From the Banting and Best Department of Medical
Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada,
¶ Ottawa Health Research Institute, Ottawa, Ontario K1H 8L6,
Canada,
Division of Pathology, Hospital for Sick Children,
Toronto, Ontario M5G 1X8, Canada, ** Department of Laboratory
Medicine and Pathobiology, University of Toronto, Banting Institute,
Toronto, Ontario M5G 1L5, Canada, and
Department of Pediatrics (Neurology),
Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
Received for publication, December 27, 2002, and in revised form, January 24, 2003
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ABSTRACT |
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Mutations in the ATP2A1 gene,
encoding isoform 1 of the sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA1), are one cause of Brody disease,
characterized in humans by exercise-induced contraction of fast twitch
(type II) skeletal muscle fibers. In an attempt to create a model for Brody disease, the mouse ATP2A1 gene was targeted to
generate a SERCA1-null mutant mouse line. In contrast to humans, term
SERCA1-null mice had progressive cyanosis and gasping respiration
and succumbed from respiratory failure shortly after birth. The
percentage of affected homozygote SERCA1 Sarco(endo)plasmic reticulum Ca2+-ATPases
(SERCAs)1 are 110-kDa
membrane proteins that catalyze the ATP-dependent transport
of Ca2+ from the cytosol to the lumen of the
sarco(endo)plasmic reticulum (1). Three different ATP2A
genes encode six different sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) proteins (2-5). SERCA1a and SERCA1b,
the developmentally regulated isoforms of the ATP2A1 gene,
arise through alternative splicing at the 3'-end of the
ATP2A1 transcript (6). SERCA1a accounts for more than 99%
of SERCA isoforms expressed in adult rat fast twitch skeletal muscle,
whereas SERCA1b is predominant in neonatal muscle (7). SERCA2a is the
major isoform in heart and slow twitch skeletal muscle, whereas SERCA2b
and SERCA3 are more ubiquitously expressed.
Brody disease is a rare inherited disorder of skeletal muscle,
resulting in exercise-induced impairment of skeletal muscle relaxation,
stiffness, and cramps (8). Sarcoplasmic reticulum Ca2+
uptake and Ca2+-ATPase activities in muscle samples
obtained from Brody's patients are reduced to levels ranging from 0 to
50% compared with activities measured in normal controls (9-13),
suggesting that Brody disease might result from defects in the
ATP2A1 gene. Sequencing of ATP2A1 DNA from Brody
disease patients has revealed a number of frameshift mutations that
truncate SERCA1 (14, 15) as well as a missense mutation (16). All of
these mutations lead to loss of SERCA1a function. However, mutations in
ATP2A1 account for only about half of Brody disease cases,
and the genetic basis for the other Brody syndrome patients remains to
be discovered (17).
Recent advances in transgenic mouse technology have made it possible to
address the physiological relevance of increases or decreases in SERCA
expression. Overexpression of SERCA2 in the myocardium resulted in
enhanced myocardial function (18-20). By contrast, the ablation of
ATP2A2 was lethal, and heterozygous SERCA2+/ The purpose of the present study was to investigate both the role of
SERCA1 in physiological functions and the mechanisms by which SERCA1
mutations could cause Brody disease. Since the ATP2A1 gene
is expressed almost exclusively in fast-twitch fibers, it was of
particular interest to investigate the effects of its disruption on the
function of muscles such as the diaphragm, where fast twitch fibers
make up a large fraction of the total fibers.
SERCA1 Preparation of the Targeting Construct--
A
The targeting construct was prepared with the pBluescript II
SK Gene Targeting and Generation of Mutant Animals--
The
construct was linearized at the NotI site in the vector and
transfected by electroporation into ES cells. Twenty-four hours after
electroporation, the cells were exposed to G418 at 200 µg/ml and 2 µM gancyclovir to select cells containing the neo gene and lacking the HSV-tk gene,
respectively. Three hundred clones resistant to both drugs were picked
and expanded 10 days after electroporation. DNA from individual ES cell
clones was digested with EcoRI and analyzed by
nonradioactive Southern blotting (24) using two different probes. Probe
1 was a 1.8-kb HindIII-XbaI fragment located 2.8 kb downstream from exon 5. Probe 2 was a 2-kb fragment specific for the
neo gene. Fourteen ES cell lines containing the disrupted
ATP2A1 gene were used for blastocyst-mediated transgenesis.
Blastocyst injection was carried out by Dr. M. Rudnicki (MacMaster
University, Hamilton, Canada). Five chimeric mice with 50-70%
chimerism were produced and mated to CD1 mice. Germ line transmission
of the recombinant gene was achieved with only one chimera, and this
mouse was used as a founder of the murine line with targeted ablation
of the ATP2A1 gene.
Genotype Analysis--
Genotyping was performed by
nonradioactive Southern blot analysis of genomic DNA from tail clips,
using probe 1 and probe 2, and by PCR analysis using a combination of
three primers that amplify both wild-type and mutant alleles. For PCR
analysis, the following primers were used: primer 1 (5'-GGTAGAGCTCCCTGCTGAGGAAGGTAAG-3'), corresponding to genomic sequence
in exon 2 that was deleted in the targeted allele; primer 2 (5'-GTCAGCCCGATAGACCTTTCCCAT-3'), complementary to sequence in exon 5;
and primer 3 (5'-GCTATTCGGCTATGACTGGGCACAACAGACAATCG-3'), corresponding
to the sequence of the neo cassette. PCR conditions for
simultaneous amplification of the wild-type 3.6-kb product and the
mutant 2.8-kb allele were as follows: denaturation at 94 °C for
30 s, annealing for 30 s at 64 °C, and extension for 3 min
at 72 °C.
