close

Вход

Забыли?

вход по аккаунту

?

402

код для вставкиСкачать
DEVELOPMENTAL DYNAMICS 210:106–116 (1997)
Transitory Expression of Alpha Cardiac Myosin Heavy
Chain in a Subpopulation of Secondary Generation
Muscle Fibers in the Pig
L. LEFAUCHEUR,1,2* R. HOFFMAN,1 C. OKAMURA,3 D. GERRARD,4 J.J. LÉGER,5
N. RUBINSTEIN,6 AND A. KELLY1
1School of Veterinary Medicine, Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania
2Station de Recherches Porcines, INRA, St-Gilles, France
3Department of Food Science, University of Missouri, Columbia, Missouri
4Department of Animal Science, Purdue University, West Lafayette, Indiana
5INSERM U300, Faculté de Pharmacie, Montpellier, France
6School of Medicine, Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT
Unlike the random distribution
of fiber types seen in skeletal muscles of most
mammals, pig muscle exhibits a rosette pattern
consisting of islets of slow fibers surrounded by
concentric circles of type IIA and IIB fibers.
Within each islet of slow fibers, one of the central
fibers is a primary myofiber, whereas all others
are secondary fibers. The present study demonstrates that a subpopulation of the slow secondary fibers transiently expresses a-myosin heavy
chain (MHC). Two cDNA libraries were made
from longissimus dorsi skeletal muscle of 14-dayold piglet and adult pig atrium; the latter muscle
is mainly composed of a-MHC. Screening of the
libraries with a human anti-a-MHC mAb (F8812F8)
demonstrated the presence of positive MHC
clones in both libraries; the nucleotide sequence
of the 38-untranslated region (38-UTR) was identical in both libraries. As this MHC 38-UTR had 75%
homology with the human a-MHC, it was identified as pig a-MHC. Using specific cRNA probes
and mAbs against pig a-cardiac and b/slow/type I
MHC, we studied the expression of these MHCs in
developing pig semitendinosus muscle by combining in situ hybridization and immunocytochemistry on serial sections at 90 days of gestation, and
at 1, 6, 35 days and 6 months of age. The results
showed that a subpopulation of secondary fibers
that directly abut primary fibers, transiently produced a-MHC, both at the levels of the protein
and its transcript. Subsequently, these fibres expressed b-MHC. At 1 day, immunocytochemistry
showed that 16% of the secondary fibers expressed a-MHC, among which 20% did not yet
express b-MHC. At 6 days, a- and b-MHCs were
mostly present in the same fibers, i.e., 23% of the
secondary fibers. Thereafter, the proportion of
secondary fibers reacting with a-MHC mAb decreased to 10% at 5 weeks and 0% at 6 months,
whereas b-MHC was still accumulating in about
38% of the secondary fibers. During the period
studied, the distribution of a- and b-MHC tranr 1997 WILEY-LISS, INC.
scripts closely matched that of the corresponding
proteins. Expression of a-MHC was not detected
in primary type I muscle fibers and slow type I
secondary fibers at the periphery of the rosettes
of slow fibers. This study is the first unequivocal
demonstration of a transitory expression of
a-MHC in a subpopulation of secondary fibers in
a limb skeletal muscle during mammalian development. Dev. Dyn. 1997;210:106–116.
r 1997 Wiley-Liss, Inc.
Key words: alpha-cardiac myosin heavy chain;
skeletal muscle; immunocytochemistry; in situ hybridization; pig
INTRODUCTION
In vertebrate skeletal muscles, myosin heavy chains
(MHC) are encoded by a highly conserved multigene
family and are subjected to developmental stage- and
muscle type-specific regulation (for review, see Pette
and Staron, 1990). Thus far, four major MHC isoforms
have been identified in adult mammalian limb muscles
(I, IIA, IIB, and IIX) (Schiaffino et al., 1989), and two
isoforms (embryonic and fetal/neonatal/perinatal) are
developmentally expressed (Whalen et al., 1981). All
these isoforms are coded by single genes that are
differentially regulated in muscle cells (Mahdavi et al.,
1987; Buckingham et al., 1986). Genes coding for types
IIA, IIB, IIX, and developmental isoforms are clustered
on the same chromosome in human (Leinwand et al.,
1983; Rappold and Vosberg, 1983; Yoon et al., 1992;
Soussi-Yanicostas et al., 1993) and mouse (Weydert et
al., 1985), whereas genes coding for b- and a-MHC are
tandemly linked on another chromosome in both human and mouse (Mahdavi et al., 1984; Saez et al., 1987;
Grant sponsor: Muscular Dystrophy Association of America.
