* Department of Molecular Genetics, Department of Microbiology and Immunology, Albert Einstein College of Medicine, New
York 10461; § Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261;
Department
of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109; ¶ Department of Molecular Cellular and
Developmental Biology, University of Colorado, Boulder, Colorado 80309
The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying null mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both null strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb null mutants are generally milder than in the MyHC-IId/x null strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for null expression of the two genes. Most striking is that while both null strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb null mice has significantly reduced ability to generate force while IId null mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.
MYOSIN heavy chains (MyHCs)1 comprise a family
of molecular motor proteins, eight of which are
expressed in mammalian cardiac and skeletal
muscle. Each is characterized by its time and site-specific expression pattern. There are two principal developmental
isoforms expressed in embryonic and neonatal skeletal
muscle (Bouvagnet et al., 1987 Members of the sarcomeric MyHC multi-gene family contain 39-40 exons (Mahdavi et al., 1986 Myosin is the major component of thick filaments within
the sarcomere. A single holomyosin molecule (~ 600 kD)
is composed of two identical heavy chains (230 kD each),
two phosphorylatable (regulatory) light chains and two alkali (essential) light chains. The COOH-terminal rods of
the two heavy chains assemble into a coiled-coil, while their
NH2-terminal sequences fold to form two globular heads
which constitute the motor domain.
Muscle contraction involves the coordinate action of
nerve stimulus, ion transport, metabolic energy substrates
and the proper functioning of the sarcomeric "engine" for
its normal activity. Abnormalities in any of these individual constituents could conceivably result in the improper
function of the assembled sarcomere. In this context, the
myosin molecule is one of the more important structural
components of the muscle sarcomere (representing ~40% of the myofibrillar protein) (Warrick and Spudich, 1987 Most vertebrate skeletal muscles express multiple MyHC
genes. The most striking examples of this are adult masseter muscle and extraocular muscle. The masseter expresses seven of the eight known muscle MyHC genes including both developmental isoforms, all three adult fast
isoforms, the The number of members in the myosin gene family and
its complex temporal and spatial patterns of expression
raise the question of whether each gene is functionally distinct or whether there is functional redundancy within the
family. There are several lines of evidence that suggest
functional diversity within the family. First, the orthologous MyHCs from different species are more related to
each other than family members within a species (Weiss
and Leinwand, 1996 To determine whether the molecular diversity in the
vertebrate myosin heavy chain gene family represents functional diversity, we initiated an effort to generate strains of
mice carrying mutations in different MyHC genes. Here
we report the successful targeted inactivation of the adult
fast skeletal myosin heavy chain genes MyHC-IIb and IId/x
in embryonic stem cells and derivation of mice carrying
the mutation. Mice which are null for the MyHC-IIb and IId/x gene are viable and fertile, but weigh less than wild
type by 10 wk of age, and this lower weight is maintained.
At young ages (4-6 wk), IIb null mice exhibit gross limb
muscle weakness which resolves with age, whereas IId/x
null mice have chronic limb weakness. Distinct physiological defects were seen in muscles from both null strains. IIb
null mice are severely impaired in the amount of force
generated/cross sectional area of muscle, whereas IId/x
null mice have normal force generation, but delayed kinetic properties. A large proportion of homozygous IId/x
null mice and a small number of heterozygotes develop severe curvature of the spine (kyphosis). Distinct histological abnormalities were found in both null strains. These results suggest that MyHC-IId/x and IIb are required for the
normal function of adult skeletal muscle and that mice
missing these gene products display distinct phenotypes.
Generation of MyHC Clone E14-1 ES cells were cultured according to Robertson (Robertson,
1987 Clones in which homologous recombination had taken place were injected into C57BL/6 recipient blastocysts which were transferred to CD-1
pseudopregnant females (Robertson, 1987 Analysis of RNA
A 90-bp PCR fragment containing the 3 The transcription reaction was performed with the Maxiscript kit (Ambion Inc., Austin, TX) according to the manufacturer's protocols. The
mouse pTRI-actin template used to make the 360-bp mouse Myofibrils, Western Blot, and High
Resolution Electrophoresis
Myofibrils were prepared from the tibialis anterior muscles of 5-mo-old
female mice. Muscles were minced with scissors and essentially prepared
as described by Solaro et al. (1971) Grip Strength Measurement
Muscle strength in mice was measured using an automated Grip Strength
Meter (Columbus Instruments, Columbus, OH). The instrument is designed to measure both fore- and hindlimb grip strength in small laboratory rodents. The instrument uses an electronic digital force gauge which
measures the peak force exerted upon it by the action of the animal.
For the forelimb grip strength measurement, the animal was held by
the base of the tail and allowed to place its forepaws on the flat wire mesh
of the pull bar connected to the force gauge. The animal was slowly pulled
away from the pull bar at a rate of ~1 inch/s until it released the pull bar.