Calcium Uptake Assays--
Skeletal muscles isolated
from hind limb or diaphragm muscle from five mice of the same genotype
were combined and homogenized using a Dounce homogenizer in ice-cold
buffer consisting of 10 mM Tris-HCl, pH 7.4, 150 mM KCl, 20 µM CaCl2, 0.25 M sucrose, 2 mM dithiothreitol, and CompleteTM
protease mixture. Homogenates were centrifuged at 4,000 × g for 20 min at 4 °C. The supernatants were used for
measurement of the Ca2+ dependence of Ca2+
transport, as described previously (25). Briefly, Ca2+
transport activity in homogenates was assayed in 150 µl of a reaction
mixture containing 20 mM MOPS-Tris-HCl, pH 6.8, 100 mM KCl, 5 mM MgCl2, 5 mM ATP, 5 mM potassium oxalate, and about 20 µg of protein. For the measurement of Ca2+ dependence of
Ca2+ transport, free Ca2+ concentrations were
calculated using the computer program of Fabiato and Fabiato (26).
45Ca2+ was present at a specific activity of
about 106 cpm/µmol. The uptake reaction was initiated by
the addition of homogenates, incubated at room temperature for 7 min,
and stopped by filtration through a 0.3-µm Millipore filter, followed
immediately by washing with 10 ml of 100 mM KCl.
Radioactivity on the filter was measured by liquid scintillation
counting. Experiments were carried out with three independent
homogenate preparations.
Western Blot Analysis--
Total homogenates like those used in
Ca2+ uptake assays were separated by SDS-PAGE and then
transferred to a nitrocellulose membrane. Primary monoclonal mouse
antibodies used were as follows: A52 (diluted 1:5000) against SERCA1;
2A7-A1 (diluted 1:500) against SERCA2a (Affinity Bioreagents Inc.);
34-C (diluted 1:1000) against ryanodine receptors (Affinity
Bioreagents); and R3F1 (diluted 1:1000) against
Na+-Ca2+ exchanger (Research Diagnostic Inc.).
Binding of primary antibody was detected by horseradish
peroxidase-conjugated goat anti-mouse secondary antibody and an
enhanced chemiluminescence kit (Pierce Super Signal). Protein
concentrations were determined by the Bio-Rad method using bovine serum
albumin as a standard.
Semiquantitative RT-PCR Analysis--
Total RNA was isolated
from diaphragm or limb muscles using Triazol reagent, following the
method described in the kit (Invitrogen). Trace genomic DNA
contamination from total RNA was removed by 2 units of DNase I
treatment (Ambion) in a 100-µl reaction for 10 min at 37 °C and
then extracted once with phenol/chloroform and twice with chloroform.
The integrity of each of the RNA samples was assessed by
electrophoresis (28 S:18 S ratio), and the concentration was estimated
by spectrophotometry using the
A260/A280. Two micrograms of total RNA from each sample were subjected to hexamer random primed
first-strand cDNA synthesis in a volume of 20 µl using Superscript II reverse transcriptase (Invitrogen), according to the
guidelines of the manufacturer. The absence of contaminating DNA from
each RNA sample was confirmed by PCR by omitting reverse transcriptase
from the RT reaction.
Equal amounts (2 µl) of the reverse transcription product were
subjected to PCR amplification of SERCA1a, SERCA1b, SERCA2a, SERCA2b,
SERCA3, phospholamban, and sarcolipin. Analysis of the expression of
the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), was carried out to normalize the level of expression of the
genes of interest. The step-cycle program was adjusted within the
linear range of amplification for specific transcripts. The PCR
amplification products were analyzed by electrophoresis on 6%
polyacrylamide gels, stained with SYBR Green (Stratagene), and signals
were detected using the Fluor-S MultiImager System (Bio-Rad) according
to the instructions of the manufacturer. Band intensities of the
amplified fragments were normalized to the corresponding amount of
GAPDH.
PCR Primers and PCR Analysis of the Fragments--
Mouse
sequences of SERCA1 (GenBankTM accession number NM_007504),
SERCA2 (GenBankTM NM_009722), SERCA3 (GenBankTM
XM_122224), sarcolipin (GenBankTM NM_025540.1),
phospholamban (GenBankTM NM_023129), and GAPDH
(GenBankTM M32599) were used to create primers for
amplification of SERCA1a and SERCA1b, using sense primer
(5'-TTCCTCATCCTCTATGTCGACC-3') and antisense primer
(5'-CTGAAGATGCATGGCTATTGG-3'); SERCA2a, using sense primer
(5'-TGATCCTCATGGATGAGACG-3') and antisense primer (5'-CCACATCACACAGTGAGTTGG-3'); SERCA2b, using sense primer
(5'-TGATCCTCATGGATGAGACG-3') and antisense primer
(5'-AGTCAAGACCAGAACATATCGC-3'); both SERCA1 and SERCA2, using sense
primer (5'-GACGAGTTTGGGGAGCAGCT-3') and antisense primer
(5'-AGGTGGTGATGACAGCAGG-3'); both SERCA2 and SERCA3, using sense primer
(5'-TGCCTGGT(G/A)GAGAAGATGAATG-3') and antisense primer
(5'-CCCTTCACAAACATCTTGC-3'); sarcolipin, using sense primer
(5'-GTCCTTCTGGAGTTCTCATCC-3') and antisense primer
(5'-GTCAGGCATTGTGAGTGTGG-3'); phospholamban, using sense primer
(5'-TGCCTTCCTGGCATAATGG-3') and antisense primer
(5'-ATGTTGCAGGTCTGGAGTGG-3'); and GAPDH, using sense primer
(5'-CTTCACCACCATGGAGAAGG-3') and antisense primer
(5'-CATGGACTGTGGTCATGAGC-3').
The enzymes and fragment sizes used to confirm and discriminate
specific transcripts are listed in Table
I. To measure the relative ratio of SERCA1a to SERCA1b expression,
SERCA1a and SERCA1b primers were used to co-amplify the SERCA1a and
SERCA1b fragments. Since the two fragments differ in length by 42 bp,
with the longer fragment representing the adult form, SERCA1a, they
could be distinguished and quantified. To determine the levels of
SERCA2a and SERCA2b, sets of specific primers were used to amplify
SERCA2a and SERCA2b individually. To measure the SERCA1 to SERCA2 and
SERCA2 to SERCA3 mRNA ratios, the co-amplified products were
digested using the specific enzymes listed in Table I to produce
digestion fragments that could be used for quantification.