*Correspondence to: Louis Lefaucheur, Station de Recherches Porcines, INRA, 35590, St. Gilles, France. E-mail: lefaucheur@stgilles.rennes.inra.fr
Received 7 February 1997; Accepted 11 June 1997
MYOFIBER DIVERSITY IN PIG DEVELOPMENT
Emerson and Bernstein, 1987; Matsuoka et al., 1991;
Gulick et al., 1991). It has been suggested that a- and
b-MHC genes originated from an evolutionarily recent
duplication of a common ancestral gene (Mahdavi et al.,
1984). Unlike the b-cardiac MHC, which is identical to
the slow/type I MHC expressed in slow-twitch skeletal
muscle fibers, a-MHC was initially thought to be exclusively expressed in the heart (Lompré et al., 1984;
Mahdavi et al., 1987). However, recent data have
demonstrated that this isoform is also present in other
adult skeletal muscles in human and rabbit, such as the
masseter, retractor mandibulae, temporalis, and extraocular muscles (Bredman et al., 1991, 1992a,b; D’Albis
et al., 1991, 1993; Pedrosa-Domellöf et al., 1992; Hämäläinen and Pette, 1994). Furthermore, it is also
present in some intrafusal fibers of human muscle
spindles (Pedrosa et al., 1990; Pedrosa-Domellöf et al.,
1992; Kucera et al., 1992; Pedrosa-Domellöf and Thornell, 1994). A transitory expression has also been
reported in the diaphragm of the rabbit at 4–5 weeks of
age (D’Albis et al., 1991). More recently, in pig semitendinosus (ST) muscle, a subpopulation of secondary
fibers in the direct vicinity of primary myotubes has
been shown to be transiently labeled with a monoclonal
antibody (mAb) raised against human a-MHC (Lefaucheur et al., 1995). However, possible cross-reaction
with an as yet uncharacterized MHC isoform could not
be ruled out. This possibility is consistent with data
suggesting the existence of new developmental isoforms such as three slow MHC isoforms in developing
human and rat skeletal muscle (Hughes et al., 1993).
The present study was undertaken to identify unequivocally the MHC isoform labeled with the human
a-cardiac mAb during development in pig ST muscle.
Because of the high degree of polymorphism in the
38-untranslated regions (38-UTR) of different MHC
mRNAs in laboratory animals and humans (Saez and
Leinwand, 1986), we used the 38-UTRs to generate
specific probes. Our objectives were (1) to clone and
sequence the 38-UTRs of the mRNAs coding for the
isoforms recognized by the anti-a and anti-b-MHC
monoclonal antibodies in the pig and (2) to combine
immunocytochemistry and in situ hybridization techniques on serial transverse sections of the pig ST
muscle during development.
MATERIALS AND METHODS
Production of the cDNA Libraries
Total RNA was extracted from frozen samples of
14-day-old longissimus dorsi (LD) muscle and adult
atrium of Yorkshire pigs by the acid-guanidinium
method (Chomczynski and Sacchi, 1987) and quantified
by measuring optical density at 260 nm. Fourteen-dayold LD muscle corresponds to a stage of intense maturation where a subpopulation of secondary fibers is transiently labeled with the anti-a-MHC mAb F8812F8 in
pig skeletal muscle, whereas adult pig atrium has been
shown to be mainly composed of a-MHC (Lefaucheur et
al., 1995). Poly(A)1 mRNA was isolated by two passages
107
over an oligo(dT)-cellulose column. First strand cDNA
synthesis was primed with an oligodT/XhoI primer/
adapter and carried out for 60 min at 42°C with 5 µg of
poly(A)1 using 250 U RNase H2 Moloney murine
leukemia virus reverse transcriptase (Stratagene, La
Jolla, CA) and 5-methyl dCTP. By construction, the
poly(A)1 tail was near the added Xho I site, which
allowed the production of a directional cDNA library.