Peak tension was recorded from the force gauge's digital readout. The
hindlimb grip strength measurement was performed in a similar manner,
but allowing the animal to grasp the pull bar with both front and hind
paws.
Metabolic Studies
Two separate metabolic cages (Nalge Co., Rochester, NY) were used to
group-house five wild-type mice and five homozygous null IId/x mice at 4 wk
of age. 4 d were allowed for acclimatization; for an additional 7 d, food
and water intake were followed in these two groups of mice.
X-Ray Analysis
One wild-type male mouse (12 wk old) and one MyHC-IId Muscle Physiology
Experimental Apparatus.
The experimental chamber consisted of a plastic
trough with a glass bottom filled with Ca2+ Krebs solution. Solutions in
the chamber were continuously bubbled with oxygen and kept at 22°C as
described previously (Metzger et al., 1985; Periasamy et al., 1984
),
and four isoforms expressed in adult fast skeletal muscle
(Weydert et al., 1983
; Parker-Thornburg et al., 1992
). One
of these genes is exclusive to adult extraocular muscle
(Wieczorek et al., 1985
). Two isoforms are expressed in cardiac muscle (Gulick et al., 1991
), one of which is also
the slow skeletal muscle isoform (Rindt et al., 1993
).
) and exhibit a striking degree of sequence conservation including the location
of intron/exon boundaries among the isoforms as well as
between such widely divergent species as rat and Caenorhabditus elegans. The MyHC genes expressed primarily in
skeletal muscle are located in a cluster on mouse and human chromosomes 11 and 17, respectively (Leinwand et al., 1983
), while the two cardiac MyHC genes (
and
) are located on mouse and human chromosomes 14 (Saez et al.,
1987
; Weydert et al., 1985
).
).
While mutations in the major cardiac MyHC gene (MyHC-
)
have been implicated in a dominant genetic heart disease,
familial hypertrophic cardiomyopathy (Marian and Roberts, 1994
), no diseases specific to skeletal muscle have yet
been ascribed to MyHC mutations.
-slow, and the
-cardiac. Extraocular muscle expresses, in addition to the seven genes described above, the extraocular isoform, MyHC-eo (Wieczorek et
al., 1985
; Pedrosa-Domellof et al., 1992
).
). Second, the contractile velocity of a
muscle has been shown to largely correlate with its myosin
content (Barany, 1967
). While the enzymatic properties of
the two cardiac MyHCs (
and
) have been shown to be
quantitatively distinct, the biochemical properties of each
skeletal myosin isoform are not yet known. Arguing
against distinct functions for each gene are the common
origin of vertebrate skeletal MyHC isoforms and their
very high degree of conservation in genomic structure and amino acid sequence. This argument may be most relevant
to the three adult fast isotypes which include the MyHC-IIa, -IIb, and -IId/x genes. These three genes are highly homologous and are broadly expressed in skeletal musculature. The fast IIb and IId/x MyHC genes show the highest
homology, although complete sequence for these three
genes is not available to allow a full comparison. Over a
segment of 613 amino acids, human MyHC-IIb and IId/x
show 94% identity (Weiss and Leinwand, 1996
). IId/x and
IIb gene transcripts can be detected between days 2 and 5 of post-natal life in the mouse and are coexpressed in
many muscle groups including masseter, tongue, quadriceps, plantaris, soleus, psoas, gastrocnemius, tibialis anterior, and extensor digitorum longus (EDL). The most
prominent MyHC in the adult skeletal musculature is IIb
where it comprises >70% of the myosin overall. MyHC-IId
is less abundantly expressed but comprises 30-60% of the
MyHC in diaphragm, tongue, masseter, plantaris, and tibialis anterior (Jansen et al., 1996
).
Materials and Methods
/
-deficient Mice
) except that the medium was supplemented with leukemia inhibitory
factor (GIBCO BRL, Gaithersburg, MD) at 1,000 U/ml. Approximately 2 × 107 E14-1 ES (Fodde, 1994
) cells were resuspended in electroporation buffer (10 mM HEPES-PBS, pH 7.05). Cells were mixed with 100 µg of
the linearized targeting constructs and subjected to a 4.1-ms pulse at 400 V,
250 uFA. Cells were divided equally onto 10 10-cm dishes containing
feeder cells and ES-cell medium and grown for 36 h before addition of 150 µg/ml G418 (Geneticin; GIBCO BRL) for positive selection. Cells were
cultured for 7-10 d, until such time as discrete G418 resistant colonies appeared on the plates. 140 individual colonies were picked, trypsinized, and transferred to individual wells of 24-well dishes containing ES-cell medium and feeder cells. DNA from individual clones was analyzed by Southern blot hybridization with probes flanking the construct integration site.
). Genotyping of mice was done
by Southern analysis as described above.