Electrical Stimulation and Muscle Contractile
Measurements--
Experiments were performed on isolated diaphragm
muscle strips from newborn homozygous ATP2A1 mice and their
wild-type littermates. Isometric contractile properties of diaphragm
muscles were measured in vitro from neonatal
mice within 0.5-5 h following delivery by caesarian section on day 19 or 20 of embryogenesis. Each diaphragm muscle was exposed, removed with
intact ribs and tendons, and placed into oxygenated Krebs solution
(95% O2, 5% CO2) containing 118 mM NaCl, 25 mM NaHCO3, 11 mM glucose, 1.2 mM KHPO4, 1.9 mM CaCl2, and 1.2 mM
MgSO4, pH 7.4, and maintained at 4-10 °C. Each diaphragm was trimmed and cut into a single strip suitable for study.
Diaphragm muscle was mounted vertically in a jacketed muscle bath
(Radnotti Glass, Monrovia, CA) containing oxygenated Krebs solution
(33 °C) between a plexiglass clamp and a servomotor (Cambridge Technologies, model 300H Dual Mode Servo) used to measure force output.
Stimulation was applied by a Grass S88 stimulator (Grass
Instruments, Quincy, MA) via closely flanking platinum wire electrodes. A supramaximal stimulation voltage was used (110-120 V) with a pulse
duration of 0.2 ms. Force data were collected on-line using a 640A
signal interface (Aurora Scientific Inc.) connected to a National
Instruments 16-bit A/D card and analyzed using the Dynamic Muscle
Control and Data Acquisition (DMC) and Dynamic Muscle Analysis (DMA)
Software (Aurora Scientific). Muscle length was adjusted to obtain
maximal isometric twitch force. Peak isometric force amplitude (g) and
the maximal rates of force development (+dF/dt)
and relaxation ( Light and Electron Microscopic Level
Analysis--
After genotyping, samples from animals in some 2 dozen
litters were collected and processed by various pathological
techniques: 1) paraffin embedding for histopathological evaluation of
entire 10-, 15-, and 18-day embryos and term neonatal mice; 2) paraffin embedding of limb and diaphragm muscles from neonatal term mice; and 3)
electron microscopy of limb and diaphragm muscles of term mice. For
paraffin studies, whole bodies of neonatal or embryonic mice were fixed
by immersion in 10% neutral buffered formalin, embedded in paraffin,
sectioned at 5 µm, stained with hematoxylin and eosin, and analyzed
by light microscopy. Diaphragm and limb muscles were dissected
immediately after death, flash-frozen in OCT blocks cooled by
isopentane in liquid nitrogen, and cut into 5-µm sections for
analysis. For electron microscopy, diaphragm and limb muscles were
fixed in situ by injection of 2% paraformaldehyde and 2%
glutaraldehyde (in 200 mM Sørensen's phosphate buffer, pH
7.4) into the thoracic and abdominal cavities immediately after death.
The diaphragm and limb muscles were then dissected into 1 × 1-mm
cubes and immersed again in the same fixative at 4 °C. Muscle was
processed for electron microscopy by postfixation with 1% osmium
tetroxide in water for 1 h, washed in water, and dehydrated with
ethanol and propylene oxide. Dehydrated tissues were embedded in
plastic, sectioned at 60 nm, and stained with 5% uranyl acetate and
1% lead. Sections were viewed and photographed on a Hitachi60 electron microscope.
Skeletal Muscle Culture--
Primary myoblast cultures were
established from the limbs of E19 SERCA1+/+ and
SERCA1 Generation of Mice with a Targeted Disruption of the ATP2A1
Gene--
The strategy illustrated in Fig.
1A was followed to disrupt the
ATP2A1 gene. The targeting construct of 7.5 kb
(middle panel of Fig. 1A) consisted of
mouse genomic sequence in which the neo gene substituted for
4.2 kb of the ATP2A1 gene, corresponding to a 3' portion of
the promoter sequence and a 5' fragment of the coding sequence, which
included exons 1-4. After electroporation of ES cells, 300 colonies
survived positive-negative selection in G418 and gancyclovir. Southern
blot analysis using probe 1 (Fig. 1B) and probe 2 (data not
shown) revealed that 14 clones contained the 6.4-kb EcoRI
fragment that was diagnostic of a targeted allele. Blastocyst-mediated
transgenesis yielded five male chimeric mice, but the progeny of only
one of these mice carried the targeted allele in its germ line
after breeding with wild-type females. After five generations of cross
breeding with CD1 strain mice, heterozygous mice were mated to produce
wild-type, heterozygous, and homozygous mutant offspring, as
demonstrated by PCR analysis of tail DNA (Fig. 1C).
Gross Phenotype and Characterization of SERCA1-deficient
Mice--
Genotype analysis of 299 offspring of heterozygous matings
yielded 81 (27%) wild-type, 144 (48%) heterozygous, and 74 (25%) homozygous mutant mice, which is almost identical to a normal 1:2:1 Mendelian ratio. All pups were born alive, and
SERCA1 Histological Analysis of SERCA1-deficient Mice--
A survey of
multiple organs from 10-, 15-, and 18-day SERCA1 Electron Microscopy of SERCA1-deficient Mice--
Electron
microscopy of limb muscles revealed central nuclei, lakes of unbound
glycogen, and prominent mitochondria, all of which were present to a
similar degree in wild-type term littermates (data not shown).
Examination of the diaphragm muscle in SERCA1 RT-PCR and Western Blot Analysis of SERCA1-deficient Mice--
To
demonstrate the loss of SERCA1 protein expression and any consequent
compensatory responses, mRNA and total homogenates prepared from
diaphragm and hind limb muscles were examined by parallel
semiquantitative RT-PCR and Western blot analysis, respectively. Fig.