After second strand synthesis, cDNAs were made blunt
by using 5 U cloned Pfu DNA polymerase (Stratagene)
and EcoR I adaptors were added. After digestion with
Xho I, cDNAs were size fractionated through a Sephacryl S-400 spin column. Fractions over 0.5 kb were
cloned into the EcoR I/Xho I sites of the expression
Uni-Zap XR vector (Stratagene). The plating of the
packaged reaction was done by infecting XL1-blue
MRF8 Escherichia coli bacteria (Stratagene). From 100
ng cDNA and 1 µg vector, 5.7 and 5.2 3 106 independent
recombinants were obtained from the 14-day-old LD
and adult atrium, respectively.
Screening of the cDNA Libraries
Amplified libraries (1.35 and 1.56 1010 plaque forming units [pfu]/ml) were screened using two mAbs on
duplicate filters from the same plates, NOQ7-5-4D,
which is specific for b-MHC (Narusawa et al., 1987),
and F8812F8, which has been shown to be specific for
a-MHC in rat, cat, and man (Dechesne et al., 1985,
1987; Léger et al., 1985; Pons et al., 1986; PedrosaDomellöf et al., 1992). The anti-a-MHC mAb (F8812F8)
has also been shown to label a very strong band on
transblots of MHC from adult pig atrium with no
reaction in 75-day gestation and adult pig ST muscle
(Lefaucheur et al., 1995). Several clones reactive with
the antibodies were recovered from the agar plates and
purified twice to homogeneity by rescreening with the
same antibodies. Excision and recircularization of the
Bluescript phagemids containing the cloned inserts
was achieved in vivo by simultaneously infecting another strain of E. coli (SOLR) with both the Uni-ZAP XR
vector and a f1 bacteriophage (ExAssisty) (Stratagene). Plasmid DNA was prepared using the QIAprepspin plasmid miniprep protocol (QIAGEN Inc., Chatsworth, CA). The size of the inserts and their restriction
map were analyzed to select the clones to be further
studied.
Sequencing and Subcloning
of the Selected Clones
Double-stranded plasmid DNA was sequenced using
the Sequenase DNA sequencing kit (U.S. Biochemical
Corp., Cleveland, OH), [35S]dATP (Amersham, Arlington Heights, IL), and the M13 primer to start the
sequencing of the insert from the poly(A) tail. Only the
38-UTR and the beginning of the coding region were
sequenced. The homology with known sequences of
MHC in human (Jaenicke et al., 1990; Liew et al., 1990;
Yamauchi-Takihara et al., 1989; Matsuoka et al., 1991;
Eller et al., 1989) and rat (DeNardi et al., 1993) was
108
LEFAUCHEUR ET AL.
analyzed using the NMATPUS program from the PC
Gene software package (PC Gene, Intelligenetics, Inc.).
The RESTRI program (PC Gene, Intelligenetics, Inc.)
was used to analyze the sequence for restriction enzyme cutting sites. The 38 ends (38 end of the coding
region 1 38-UTR) of the cDNA inserts from selected
clones were subcloned by PCR into Bluescript (Stratagene, Mountain View, CA) to get rid of the poly(A) tail
before making the corresponding riboprobes.
Riboprobe Synthesis
Vectors containing subcloned cDNAs specific to band a-MHC mRNAs were linearized with StyI and
MaeIII, respectively. The complementary RNA probes
were transcribed using T3 RNA polymerase (Promega,
Madison, WI) and 35S-UTP (New England Nuclear,
Boston, MA) as the labeled nucleotide. The b-MHC
probe length was 202 nucleotides, of which 19 nucleotides correspond to 38 end of the coding region, 115
nucleotides to the 58 end of the 38-UTR, and 68 nucleotides to vector sequence. The a-MHC probe length was
125 nucleotides, of which 57 nucleotides correspond to
the 58 end of the 38-UTR and 68 nucleotides to vector
sequence.