UTR of the MyHC-IId/x gene
was generated from genomic mouse DNA using a specific primer pair
generated based on published mouse sequences. They are RK-1509 (5
-GCCATGTTAATGTGGAGACC-3
) and RK-1510 (5
-CATGCTTCCCTACCTTCTC-3
). Similarly, the probe for the MyHC-IIb gene was
constructed using a 120-bp PCR fragment corresponding to the 3
UTR.
The primer pair used to amplify this fragment from mouse genomic DNA
are IIb3F (5
-ACAAGCTGCGGGTGAAGAGC-3
) and IIb3R (5
-CAGGACAGTGACAAAGAACG-3
). The product was blunt-ended using T4 Polymerase (GIBCO BRL), and ligated into EcoRV digested
Bluescript SK+/
cloning vector (Stratagene Inc., La Jolla, CA) thus
placing them under the control of the T7 and T3 promoters. The transcriptional orientation of the insert in each probe was assessed by sequencing
individual clones.
-actin antisense probe was provided with the kit. RNA transcript of high specific activity was generated either by digesting the plasmid with XbaI and transcribing with the T7 polymerase enzyme in the presence of [32P]UTP, or
by cutting the plasmid with XhoI and transcribing with the T3 polymerase
enzyme in the presence of [32P]UTP. Ribonuclease protection assays were
performed with the RPA II kit (Ambion) according to the manufacturer's
protocol.
. Tissues were homogenized in 1 ml of
buffer A (50 mM KCl, 10 mM KPO4, 2 mM MgCl2, 0.5 mM EDTA, 2 mM
DTT, pH 7.0). The homogenate was separated by centrifugation at 15,800 g
for 15 min at 4°C. The resulting pellets were resuspended in 1 ml of buffer
C (buffer A + 1% Triton X-100) and again separated by centrifugation
(15,800 g for 15 min at 4°C). Pellets were resuspended in 1 ml of buffer (60 mM KCl, 30 mM imidazole, 2 mM MgCl2, 1 mM DTT, pH 7.0) and stored at
70°C. 5 µg of total protein was either separated on a 10% SDS-polyacrylamide gel or blotted onto nitrocellulose (Bio-Dot; Bio Rad Labs., Hercules, CA). The gel was stained with Coomassie blue and the nitrocellulose was probed with an antibody specific to MyHC-IIb (BFF3)
(Schiaffino et al., 1986
). High resolution gel electrophoresis was performed according to Talmadge and Roy (1993)
after homogenication of
tongue and preparation of myofibrils as described above.
/
male
mouse which displayed visible kyophosis (8 wk old) were chosen for x-ray
analysis. The mice were anesthetized and x-rayed by a veterinarian.
). Muscles were mounted in the
chamber between a force transducer (model 400; Cambridge Technology,
Inc., Watertown, MA) and a moving coil galvanometer (Cambridge Technology, Inc.) was used to rapidly release the muscle. Both the force transducer and the galvanometer were mounted on three-way positioners to allow the muscle strips to be properly stretched and aligned. Muscle strips
were electrically stimulated through the use of platinum electrodes glued
to the sides of the trough.
/
mouse. Mice were anesthetized by injection with nembutal. The tendons of the EDL in one leg were exposed and tied with 4-0 suture. The muscle was then isolated, the tendons cut, and the muscle removed from the animal and placed in Ca2+ Krebs solution on ice. The
Krebs solution was continuously bubbled with oxygen. The EDL from the
other leg was then removed in a similar manner. The EDL was mounted
in the experimental chamber between the force transducer and a moving
coil galvanometer by means of hooks pushed through the tendons at the
level of the ties. The fiber length and the simulation voltage were then adjusted to produce a maximum twitch force response, and the stimulation frequency necessary to produce a fused tetanus determined. At the end of
the experiment the length of their muscle was measured in the experimental chamber. The muscle was then cut out and stored in SDS buffer for
later analysis of the protein composition. Cross-sectional area (CSA) was
determined by dividing the mass by the length and density of the muscle.
Muscle density was assumed to be 1.06 mg/mm3 (Hill, 1931
).
Diaphragm Muscle Strips.
Mice were anaesthetized by injection with
Nembutal and the diaphragm, along with the adjoining portion of the rib
cage, was removed. The diaphragm was immediately placed in a small
Petri dish filled with Ca2+ Krebs on ice. The solution was continuously
bubbled with oxygen. The diaphragm was then cut into 6-8 strips ~0.5-0.1
mm in width with a piece of the central tendon and the adjoining rib intact. These muscle strips were slightly stretched, pinned down, and kept
on ice until transfer to the experimental chamber. Holes were pushed
through the central tendon and the rib to allow the muscle strip to be
mounted in the experimental chamber on hooks attached to the force
transducer and galvanometer. The optimum length and stimulus voltage
were then determined by monitoring the twitch force response, and the dimensions of the muscle strip determined. After the experiment, the diaphragm strip was removed from the chamber, the tendons carefully
removed and the strip weighed. A small segment from the center of the
bundle was then placed in SDS buffer and stored for later analysis of
the protein composition. CSA was determined by dividing the mass by the
length and density of the muscle strip. Muscle density was assumed to be
1.06 mg/mm3 (Hill, 1931).