4A shows through RT-PCR
analysis that SERCA1a and -b mRNAs are expressed in diaphragm and
hind limb muscles from SERCA1+/+ mice but are absent in
SERCA1 Ca2+ Uptake Activity in Sarcoplasmic Reticulum
Preparations from Skeletal Muscle from SERCA1-deficient
Mice--
Homogenates were prepared from diaphragm and hind limb
muscle from SERCA1+/+ and SERCA1 Contractile Activities in Diaphragm Preparations from
SERCA1-deficient Mice--
Isometric contractile properties of
skeletal muscle of SERCA1 knockout mice were studied on muscle strips
dissected from the diaphragm of newborn SERCA1 Differentiation in Vitro of Myoblasts from SERCA1-deficient
Mice--
Primary myoblast cultures (Fig.
7, A and B) derived
from the limbs of E19 embryos of SERCA1+/+ and
SERCA1 SERCA1 is a member of a family of Ca2+ pumps expressed
in the sarco(endo)plasmic reticulum of mammalian tissues.
ATP2A1, which encodes SERCA1a and SERCA1b, is highly
up-regulated during myoblast differentiation, although its expression
is largely limited to fast twitch muscle (2-5). Frameshift or missense
mutations in the ATP2A1 gene that lead to loss of SERCA1
function, often with loss of expression of SERCA1 protein, have been
shown to be causal of the human muscle disorder Brody disease (14, 28).
Brody disease is not life-threatening in humans but is characterized by
a lifelong history of exercise-induced muscle contracture and painless
muscle cramping without myotonia (9-13).
In the present study, targeted ablation of the ATP2A1 gene
produced mice lacking SERCA1 in skeletal muscle so that structural and
functional consequences of the elimination of SERCA1 could be assessed
in vivo. The absence of detectable mRNA encoding SERCA1a and -b (Fig. 4A) and the absence of SERCA1 protein (Fig.
4B) in homozygous SERCA1 Sections of hind limb and cardiac muscle from both
SERCA1+/+ and SERCA1 To evaluate compensatory responses of SERCA1 deficiency in our mouse
model, semiquantitative RT-PCR and Western blot analysis of
SERCA1 In response to chronic low frequency muscle stimulation, adaptive
responses were observed in fast twitch skeletal muscle that included a
shift in SERCA isoform expression from SERCA1 to SERCA2 (30, 31). It
was hypothesized that the adaptive responses resulted from the
elevation in cytosolic free Ca2+ that occurred with this
chronic low frequency muscle stimulation model. From our studies, we
can conclude that such adaptive processes had not been initiated in
SERCA1 If we compare the physiology of humans and mice, we note that 8-day-old
mice respire at a rate of 370 breaths/min (32) versus the
standard of 45 for human infants (33). This is in line with a much
higher heart rate in neonatal mice of about 500 beats/min (34)
versus the standard 125 in neonatal humans. The percentage of fast twitch type II fibers in mouse diaphragm at birth is
significantly higher than the percentage of slow twitch type II fibers
in human diaphragm. Over 90% of muscle fibers in the mouse diaphragm
are fast twitch type II fibers, and only 10% are type I (35). By comparison, in humans and in rats, fast twitch type II fibers account
for about 60% of fibers in the diaphragm, and 40% are slow twitch
type I (36). In line with these observations, SERCA2 mRNA
(expressed in slow twitch fibers) represents ~43% of the total SERCA
mRNA measured in diaphragm in rats and presumably also in humans
(37). We suggest that the levels of SERCA2 and SERCA3 that were
detected in the mouse diaphragm did not contribute enough functional
activity to rescue contraction and relaxation in the diaphragmatic
muscle of SERCA1 The fact that only the diaphragm muscle showed the defect, whereas hind
limb muscle was histologically normal in SERCA1 To test the role of SERCA1 in an early stage of muscle development, we
studied myotube formation in primary cultures from hind limb muscles
from SERCA1 We cannot rule out the possibility, however, that SERCA1 plays a role
in lung development in the very late prenatal stage. The preterm mouse
lung is normally very cellular and "cuboidal," and this morphology
evolves rapidly to adult respiratory epithelium over several days.
SERCA1 could be involved either directly or indirectly with the final
"terminal sac phase," which occurs between day 18 and term.
The Vmax of Ca2+ transport activity
in both total diaphragm and limb muscle homogenates of
SERCA1 The depression of the maximal rates of force relaxation (Fig. 6,
A and D) of the response to electrical
stimulation observed in SERCA1 In addition to alterations in diaphragm contractile responses that are
directly related to disruption of the Ca2+ signal,
disruption of metabolic processes may occur as a consequence of loss of
Ca2+ homeostasis, thereby contributing to a decline in
muscle function. Increases in resting free Ca2+ levels
would stimulate glucose transport into skeletal muscle (44), inhibit
glycogenolysis, stimulate pyruvate dehydrogenase, and activate
mitochondrial function (41). Structural and morphological degradation
after exercise has also been proposed to result from increases in the
cytosolic Ca2+ concentration. Several
Ca2+-dependent proteases have been found to be
activated after exercise (45), and myofibrillar proteins have been
degraded by lysosomal proteases (46). All of these processes need to be
evaluated further in our SERCA1/
mice was
consistent with predicted Mendelian inheritance. A survey of multiple
organs from 10-, 15-, and 18-day embryos revealed no morphological
abnormalities, but analysis of the lungs in term mice revealed diffuse
congestion and epithelial hypercellularity and studies of the diaphragm
muscle revealed prominent hypercontracted regions in scattered fibers
and increased fiber size variability. The Vmax
of Ca2+ transport activity in mutant diaphragm and skeletal
muscle was reduced by 80% compared with wild-type muscle, and the
contractile response to electrical stimulation under physiological
conditions was reduced dramatically in mutant diaphragm muscle. No
compensatory responses were detected in analysis of mRNAs encoding
other Ca2+ handling proteins or of protein levels.