Histological Analysis
In situ hybridization with riboprobes, immunocytochemistry with anti-MHC mAbs, and conventional histochemistry were performed on the deep portion of
semitendinosus (ST) muscle from 90-day-old fetus,
from 1-, 6-, and 35-day-old piglets and from 6-monthold pig of the Yorkshire breed. Muscle samples were
frozen in isopentane cooled with liquid nitrogen and
stored at 280°C until further analysis. Transverse
serial sections of 10 µm were cut on a cryostat (Microm
505M, Walldorf, Germany) at 220°C, dried at room
temperature, and stored at 280°C. Cryosections were
fixed for 15 min with 10% formalin in PBS and subsequently processed for in situ hybridization as described
by Lyons et al. (1990), except that sections were treated
for 7.5 min with 5 µg/ml instead of 20 µg/ml proteinase
K. Sections were hybridized overnight at 50°C in 50%
deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl, pH
7.4, 5 mM EDTA, 10 mM NaH2PO4, 10% (w/v) dextran
sulfate, 1 3 Denhardt’s, 0.5 mg/ml yeast tRNA. Riboprobes were used at a final concentration of 20,000
cpm/µl. Slides were washed at RT for 30 min in 4 3 SSC
containing 10 mM DTT; followed by washings at RT for
10 min in 4 3 SSC and two times at 37°C for 10 min in
1 3 Washing Solution (WS) (10 mM Tris-HCl, pH 7.5,
400 mM NaCl, 5 mM EDTA). After treatment with 20
µg/ml RNAse A at 37°C for 30 min in 1 3 WS, slides
were rinsed at 37°C for 5 min in 1 3 WS, followed by
washes for 15 min at 37°C in 2 3 SSC and for 45 min at
43°C in 0.1 3 SSC. Slides were air dried and dipped in
Kodak NTB-2 nuclear track emulsion and exposed for 1
week in light-tight boxes with desiccant at 4°C. After
development in Kodak D-19, slides were analyzed with
bright-field, phase-contrast, and dark-field optics of a
Nikon microscope (Nikon microphot-FXA). In addition
to in situ hybridization, unfixed serial cryosections
were processed for immunocytochemistry using the
immunofluorescence technique described by Pons et al.
(1986). The reaction was carried out with the same
antibodies used for the screening of the libraries:
NOQ7-5-4D, reactive with b-MHC (Narusawa et al.,
1987) and F8812F8, reactive with a-MHC. Monoclonal
Ab NOQ7-5-4D was a generous gift from Dr. R.B.
Fitzsimons (Med. Res. Council Lab. Mol. Biol., Cambridge, UK) and F8812F8 was obtained from Dr. J.
Léger (INSERM, U300, Montpellier, France). Finally,
two serial sections were stained for Ca21 actomyosin
ATPase activity after preincubation at pH 4.6 and 10.4
(Brooke and Kaiser, 1970; Guth and Samaha, 1970).
At birth, primary generation fibers can be identified
by a combination of characteristics: larger size, central
location within the islets, high expression of slow MHC,
and absence of fetal MHC (Lefaucheur et al., 1995). At 6
days postpartum, primary myotubes are still identifiable by these criteria; later, however, primary myotubes
are not distinguishable from secondary slow fibers any
longer.
RESULTS
Isolation of b- and a-MHC Clones
For both libraries, double screening with NOQ7-5-4D
(b-MHC) and F8812F8 (a-MHC) mAbs showed that
reactive clones were mutually exclusive between the
two mAbs. In both libraries, several positive clones
were identified with each antibody. However, the ratios
between the number of positive clones with a- and
b-MHC mAbs on serial filters were 12:88 and 93:7 for
the 14-day-old LD and adult atrium cDNA libraries,
respectively.