Histology
Muscle tissues were isolated from male mice at 4-6 wk of age and rapidly frozen in liquid nitrogen cooled isopentane. 10-µm sections were cut on a Tissue-Tek cryostat and mounted on poly-lysine-coated cover-slips. Hematoxylin and eosin, Masson trichrome, and NADH-tetrazolium reductase (NADH-TR) staining were performed according to standard procedures.
Targeted Disruption of MyHC-IIb and MyHC-IId/x in the Mouse
A 2.6-kb probe consisting of the 5 half of a full-length human perinatal MyHC cDNA clone (Karsch-Mizrachi et
al., 1990
) was used to screen a mouse genomic library (129/
Ola, gift from Dr. O. Smithies, University of North Carolina, Chapel Hill, NC). One of the resulting clones,
19a,
contained sequences identical to the 3
-end of the previously characterized MyHC-IIa gene (Parker-Thornburg et al., 1992
); this gene is known to lie 4 kb upstream of the MyHC-IId/x gene whose first five exons were also present
on
19a. A second clone,
10a was found to have 99%
identity over a stretch of 383 bp to noncoding sequence
(data not shown) previously identified in an isolated
cosmid clone as the promoter region of the adult mouse
MyHC-IIb gene (Takeda et al., 1992
).
The MyHC-IIb targeting vector (Fig. 1 A) was constructed from a genomic fragment which contained part of
the 5 end of the MyHC-IIb gene, including the first two
non-coding (exons 1 and 2), and the first three coding exons (exons 3-5). A 1.8-kb fragment containing the PGK-Neo cassette was inserted into a BspEI site in the third
exon of the MyHC-IIb gene. The transcriptional orientation of the Neo cassette was opposite to that of the MyHC
gene. A PGK-TK cassette was inserted downstream of the
modified gene for negative selection.
The MyHC-IId/x targeting vector (Fig. 1 B) was constructed from an 11-kb genomic fragment which contained
the 5 end of the MyHC-IId/x gene including its first two
noncoding exons and the first three coding exons. This
fragment contains a unique BspEI site which maps to the
third exon of the IId/x gene. A 1.8-kb fragment containing
the PGK-Neo cassette was inserted into the BspEI site.
The transcriptional orientation of the Neo cassette was the
same as that of the MyHC gene.
The targeting constructs were introduced into E14-1 ES
cells by electroporation and individual G418 resistant clones
were isolated and genotyped (Hooper et al., 1987). 144 drug-resistant clones were screened for targeted disruption of MyHC-IIb using a 2.5-kb EcoRV/HindIII genomic
fragment not included in the targeting vector. This probe
detects a 10-kb EcoRI fragment in wild-type cells and 6.5-kb
fragment from properly targeted ES cells. 2 of 144 clones
analyzed were found to have the appropriately modified locus.
180 drug-resistant clones were screened for targeted disruption of MyHC-IId/x using a 1.6-kb EcoRI genomic fragment not included in the targeting vector. This probe detects a 17-kb BglII fragment in wild-type cells and 14-kb fragment from properly targeted ES cells. 42 of 180 (23%) of the clones analyzed were found to have the appropriately modified locus.
Two clones (F2b c.4 and c.7) heterozygous for the MyHC-IIb mutation were injected into C57Bl/6J blastocysts. Chimeric male animals derived from the F2b c.7 ES cell clone
transmitted the mutation to their offspring when bred to
C57Bl/6J females. F1 heterozygous animals were interbred. The resulting offspring were genotyped by Southern
blot analysis using the 2.5-kb EcoRV/HindIII fragment of
the probe. Representative results are shown in the first
panel of Fig. 1 C. Of the 59 mice examined, 12 were IIb+/+, 29 were IIb+/, and 17 were IIb
/
.
Four clones heterozygous for the MyHC-IId/x mutation
were injected into C57BL/6J (B6) blastocysts. Chimeric male
animals derived from three independent ES cell clones
transmitted the mutation to their offspring when bred to
B6 females. F1 heterozygous animals were interbred. The
resulting offspring were genotyped by Southern blot analysis using the 1.6-kb EcoRI fragment as the probe. Representative results are shown in the second panel of Fig.
1 C. Of the 114 mice examined, 22 were IId/x+/+, 55 were
IId/x+/, and 37 were IId/x
/
. These results indicate
that the modified loci are transmitted in a Mendelian fashion and the modifications do not appear to affect normal
prenatal development.