Expression of ATP2A1 is largely restricted to type II
fibers, which predominate in normal mouse diaphragm. The absence of
SERCA1 in type II fibers, and the absence of compensatory increases in
other Ca2+ handling proteins, coupled with the marked
increase in contractile function required of the diaphragm muscle to
support postnatal respiration, can account for respiratory failure in
term SERCA1-null mice.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mice manifested impaired cardiac contractility and delayed
cardiomyocyte relaxation (21). Ablation of the ATP2A3 gene
encoding SERCA3 was not lethal, but defects were noted in
endothelium-dependent relaxation of vascular smooth muscle
and endothelial cell Ca2+ signaling in SERCA3-null mice
(22).
/
mice were born with normal body weight and
normal gross morphology. However, affected mice developed cyanosis and
gasping respiration and died shortly after birth. Histopathological
analysis of mice at term revealed congestion and hypercellularity of
the lung, consistent with failure of the respiratory musculature to produce sufficient chest expansion to open the lung alveoli after birth. Analysis of the diaphragm muscle revealed increased fiber size
variability and prominent hypercontracted regions in scattered muscle
fibers. The demonstrable loss of SERCA1 protein from diaphragm and
skeletal muscles was accompanied by a dramatic reduction in Ca2+ uptake activity in homogenates from these tissues. No
compensatory responses were detected in mRNA or protein levels for
other Ca2+ regulatory proteins. A significant impairment of
the contractile response of isolated diaphragm muscles to electrical
stimulation was also measured. These results demonstrate that the loss
of SERCA1 in the diaphragm of mice leads to neonatal death from
respiratory failure.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage clone
containing part of the mouse ATP2A1 gene was isolated from a
strain 129/SvJ genomic library (a gift of Dr. J. Rossant, Samuel
Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada)
and partially characterized by Southern blot analysis, restriction
mapping, and DNA sequence analysis. The cloned 15-kb fragment contained
the 3'-end of the promoter and the sequence corresponding to exons 1-9
of the human ATP2A1 gene, which encode amino acids 1-365.
In over 154 codons sequenced, no amino acid substitutions were observed
in the mouse sequence relative to that of human SERCA1, thereby
confirming the identity of the gene (23). This conclusion was
strengthened when a partial sequence of the corresponding region was
retrieved from the murine genome via the Ensembl Genome Browser
(available on the World Wide Web at www.ensembl.org). Strikingly, the
similarity between the human and mouse ATP2A1 gene regions
extended beyond the conserved sequences within the exons; the overall
size of the introns was very similar in the two species.
vector as a backbone. The PGK-Neo and PGK-TK cassettes
in plasmids pGEM7(KJ1)SalI-R and pGEM7(TK)SalI,
respectively, were used in construction. The targeting vector was
constructed by replacing ~4.2 kb of genomic sequence with the PGK-Neo
expression cassette (see Fig. 1A). The cassette was inserted
between a 5.2-kb EcoRI-AvrII gene fragment
located 1.8 kb upstream of exon 1 and a 0.9-kb
BamHI-HindIII fragment containing exon 5. The
orientation of the PGK-Neo cassette was the same as that of the
ATP2A1 gene. The PGK-TK expression cassette was inserted at
the HindIII site 0.7 kb downstream from exon 5.
Amplified fragments and enzymes used to confirm and discriminate
specific transcripts by semiquantitative RT-PCR
dF/dt) were determined during a
twitch and across a range of stimulation frequencies from 10 to 100 Hz. Diaphragm fatigability was also assessed using a 3-min stimulation protocol consisting of 350-ms contractions at 100 Hz, once per second
Fatigue data are expressed as a percentage of initial force data. After
the contractile and fatigue properties were measured, the diaphragm
muscle strips were trimmed of the remnants of the central tendon and
rib, blotted on filter paper, and weighed on an analytical balance.
Total muscle fiber cross-sectional area of each muscle strip was
determined by dividing the muscle mass (mg) by the product of muscle
length and 1.06 mg/mm3, the density of mammalian skeletal
muscle. Force data were normalized for total muscle fiber
cross-sectional area.
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embryos, as described in Ref. 27. Muscle tissue
dissected from all four limbs was dissociated by fine mincing with
microdissecting scissors, followed by treatment with 0.125% trypsin
and 0.5% pancreatin and 0.01% of DNase (Life Technologies, Inc.) for
30 min at 37 °C. The enzymatic digestion was stopped by the addition
of Ham's F-10 medium containing 20% fetal bovine serum. Contaminating
fibroblasts were removed selectively by preplating the cell suspension
for 1 h at 37 °C. Suspensions enriched in myoblasts were then
plated on dishes coated with 0.1% gelatin and grown in 20% fetal
bovine serum in Ham's F-10 medium, supplemented with 5 nM
basic fibroblast growth factor. Cells were fed the following day and
every third day thereafter. To induce differentiation, the growth
medium was changed to 2% horse serum in Ham's F-10.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
ATP2A1 gene targeting strategy to
disrupt SERCA1, verification of targeting, and characterization of
SERCA1 /
mice. A, targeting strategy. Top,
organization of the relevant region of the wild-type gene with a
partial restriction map of the locus. Middle, targeting
construct with the PGK-Neo expression cassette replacing ~4.2 kb of
genomic sequence including the first four exons of the
ATP2A1 gene. The herpes simplex virus thymidine kinase gene
(TK) was included for negative selection of ES cell clones.
Bottom, targeted ATP2A1 gene and location of
probe 1 and probe 2 for Southern blot analysis. The EcoRI
fragments (14.2 kb for wild-type allele; 6.4 kb for mutant allele)
detected by Southern blot analysis are indicated. Restriction enzyme
sites are as follows. A, AvrII; B,
BamHI; E, EcoRI; H,
HindIII; X, XbaI. Black
boxes represent exons 1-9. The arrows indicate
the transcriptional orientation of the inserted expression cassettes.