Partial sequencing from the 38 end was used to
confirm the nature of the clones as MHC cDNAs, based
on the presence of the poly(A) tail and the high degree of
homology of the coding region with that of the human
embryonic MHC (Eller et al., 1989). Two groups of
clones were identified in each library on the basis of
restriction endonuclease maps and 38 end sequences
(Figs. 1, 2, and 3). The two groups will be referred to as
a- and b-MHC cDNAs, based on the mAbs used to
identify them, i.e., F8812F8 and NOQ7-5-4D, respectively. The size of the a- and b-MHC clones ranged from
3.0 to 5.8 kb and 3.2 to 5.2 kb, respectively. The 38 end
sequences of the a and b clones are closely related to the
human a- (Yamauchi-Takihara et al., 1989) and human
b- (Jaenicke et al., 1990) MHC cDNA sequences, respectively (Table 1). The first 48–50 carboxy-terminal amino
acids are identical between pig and human for both
isoforms with a high divergence between the a- and
b-MHC isoforms for the last 9 (a-MHC)/7 (b-MHC)
amino acids from the carboxy-terminus. The 38-UTRs of
the pig a- and b-MHC mRNAs show 75.0 and 79.6%
similarity with the human a- and b-MHC mRNAs,
respectively (Table 1).
MYOFIBER DIVERSITY IN PIG DEVELOPMENT
109
TABLE 1. 38 End Sequence Homology of a- and b-MHC
cDNAs Between Pig (P), Human (H), and Rat (R)
(Calculated From Figure 2)a
Coding region (%)
AA
N
a-MHC
P vs H
P vs R
H vs R
b-MHC
P vs H
P vs R
H vs R
38-UTR (N, %)
100
100
100
94.7
93.3
93.3
75.0
73.1
70.0
100
97.9
97.9
94.7
91.7
94.4
79.6
63.7
66.4
Fig. 1. Restriction maps of pig a- and b-MHC cDNA clones. Black
area, 38 end of the coding region; hatched area, 38-untranslated region;
open area, poly-(A) tail. Restriction enzyme abbreviations: B, Bst NI; D,
Dde I; Ha, Hae III; Hd, Hind III; M, Mae III; N, Nla III; S, Sty I.
aAA, amino acids; N, nucleotides; 38-UTR, 38-untranslated
region.
Expression of b- and a-MHC in the Deep Medial
Portion of Pig Semitendinosus Muscle
at 6 Days of Age
Serial sections in the deep medial portion of ST
muscle at 6 days of age were processed for acto-myosin
ATPase, reacted with NOQ7-5-4D and F8812F8 mAbs,
and hybridized with RNA probes complementary to
38-UTRs of a- and b-MHC transcripts (Fig. 4). The
b-MHC cRNA probe specifically hybridized with the
islets of type I fibers as evidenced by ATPase staining
and reaction with NOQ7-5-4D (Fig. 4a,c,e). Primary
myotubes (labelled P on Fig. 4) were strongly reactive
with NOQ7-5-4D mAb (Fig. 4c) and unreactive with
F8812F8 (Fig. 4d), whereas a subpopulation of secondary fibers, in the direct vicinity of primary myotubes,
were reactive with both mAbs (Fig. 4c,d). Fast-twitch
secondary fibers surrounding the islets of slow fibers
did not react with any of these mAbs. Figure 4e and f
shows that the distribution of b- and a-MHC transcripts closely matches that of the corresponding proteins (Fig. 4c,d). In particular, primary myotubes, labelled P on Figure 4e and f, were labeled with the
b-MHC probe (Fig. 4e) and unlabeled with the a-MHC
probe (Fig. 4f).
Developmental Pattern of a-MHC Expression
Alpha-MHC transcripts could be detected using PCR
in RNA from fetal muscle at 70 days of gestation, but
this technique cannot discriminate between expression
from intrafusal (bag) fibers and extrafusal muscle
fibers (data not shown). We detected a-MHC in intrafusal fibers by immunocytochemistry as early as 90
days of gestation, but not in extrafusal fibers. At this
stage, b-MHC transcript and protein were only expressed in primary myofibers (Fig. 5a,b), not in secondary fibers. At 103 days of gestation, the first a-MHC was
detected in a few extrafusal fibers spread out in small
areas of the muscle (data not shown). Thereafter, the
number of fibers staining positively with F8812F8 mAb
increased through late gestation and birth (113 days
gestation).