Analysis of MyHC Gene Expression in Null Mice
We used two different methods to determine the effect of
the genetic modification on MyHC-IIb and IId/x gene expression. RNase protection was chosen for the analysis of
MyHC mRNA expression levels. RNA samples from five
different mouse muscle tissues (heart, diaphragm, whole
lower leg, masseter, and tongue) were studied using this
method. Representative results are shown in Fig. 2 of RNase
protection analysis of mRNA from masseter and lower-leg tissue using a probe specific to the 3-UTRs of the two
MyHC genes. These results show that while +/+ and +/
animals express the MyHC-IIb and IId/x genes, MyHC-IIb
and IId/x
/
animals had little or no detectable MyHC-IIb or IId/x mRNA expression, respectively. This was true
for all five muscle sources tested (data not shown). Analysis of the same samples with a
-actin probe revealed approximately equal expression levels of these mRNAs in
samples from all three groups of animals (Fig. 2). To confirm that the homologous recombination event resulted in
a null allele for protein expression, myofibrils were purified from tibialis anterior, which in +/+ mice expresses high levels of MyHC-IId/x and IIb. To confirm that the IIb
mice are null for IIb protein, immunoblot analysis was carried out with a mAb specific for MyHC-IIb. Fig. 3 A shows
myofibrils from the tibialis anterior muscles of IIb mice
separated by SDS-PAGE. The same amounts of protein as
in Fig. 3 A (lanes 2-4) were blotted onto nitrocellulose and
probed with an antibody specific to MyHC-IIb (Fig. 3 B).
Whereas IIb+/+ and IIb+/
mice contain IIb protein, there is no detectable MyHC-IIb protein in
/
mice. An
additional point shown by Fig. 3 A is that the normal actin/
myosin ratio appears normal. Because there is no antibody
specific for MyHC-IId/x, high resolution protein gel expression was carried out on myosin from tongue, which in
+/+ mice, contains MyHC-IIB (faster migrating species)
and MyHC-II/x (slower migrating species). As shown in Fig. 3 C, there is no detectable MyHC-IId/x in
/
mice.
Reduced Whole Body Weight in MyHC Null Mutants
Both MyHC-IIb/
and MyHC-IId/x
/
animals were
significantly smaller in size than their +/
and +/+ littermates by 10 wk of age. wild-type, heterozygous, and homozygous male mice were weighed once a week for a period of 100 d. The data, shown in Fig. 4, reveal that, by 40 d
of postnatal life, the
/
mice begin to show consistent
growth retardation. By 100 d of age, they have an average
weight ~20% less than their +/+ and +/+ littermates. To
determine whether the lower body weight was due to decreased muscle mass, the masses of 8 muscle groups were
determined for
/
, +/
, and +/+ animals from the
MyHC-IId/x line at 6 wk of age. Tibial lengths were also
determined. These data are shown in Table I. The tibial
lengths for all three groups in the IId/x mice were nearly
identical, indicating that the differences in body weight
were not due to a bony skeletal developmental defect, or
retardation in bone growth. Three of the eight muscle
groups had significantly lower muscle mass than the +/
mice; however, no quantitative correlation could be made
between the decreased muscle mass and the reported IId/x
MyHC content of wild-type muscle. In the case of the IIb
mice, there was also nonuniform decrease in muscle mass.
However, different muscles relative to the IId/x
/
were
affected and to a greater degree than in IId
/
mice. For example, gastrocnemius and vastus lateralis in IIb
/
mice were both <+/
the mass of +/+, while the tibialis
anterior was of normal mass (not shown). This is despite
the fact that all three muscles have high MyHC-IIb content in the wild type mouse.
Table I. Muscle Mass and Tibial Length Measurements from 6-wk-old Male Mice |
Based on the high expression levels of IId/x in masseter, tongue and diaphragm in wild-type animals, it seemed possible that the weight loss observed in these animals may be related to problems the animals may experience with breathing and eating. To address the possibility that smaller mass in these animals may, in part, be a consequence of malnutrition, studies geared to detect possible anomalies in food and water intake in these animals were initiated. Over a course of 7 d, wild-type male mice consumed an average of 16.3 ± 1.6 gm of dry food per day (n = 5), while homozygous null IId/x mice consumed 14.0 ± 2.0 gm of food per day (n = 5), or ~13% less than wild-type (P < 0.05). Similarly, the wild-type cohort consumed an average of 28.7 ± 1.5 ml of water per day while homozygotes consumed 20 ± 2.8 gm of water per day, or 30% less than wild-type (P < 0.001).