B, Southern blot analysis of EcoRI-digested
genomic DNA from ES cells (genomic DNA from tail clips of the offspring
from heterozygous matings was hybridized with probe 1, as indicated).
C, PCR of genomic DNA from tail clips from mice of all three
genotypes with the primers specific to wild-type (lanes
1, 3, and 5) or mutant
(lanes 2, 4, and 6)
alleles. The PCR product generated by the mutant allele is 1.4 kb, and
that generated by the wild-type allele is 3 kb. D, lethality
of homozygous SERCA1
/
newborn mice shortly after birth.
A mating between SERCA1+/
parents yielded the
littermate progeny with gross phenotypes representative of
SERCA1+/+, SERCA1+/
, and
SERCA1
/
newborn mice. Note the similar size and normal
appearance of the littermates, except that the two
SERCA1
/
mice are cyanotic and appear light
purple in color.
/
mice were indistinguishable from wild-type
SERCA1+/+ littermates in their gross appearance at birth.
The average birth weight (g) of SERCA1+/+ (1.4 ± 0.08), SERCA1+/
(1.37 ± 0.11), and
SERCA1
/
(1.38 ± 0.13) mice did not differ
significantly. Within minutes, SERCA1
/
animals
displayed abnormal signs that were characterized by gasping respiration, limited chest wall movements, and progressive cyanosis. Affected mice demonstrated slow limb movements and delayed relaxation of skeletal muscles that was reminiscent of the cramping that characterizes Brody patients. Unfortunately, contractile measurements on skeletal muscles in these mice were not feasible due to the fragile
texture and small size of the limb muscles. We assume that the
contractile properties of the limb muscles would be similar to those
observed with diaphragm muscle and would, therefore, be consistent with
our observations on the movements of the live animals.
SERCA1
/
mice died within 30 min to 2 h after birth.
/
embryos revealed no significant morphological abnormalities (data not
shown). Studies of term SERCA1
/
mice that died shortly
after birth of respiratory failure revealed prominent hypercontracted
regions in scattered fibers (Figs. 2, G and H) and increased fiber size variability
(Fig. 2H) in diaphragm muscle. Morphological analysis of
hind limb muscles of SERCA1
/
and SERCA1+/+
mice revealed uniform fiber size and no pathological alterations in
myofiber architecture (data not shown). Analysis of the lung of
SERCA1
/
mice revealed diffuse congestion and
hypercellularity (Fig. 2F). In contrast, analysis of
SERCA1+/+ animals revealed uniformly open alveolar spaces
in lungs (Fig. 2B), absence of regional hypercontraction and
a uniform size of muscle fibers in the diaphragm (Fig. 2D).
Analysis of cardiac muscle of SERCA1
/
and
SERCA1+/+ mice revealed no pathological abnormalities (Fig.
2, A and E).
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Fig. 2.
Histological analysis of multiple organs in
SERCA1 /
newborn mice. Histological analysis of heart (A and
E), lung (B and F), longitudinal
sections of diaphragm (C and G), and
cross-sections of diaphragm (D and H) are
presented from SERCA1+/+ (A-D) and
SERCA1
/
(E-H) newborn mice. Note that a
lack of SERCA1 expression in SERCA1
/
mice results in a
congested and hypercellular appearance in lung
(F), scattered degeneration and focal hypercontraction in
diaphragm (G), and variation in diaphragm fiber size
(H). Heart sections exhibit histology that is similar in
newborn mice of both genotypes (A and E).
/
newborn
mice revealed prominent hypercontracted regions in scattered fibers
(Fig. 3, A and B).
The hypercontractility in some sections was profound, decreasing
sarcomere length to as short as 600 nm. At this length, both thick and
thin filaments were forced into the Z-band region, leading to increased
Z-band density. Thus, abnormal findings were restricted to changes in
diaphragm muscle architecture in affected mice.
View larger version (124K):
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Fig. 3.
Electron microscopic analysis of diaphragm in
SERCA1 /
newborn mice. A, longitudinally oriented diaphragm
muscle demonstrating a region of normal myofiber alignment immediately
adjacent to a region of hypercontraction. In the regions of
hypercontraction, the distance between Z-bands was ~1.3 µm compared
with about 2 µm in the normally contracted regions. Note the
prominence of the Z-bands within the regions of hypercontraction.
B, an enlargement of a longitudinally oriented myofiber with
marked hypercontraction, prominent Z-bands, complete overlap of thick
and thin filaments, and intrusion of filaments into the Z-band
region.
/
mice. It also shows that the expression of
SERCA2a, -2b, and -3 and their regulators, sarcolipin (SLN) and
phospholamban (PLN), was not affected in SERCA1
/
mice.
Fig. 4B shows, through semiquantitative Western blotting, that SERCA1 protein is undetectable in SERCA1
/
mice,
whereas expression of SERCA2a and other Ca2+ regulatory
membrane proteins, including the ryanodine receptor and
Na+/Ca2+ exchanger did not differ between
wild-type and affected neonatal mice.
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Fig. 4.
Quantification of mRNA and protein for
SERCA isoforms and other Ca2+ regulatory proteins in
diaphragm and hind limb muscles from SERCA1+/+ and
SERCA1 /
newborn mice. Total RNA and total homogenate preparations were
subjected to semiquantitative RT-PCR or Western blotting, respectively,
as described under "Experimental Procedures." A,
transcripts of SERCA1a and 1b (first panel);
coamplified SERCA1 and SERCA2 (second panel);
SERCA2a (third panel); SERCA2b (fourth
panel); coamplified SERCA2 and SERCA3, following
PvuII digestion, which discriminates SERCA2 from SERCA3
(fifth panel); sarcolipin (sixth
panel); phospholamban (seventh panel);
and control GAPDH were detected by semiquantitative RT-PCR (linear
range PCR amplification) using the primer pairs described under
"Experimental Procedures." B, expression of SERCA1 (3 µg), SERCA2a (40 µg), ryanodine receptors (20 µg), and
Na+-Ca2+ exchanger (20 µg) was detected with
antibody A52, 2A7-A1, 34C, and R3F1, respectively, in total
homogenates.
