expression, there is some block to complete transformation into skeletal muscle. Clearly, normal cardiac structure and function are not compatible with expression of the skeletal muscle MDFs. It is also unknown whether the pathology seen in the bmyf5 hearts is the result of an internal sarcomeric organization event or the result of an extracellular event. Both may ultimately be involved, as suggested by the two different patterns of skeletal muscle gene expression: patchy and localized versus diffuse. The EM photographs show that cells containing newly formed sarcomeres are adjacent to cells containing normal sarcomeres. Speculatively, the initial synthesis of skeletal muscle proteins within a myocardial cell could lead to dysfunction and perhaps necrosis. This, in turn, may lead to a compensatory synthesis of other proteins that have been associated with hypertrophy. Alternatively, the induction of the skeletal muscle proteins may not be balanced in the small number of cells expressing the transgene, and the overexpression of specific proteins may cause the formation of disorganized sarcomeres.
Inappropriate expression patterns of structural proteins may be the
underlying cause of the cardiopathology observed. The present
results are similar to observations made by Miner et al,20
who examined the effects of ectopic expression of MyoD in the hearts of
transgenic mice. Embryonic skeletal muscle MHC gene expression was
observed in a patchy pattern. However, in that study, the hearts were
misshapen, failed to pump effectively, and proved to be lethal during
gestation. In the present study, the animals were viable into
adulthood. The theoretical reasons for these differences in viability
could be gene dosage or the onset of transgene expression. The MyoD
transgene was under the control of the MCK promoter, whereas the
bmyf5 transgene was under the control of the MSV LTR. In the
mouse, the MCK gene is initially expressed at 
day 13.44
Expression of cardiac-specific proteins is observed much earlier
than this, with a beating heart observed by day 9.0 PC.9
In the present study, bmyf5 transgene expression was
observed before the onset of cardiac formation. However, the degree of
pathology appears to be less than that observed by Miner et
al,20 consistent with the hypothesis that the
level of expression may play a major role in phenotypic differences.
Nonetheless, in both cases, the atria appeared to be preferentially
hypertrophied. The present study suggests that the expression of
the bmyf5 MDF does not necessarily interfere with early
heart development but that activation of skeletal muscle genes induces
pathology very early in postnatal life. Expression of mutant
contractile proteins has been linked to hypertrophic
cardiomyopathy in patients and in transgenic mice
(References 43 and 45, and K.L. Vikstrom, S. Factor, and L. Leinwand,
unpublished data, 1995). Collectively, these studies suggest that the
expression of sarcomeric proteins not normally found in the
myocardium is not tolerated.
Cardiac pathology was evident 2 days after birth. Large numbers of infiltrating desmin-negative cells were evident, suggesting that they were not of muscle origin. The cells observed would not have been due to infiltration of macrophages or leukocytes, because the immune system is undeveloped in such young animals. By 6 weeks of age, the number of infiltrating cells was decreased between the two groups, and the degree of fibrosis appeared to be greater. This suggests that events occurring early in life were resolved as the animals matured.
When we examined the functional consequences of bmyf5
expression on cardiac biochemistry, we obtained seemingly paradoxical
observations. Given the pathology observed, the increase in
myofibrillar ATPase activity was surprising. There are several possible
mechanisms responsible for an increase in the ATPase activity including
(1) a shift in the phosphorylation state of several
contractile proteins, (2) a change in the Ca2+ dynamics,
and (3) an increase in the expression of -skeletal actin. Hewett
et al,39 using a BALB/c mouse strain that constitutively
expresses -skeletal actin in the heart, showed a significant
positive correlation between the percentage of -skeletal actin
mRNA and +dP/dt, suggesting that the shift in the actin isoform
influenced the contractility within those hearts. These
changes occurred in the absence of hypertrophy or any
remarkable pathology. This suggests that the response is not inherently
pathological but represents an adaptive response by the
myocardium. Increased -skeletal actin expression is
observed in response to many physiological stimuli,
including various forms of overload, adrenergic stimulation, and
thyroid hormone.46 Within the bmyf5 hearts, the
spatial distribution of -skeletal actin expression was greater
than that of the transgene. Potentially, this represents a
compensatory change by the healthy cardiomyocytes in
response to the increased stress due to the pathology present.
Similarly, the increases in ANF expression and the concomitant
production and release of ANF would serve to decrease the work
of the heart, a compensatory response to the pathology.
In summary, ectopic cardiac expression of bmyf5 induces the expression of several skeletal muscle–specific gene products. Although the onset of transgene expression preceded the heart's formation, it did not appear to interfere with embryonic developmental patterns, but pathology was observed soon after birth. These results indicate the incompatibility of the cardiac and skeletal muscle gene programs, demonstrating the need for tight regulation to maintain normal cardiac function.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This research was supported by National Institutes of Health grants HL-2R01-GM-29090 (Dr Leinwand) and grants from the Muscular Dystrophy Association (Dr Lyons) and the American Heart Association (92-GB-27). The authors are grateful to R. Santerre and C. Smith for providing us with the myf5 transgenic mice. The authors are grateful for the technical help from J. Rivera, L. Guther, S. Schailis, S. Maithias, and Yvonne Cress.
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Footnotes |
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This manuscript was sent to Robert J. Lefkowitz, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received July 13, 1995; accepted December 4, 1995.
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