Muscle Weakness in MyHC-IId/x and IIb Mutants
Based on an empirically assessed observation of muscle
weakness in both MyHC null mice at 4-6 wk of age, a
study of the comparative fore- and hindlimb grip strengths
of MyHC-IIb and IId/x mice was conducted. Five peak
grip strength measurements for each mouse for fore- and
hindlimbs were performed on +/+, +/, and
/
mice at
6 wk of age, and at 6 mo of age. Fig. 5 shows the hindlimb measurements for wild-type and null animals at both time
points. Heterozygotes were indistinguishable from wild-type (not shown). MyHC-IIb null mice showed decreased
grip strength in both sets of limbs at 6 wk of age, but by
6 mo of age, they were indistinguishable from wild type.
At 6 wk, this difference was 20% for forelimb strength and
30% for hindlimb strength. For the MyHC-IId/x line, mice
of both age groups showed significant sustained muscle
weakness. At 6 wk, this difference was 25% for forelimb
strength and 40% for hindlimb strength. This difference
was maintained with age.
Kyphosis in MyHC-IId/x Mutants
Approximately one-third (8/26) of IId/x/
knockout
mice (males and females) developed kyphosis, a curvature
of the spinal column that is commonly referred to as
"hunchback." This deformity is located in the thoracic vertebrae, and presented by as early as 6 wk of age and has
been occasionally seen in heterozygous animals. No wild-type littermates developed this phenotype. This phenotype was detectable by visual examination (not shown) or by x-ray (Fig. 6).
Histologic Analysis of Muscles from
MyHC/
Mutants
Tibialis anterior muscle was isolated from mice 4-6 wk of
age, and analyzed histologically. This muscle was chosen
because it expresses relatively high levels of both MyHC-IIb and IId/x and therefore would allow us to compare histological changes between the two null strains (Pette and
Staron, 1990). The results of this experiment are shown in
Fig. 7, where sections were stained with hematoxylin and
eosin, Masson trichrome and NADH-TR.
There is a notable difference in fiber size between sections of wild-type (Fig. 7, A-C) mice and homozygous null
mice (Fig. 7, D-I). It is also apparent that the histological
phenotypes of the two null strains are quite different from
each other. The mean fiber size increases dramatically in
the IIb/
mice. The sizes in wild-type mice range from
1,200 µm2 (fast glycolytic) to 2,500 µm2 (fast oxidative). In
IIb
/
mice, the mean fiber size is substantially increased
to >4,000 µm2 and the fibers are generally uniform in size.
In the case of the IId/x null mice, there was a major shift in
the geometry and size of the fibers. There was an increase
in the number of very small (1-500 µm2) and large(>2,500
µm2) fibers relative to the +/
and +/+ mice. For example, ~38% of the fibers in the tibialis anterior of IId/x
/
mice were <500 µm2, compared with 11 and 16% in +/
and +/+ mice, respectively. The changes in fiber diameter
in both lines of mice are statistically significant. Muscles
from MyHC-IIb and IIdx
/
mice show increased interstitial fibrosis compared with muscles from wild-type mice (Fig. 7, E and H). In both null strains, there is increased
spacing between fibers, but it is more notable in the IIb
null mice. Fibers from IId/x
/
mice were ultrastructurally abnormal with a disorganized appearance of the intermyofibrillar network which can be visualized on NADH-TR
stained section (Fig. 7 I). Contrast this phenotype with
that seen in NADH-TR staining of the IIb
/
section
(Fig. 7 F), where the staining is much more uniform when
compared with either +/+ (Fig. 7 C) or IId/x
/
(Fig. 7
I). The masseter and psoas also showed histological abnormalities, including exaggerated interstitial fibrosis characterized by a dramatic increase in the number of nuclei in
the interstitium. We did not see any indication of central
nuclei, which would be an indication of regeneration.
Physiologic Analysis of Muscle from MyHC Mutants
The reduced grip strength found in null mice could be explained by an alteration of contractile function in the striated muscles from these animals. To directly test whether
maximum force generating capacity is affected in /
animals, we examined peak isometric tetanic tension in EDL
and diaphragm muscle strips isolated from 5-7-mo-old IIb
and IId/x
/
animals, respectively. These muscles were selected for study because of their significant content of IIb
and IId/x myosin, respectively in wild-type mice (Jansen et al., 1996
). Results of this analysis are summarized in Table
II and graphed in Fig. 8.
Table II. Summary of Physiologic Studies |
The maximum tetanic force produced by EDL from homozygous mutant IIb mice was significantly reduced. This reduction is due to two factors. First, CSA is reduced from 0.83 ± 0.06 mm2 (mean ± SEM, n = 5) obtained in control animals to 0.44 ± 0.03 mm2 (n = 6) homozygous mutant mice. Second, the force/CSA (F/CSA) is significantly reduced from the control value of 262.8 ± 20.6 kN/m2 (n = 5) to a value of 138.2 ± 27.9 kilonewton/m2 (n = 6) in homozygous mutant animals. EDL from heterozygous mutant animals gave results intermediate between the control and homozygous mutants. Thus, the absence of type IIb fibers has significant effects on the ability of EDL to generate force independently of muscle size. Whether this difference is manifested by a reduction in the percentage of the CSA occupied by functional muscle fibers or a lower level of force produced by those fibers which are present cannot be determined from these data.