/
newborn
mice and assayed for Ca2+ dependence of Ca2+
uptake by the sarcoplasmic reticulum. As expected with the ablation of
SERCA1, maximal Ca2+ uptake activity was reduced
(p < 0.05) by 80% in diaphragm and hind limb muscle
homogenates of SERCA1
/
mice compared with
SERCA1+/+ mice (9.72 ± 0.2 and 1.8 ± 0.1 nmol
of Ca2+/mg of protein/min versus 2.26 ± 0.2 and 0.49 ± 0.1 nmol of Ca2+/mg of protein/min,
respectively, mean ± S.E.) (Fig.
5A). This decrease in
Ca2+ uptake by the sarcoplasmic reticulum is consistent
with the decreased overall SERCA1 protein levels in
SERCA1
/
mice. In diaphragm, the apparent
Ca2+ affinity, expressed as KCa in
pCa units, was decreased (p < 0.05) in
samples from SERCA1
/
mice, compared with samples from
SERCA1+/+ mice (5.83 ± 0.12 versus
6.31 ± 0.09, mean ± S.E.) (Fig. 5B).
View larger version (16K):
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Fig. 5.
Ca2+ dependence of
Ca2+ transport activity in diaphragm and hind limb muscles
from SERCA1+/+ and
SERCA1 /
newborn mice. Total homogenates of pooled diaphragm or pooled hind
limb muscles from newborn mice were subjected to Ca2+
uptake assays. A, Ca2+ uptake rate (nmol of
Ca2+/mg of protein/min) at varying Ca2+
concentration. B, Ca2+ dependence of
Ca2+ uptake mean values at each Ca2+
concentration are from experiments shown in A and are
expressed as percentage of Ca2+ uptake rates at 5.62 µmol/liter Ca2+. The data are from experiments with three
independent homogenate preparations made from pooled samples from five
newborn mice in each case. *, p < 0.05.
/
and
SERCA1+/+ mice. Fig.
6A shows representative
records of force obtained from 30-Hz tetani produced in a muscle strip
from a SERCA1+/+ and a SERCA1
/
mouse.
Tetanic force was dramatically lower in the SERCA1
/
muscle strip. Time of relaxation was markedly longer in the
SERCA1
/
muscle strip. The force-frequency curve was
shifted downward significantly in SERCA1
/
muscle strip
compared with a SERCA1+/+ muscle strip at all frequencies
of stimulation, with the exception of 10 Hz (Fig. 6B). The
maximum corrected tetanic force output from SERCA1+/+
muscle strips was 8.5 ± 1.5 g/mm2, compared with
2.6 ± 0.4 g/mm2 from SERCA1
/
muscle
strips. The frequency-dependent maximal rates of
contraction (+dF/dt) and relaxation
(
dF/dt) are shown in Fig. 6, C and
D, respectively. At all stimulation frequencies, both
+dF/dt and
dF/dt were
significantly (p < 0.05) depressed in
SERCA1
/
mice compared with SERCA1+/+ mice.
The response to a stimulation protocol that measures fatigue (not
shown) also showed an increased susceptibility to fatigue in diaphragm
muscle from SERCA1
/
mice.
View larger version (27K):
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Fig. 6.
Contractile properties of diaphragm strips
from newborn
SERCA1 /
mice (empty symbols and
columns) and SERCA1+/+ mice
(filled symbols and
columns). A, representative traces of
tension developed during tetanic stimulations at 30 Hz. Force-frequency
relationships (B), maximal rate of force development
(C), and maximal rate of relaxation (D) at
different Hz stimulations are shown. *, p < 0.05.
/
mice were cultured in vitro under
differentiation conditions for 5 days and 20 days. The rate of fusion
and extent of differentiation of myoblasts isolated from
SERCA1+/+ and SERCA1
/
mice were similar
(Fig. 7, C-F).
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Fig. 7.
Analysis of myoblast differentiation from
SERCA1 /
mice in vitro. Primary myoblasts (A
and B) were isolated from limb muscles of E19 embryos of
SERCA1+/+ (A, C, and E)
and SERCA1
/
(B, D, and
F) genotype and induced to form myotubes under
differentiation conditions for 5 days (C and D)
and 20 days (E and F).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice confirmed
that our targeting strategy had produced a null mutation.
SERCA1
/
mice were born alive in the predicted Mendelian
ratio, demonstrating that SERCA1 ablation is not embryonic lethal.
SERCA1
/
neonatal mice were indistinguishable from
wild-type SERCA1+/+ littermates in their gross appearance
and in their body weight at birth (Fig. 1D). Within minutes,
however, SERCA1
/
mice displayed abnormal signs that
were characterized by infrequent gasping respiration, limited chest
wall excursions, and progressive cyanosis. In addition,
SERCA1
/
mice exhibited slow limb movements and apparent
contracture that was reminiscent of the contractures observed in the
skeletal muscle of Brody patients. Affected mice died within 2 h
after birth of respiratory failure. Thus, in contrast to humans, SERCA1
is essential for survival in mice.
/
newborn mice did not
differ in morphology from wild-type littermates (Fig. 2, A
and E). However, examination of the diaphragm muscle in
newborn SERCA1
/
mice revealed prominent hypercontracted
regions in scattered fibers, indicative of impaired muscle fiber
relaxation (Fig. 2, G and H). Analysis of the
lung revealed pulmonary congestion, hypercellularity of the alveolar
epithelium, and atelectasis, indicating failure of expansion of alveoli
due to impaired respiration. These findings indicate that absence of
SERCA1 in mice leads to respiratory failure due to profound impairment
of diaphragmatic function. There are no pathological studies of
diaphragm muscle in human Brody disease patients. However, respiratory
failure is not a clinical feature of the disease. Thus,
there is a marked discrepancy in severity of the SERCA1a deficiency
between affected humans and mice, which suggests either that superior
compensatory mechanisms for Ca2+ regulation occur in human
Brody disease patients or that significant physiological differences
exist between mice and humans. Compensatory changes may involve
Ca2+ removal by the plasma membrane Ca2+-ATPase
pump or by Na+/Ca2+ exchangers in the plasma
membrane, mitochondrial Ca2+ uptake, or proliferation of
the sarcoplasmic reticulum containing compensatory levels of SERCA2 or
SERCA3. Of these possible compensatory processes, only the last would
be predicted to result in Ca2+ loading of the sarcoplasmic
reticulum, a process necessary for subsequent muscle contraction
(29).
/
were used. These studies revealed that SERCA1
mRNA and protein were completely absent in SERCA1
/
diaphragm and limb muscle (Fig. 4), but mRNA levels of SERCA2a, SERCA2b, SERCA3, PLN, and SLN were unchanged, and protein levels of
SERCA2, plasma membrane Ca2+-ATPase, RYRs, and
Na+/Ca2+ exchangers did not differ between
wild-type and affected mice. (Fig. 4). These results indicate that
there was no compensatory adaptation to the loss of SERCA1 in diaphragm muscle.
/
mice in utero. This is somewhat
surprising, since loss of Ca2+ homeostasis would be
expected in developing myotubes in SERCA1
/
neonatal
mice. If compensatory adaptations depend on increased neuromuscular
activity, in utero diaphragm muscle activity must be low,
and neonatal SERCA1
/
mice may not have survived long
enough to initiate the process of adaptation after birth. The
SERCA1
/
mice are thus incapable of supporting the
dramatic increase in neuromuscular activity required of respiratory
muscles immediately after birth.
/
mice, but the greater numbers of
slow-twitch type I fibers, expressing the SERCA2 isoform, may do so in humans.
/
mice,
may be due to the diaphragm being the most differentiated muscle at
birth (38). Consistent with this view (6, 39, 40), we also demonstrated
that the total protein content of SERCA1 in wild-type newborn mice was
5-fold higher in diaphragm than in hind limb muscle by Western blot
analysis (Fig. 4B). Our semiquantitative RT-PCR experiments
showed that, in the diaphragm, about 50% of the total SERCA1 was the
adult SERCA1a isoform, but in the hind limb muscle, SERCA1a accounted
for only 20% of total SERCA1 (Fig. 4A). Furthermore, the
Vmax of Ca2+ transport activity was
about 5 times higher in total diaphragm (9.72 ± 0.2 nmol of
Ca2+/mg of protein/min) than in total hind limb homogenates
(2.26 ± 0.2 nmol of Ca2+/mg of protein/min). This
suggests that a high expression of the adult form of SERCA1 (SERCA1a)
in the diaphragm at birth is crucial for the diaphragm to generate a
force sufficient for respiration after birth.
/
newborn mice. We did not detect any
differences in the extent or rate of growth or differentiation of
primary myoblasts into myotubes in cultures prepared from muscle
obtained from either SERCA1+/+ or SERCA1
/
(Fig. 7). When histological analysis of skeletal muscles was carried
out in SERCA1
/
mice during embryo development at days
10, 15, and 18, no obvious changes were found. This suggests that
SERCA1 is not required for the early stages of muscle development. This
is consistent with observations (6, 39, 40) in which SERCA1 was only detected at a late stage of muscle development.
/
mice was reduced to roughly the same extent as
the SERCA1 protein levels, confirming a direct relationship between
SERCA protein level and the maximal rate of Ca2+
sequestration. We also observed a lower apparent Ca2+
affinity for SERCA in homogenates prepared from SERCA1
/
diaphragm. This may be due to an increased ratio of PLN and SLN to
SERCA in SERCA1
/
mice due to the unchanged expression
of PLN and SLN, as determined by semiquantitative RT-PCR in
SERCA1
/
mice (Fig. 4A). Thus, in the
SERCA1-deficient mice, not only is there a reduction in the total
content of SERCA pumps, but those pumps that remain are more highly inhibited.
/
diaphragm strips is
fully consistent with the slow rate of Ca2+ pumping into
the sarcoplasmic reticulum in SERCA1-deficient mice. The depression of
the maximal rates of force development (Fig. 6C) and force
output (Fig. 6, A and B) is also fully consistent with a decrease in Ca2+ release from the sarcoplasmic
reticulum due to a diminished Ca2+ store in the
sarcoplasmic reticulum. The impairment of excitation-contraction coupling may lead to elevated resting [Ca2+]i,
which might initiate transient structure alterations and depressed
Ca2+ release during contraction (41-43).
/
mouse model, which
will be valuable for elucidating the mechanism of pathological changes
due to the disruption of Ca2+ homeostasis.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Alexander Kraev for invaluable advice on the manipulation, cloning, and analysis of genomic DNA and Dr. Nancy H. McKee for access to her laboratory and use of the rat contractile apparatus and Grass S88 stimulator.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Canadian Genetic Diseases Network of Centers of Excellence (to D. H. M.) and by Canadian Institutes for Health Research Grants MOP-49493 and MT-12545.
§ Postdoctoral fellows of the Heart and Stroke Foundation of Canada.
§§ To whom correspondence should be addressed: Banting and Best Dept. of Medical Research, University of Toronto, Charles H. Best Institute, 112 College St., Toronto, Ontario M5G 1L6, Canada. Tel.: 416-978-5008; Fax: 416-978-8528; E-mail: david.maclennan@utoronto.ca.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M213228200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; SLN, sarcolipin; PLN, phospholamban; MOPS, 4-morpholinepropanesulfonic acid; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ES, embryonic stem.
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