Diaphragm muscle strips from homozygous IId/x null
mutants displayed wild-type maximum normalized force.
The values of F/CSA of diaphragm muscle obtained in this
study are somewhat lower that those reported previously
by us and other investigators using rodent diaphragm muscle strips (Metzger et al., 1985). This difference likely arises
from the smaller width diaphragm strips used in the
present study (1 mm) which was done to assure the parallel alignment of muscle fibers within the isolated muscle
strips. Despite normal force generation, there were significant alterations in the time-course and kinetics of force development in IId/x
/
diaphragm. Specifically, the time
to peak tension was significantly longer as was the time to
relaxation (Table II). In addition, the peak rate of rise of
tetanic tension and rate constant for relaxation were significantly reduced in the diaphragm muscle strips from
IId/x
/
as compared with IId/x+/+ mice.
To examine the function of the adult fast skeletal myosin heavy chain isoforms in normal muscle development and function, we generated MyHC-IIb- and MyHC-IId/x-deficient mice. Survival appeared not to be affected in either strain, although we have not examined mice beyond ~14 mo of age. Common and distinct features of the two strains are summarized in Table III. Common features include normal fertility and viability, lower body mass, and decreased limb strength in young animals. Distinct features include kyphosis and sustained grip strength deficits in only IId/x null mice. Both null strains had distinct physiological and histological abnormalities. These phenotypes are not ones that could have been predicted based on the abundance and distribution of these two motor proteins.
Table III. Comparison of MyHC-IIb and IId/x Null Mice |
MyHC/
Mice Have Reduced Growth Rates and
Adult Body Weights
Skeletal muscle typically accounts for ~40-50% of the
body-weight of a mammal (Berne and Levy, 1996) and
myosin protein accounts for ~40% of the total muscle
protein. In 100-d-old mice, there was a decrease in the average body weight of homozygous IIb and IId/x null mice
relative to wild-type. When muscle masses were determined from 6-wk-old IId/x null mice (Table I) several, but not all, muscle groups showed 20-30% decreases in mass.
The food and water intake of IId/x
/
mice was significantly less than that of IId/x+/+ mice. It seems possible
that the MyHC-IId null mice have impaired ability to feed
on mouse chow due to reduced function of the jaw muscles. Consistent with that hypothesis is the observation
that
/
mice cannot be distinguished by weight from
their +/+ and +/
littermates until after weaning. Experiments are in progress to determine whether the reduced
body weights of the mice can be prevented, in part, by soft
food diet. In the case of the IIb null mice, the alterations in
muscle mass are quite dramatic, but once again, not always
correlated with their IIb content in wild-type mice. Further metabolic studies are required to assess this phenotype in both null strains; but it is quite clear that this is a
complex phenotype that may result from structural problems of the muscle as well as secondary and tertiary sequelae.
Possible Basis of the Kyphosis
There is a clear link between spinal curvature and abnormalities in both para-spinal and non-spine-related musculature. A number of human neuromuscular diseases have
been described with kypho-scoliotic phenotypes; these include diseases such as spinal muscular atrophy, limb-girdle
muscular dystrophy, myotonic dystrophy, and Duchenne
muscular dystrophy, to name only four of more than a
dozen muscular diseases that often result in spine-deforming complications (Carter et al., 1995; Johnson et al., 1995
;
McDonald et al., 1995
). Thus, many distinct primary defects can result in kyphosis. An autosomal recessive mutation in the mouse, ky, causes kyphoscoliosis (Bridges et al.,
1992
). Muscle in ky mice appears to be necrotic and regenerating. EDL muscle from these mice shows a significant
defect in force generation. Despite some similarities with
the MyHC-IId null strain reported here, there is no apparent defect in MyHC composition and this disease is thought
to be neuromuscular in origin. Kyphosis has also been found in a number of genetically manipulated mice including MyoD null/mdx mice which lack both dystrophin and
MyoD (Megeney et al., 1996
). In these mice, the paraspinal muscles were shown to be much smaller than in the
wild-type animals. Kyphosis was also seen in newborn pups
of myogenin
/
mice which do not develop any skeletal
muscle (Hasty, 1993
). The fact that the psoas muscle is significantly lower in mass in the
/
IId MyHC mice may
be an indication that the back musculature is generally not
sufficient to hold the spine in place. However, this phenotype was not seen in IIb null mice despite the much greater
prevalence of IIb MyHC throughout the skeletal musculature in the wild-type mouse.
Histologic Changes in MyHC/
Muscle
The histological features of the two different MyHC/
mice are distinct relative to each other. However, none of
the changes seen here are specific to the MyHC gene family since many different human diseases can present with
similar phenotypes. For example, increased cellularity and
fibrosis in the interstitium, as was seen in this report, have
been described in Duchenne muscular dystrophy, limb-girdle dystrophies and neurogenic atrophies.
The disorganization of the intermyofibrillar network
seen in the MyHC-IId/x/
mice is very similar to a clinical syndrome known as "moth-eaten" fibers which, once
again, is observed in a number of myopathies. The most
striking pathology in IId null mice was seen in the TA with
a very large population of small fibers and disorganization
of the intermyofibrillar network. It remains to be determined whether this phenotype is the result of muscular atrophy or represents a steady state. In contrast, MyHC-IIb
null mice show a dramatic hypertrophy of all fibers in this
muscle and do not display substantial disorganization. This phenotypic difference is somewhat surprising given the
fact that >90% of the MyHC in this muscle consists of IIb
and IId/x protein in wild-type animals.
Muscle Contractility
The normalized peak force generating capacity of the diaphragm is not altered in homozygous MyHC-IId/x mice. This
is a somewhat surprising finding considering that IId/x fibers represent ~50% of the fiber type population in normal diaphragm muscles of mice (Jansen et al., 1996). The
most likely explanation for this finding is that the IId/x fibers were at least partially rescued by activation of one or
several of the other skeletal myosin genes with similar force
generating capabilities. If functional compensation is the case, the normalized maximum force would not be expected to change significantly. We have preliminary data
which suggest that the MyHC-IIa gene is compensating for
the absence of IId/x protein. Examination of potential
compensatory changes is obviously a subject for future investigation. While maximum force generating capacity was
not changed, we did find alterations in the kinetics of tension generation that tended to slow the contractile behavior of the diaphragm strips from
/
animals. These alterations may have influenced the function of the diaphragm
in vivo, and in this case, could underlie, at least in part,
some the overall weakness observed in these animals. In
contrast, the EDL muscle, which in wild-type mice has a
high MyHC-IIb content, shows a significant decrease in
force generated/cross section but no alterations in the kinetic properties of the muscle. Once again, it will be very interesting to determine the properties of individual muscle fibers and their MyHC content.
Implications for human skeletal muscle disease. Studies
of the skeletal genes in the context of muscle pathology in
C. elegans and D. melanogaster yielded a wealth of information about their function in the muscle sarcomere and
established a clear correlation between MyHC gene mutations and skeletal muscle disease among invertebrates
(Dibb et al., 1985; Chun and Falkenthal, 1988
). Our study
presents evidence for the involvement of a noncardiac MyHC gene in skeletal muscle disease in a higher vertebrate. A number of similarities exist between phenotypes
observed in the MyHC mice with those seen in known human congenital myopathies. Muscle weakness and swallowing difficulty from birth along with scoliosis and histologic variation in fiber size and number are phenotypes
characteristic of a number of congenital myopathies, including congenital fiber-type disproportion and Escobar
syndrome (Yokochi et al., 1985). It would be useful to examine the genetic status of the skeletal MyHC genes in inherited forms of these and other muscle disorders.
We have found that targeted disruption of adult skeletal myosin heavy chain genes results in varying degrees of skeletal muscle related pathology in mice homozygous for these mutations but that the phenotypes are very different and not correlated with the abundance and distribution of the two gene products. This suggests that despite the common origin and high degree of sequence conservation between the members of the adult skeletal MyHC genes, these genes appear to have diverged significantly enough to have acquired distinct and unique roles in skeletal muscle contraction.
Address correspondence to L.A. Leinwand, Molecular Cellular and Developmental Biology, University of Colorado, Campus Box 347, Boulder, Colorado 80309-0347. Tel.: (303) 492-7606. Fax: (303) 492-8907.
Received for publication 7 July 1997 and in revised form 25 August 1997.
1. Abbreviations used in this paper: CSA, cross-sectional area; EDL, extensor digitorum longus; NADH-TR, NADH-tetrazolium reductase; MyHC, myosin heavy chain.We thank Stefano Schiaffino for his gift of the BFF3 antibody. We acknowledge the assistance of Harry Hou, Jr. for blastocyst injections, Brian Tompkins for figure preparation, Jill Jones for manuscript preparation, Renee Jacobsen for high resolution protein electrophoresis, Bill Byrnes for advice and help with histology, and Remy Burcelin for his invaluable assistance with the metabolic studies.
This work is supported by grants from the National Institutes of Health (GM29090 to L.A. Leinwand), the Human Genetics Program at AECOM and a center grant (CA13330). J.M. Metzger is an Established Investigator of the American Heart Association. C. Sartorius was supported by a grant 1F32AR08443 from NIH; B.D. Lu was supported by a grant T32GM07288 from NIH; and L.J.R. Acakpo-Satchivi was supported by a grant 5F31 HG00064 from NIH.
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