At 1 day postpartum, the primary fibers displayed
the same reactivity as at 90 days gestation whereas
some secondary fibers, in the direct vicinity of the
primary fibers, began to express a- and sometimes
b-MHC transcripts and proteins (Fig. 5e–h). At 1 day,
immunocytochemistry showed that 16% of the secondary fibers expressed a-MHC, among which 20% did not
yet express b-MHC. The proportion of secondary fibers
reacting with anti-a-MHC mAb reached a maximum
value of 23% at 6 days (Fig. 5k), plateaued until 11 days
(not shown), and thereafter decreased to 10% at 5
weeks (Fig. 5o) and 0% at 6 months (Fig. 5s). By
contrast, the proportion of secondary fibers reacting
with anti-b-MHC mAb increased to reach a plateau of
37% from 5 weeks onwards. Therefore, only 60% (i.e.,
23/37%) of the secondary fibers maturing to type I
fibers, those located in the direct vicinity of primary
myotubes, transiently expressed a-MHC. Alpha-MHC
was never expressed in primary myotubes nor in fast
secondary fibers at the periphery of the rosettes. During development, the distribution of b- (Fig. 5b, f, j, n,r)
and a- (Fig. 5d,h,l,p,t) MHC transcripts closely matched
that of their corresponding MHC proteins (Fig.
5a,e,i,m,q and Fig. 5c,g,k,o,s, respectively).
DISCUSSION
a-MHC Is Expressed in Pig Semitendinosus
During Development
This study is the first to demonstrate that a MHC
isoform that is identical to a-MHC is transiently expressed during development in a mammalian hind limb
muscle. The clones screened with F8812F8 mAB (aMHC) in the 14-day-old LD cDNA library are identical
to those screened in the library made from adult
atrium, a muscle mainly composed of a-MHC (Lefaucheur et al., 1995). The sequence of the a-MHC 38-UTR
is very different from that of the b- (Fig. 3a,b), embryonic, perinatal, fast IIA, IIB, and IIX mRNAs (Lefaucheur et al., unpublished data). The amino acid and
nucleotide sequences of the pig a-MHC show remarkable homology with that of human a-isoform (YamauchiTakihara et al., 1989; Matsuoka et al., 1991), which
confirmed that this isoform does correspond to the pig
a-MHC. As in human, the extreme carboxyl terminus
amino acid sequence is highly divergent between a- and
b-MHC isoforms (Fig. 2).
110
LEFAUCHEUR ET AL.
Fig. 2. Nucleotide and amino acid sequences of the 38 ends of a- and
b-MHC coding regions. Stop codons are underlined. A: a-MHC. B:
b-MHC. Bottom lines correspond to published nucleotide sequences for
human (h) (Yamauchi-Takihara et al., 1989; Matsuoka et al., 1991;
Jaenicke et al., 1990) and rat (r) (Mahdavi et al., 1982, 1984) MHC. Dots
and asterisks indicate identical nucleotides and amino acids with pig,
respectively.
MYOFIBER DIVERSITY IN PIG DEVELOPMENT
111
Fig. 3. Nucleotide sequences of the 38 untranslated regions of a- and
b-MHC clones. A: a-MHC 38-UTR. B: b-MHC 38-UTR. Bottom lines
correspond to published nucleotide sequences for human (h) (YamauchiTakihara et al., 1989; Matsuoka et al., 1991; Jaenicke et al., 1990) and rat
(r) (Mahdavi et al., 1982, 1984) MHC. Dots indicate identical nucleotides
with pig, blanks indicate missing nucleotides. Polyadenylation signals are
underlined.
Alpha-MHC Is Under Spatial
and Temporal Regulation
ATPase activity, which is, in turn, strongly correlated
with MHC composition (Barany, 1967; Schwartz et al.,
1981; Reiser et al., 1985). Thus, it seems likely that
muscle fibers containing a-MHC would exhibit an
intermediate speed of contraction between those containing b- and adult fast type II MHCs.
A similar transitory expression of a-MHC has also
been reported to occur in the pig ventricle during the
first 3 weeks after birth (Lompré et al., 1981) suggesting common regulatory factors.
During normal skeletal and cardiac development, it
has been well established that although some of the
myosin heavy chain transitions are nerve dependent
(Jolesz and Sreter, 1981), others are regulated by the
level of thyroid hormones (Ianuzzo et al., 1977; Gambke
et al., 1983; Izumo et al., 1986; Butler-Browne et al.,
1987; D’Albis et al., 1987). In particular, the regulation
of the a-MHC gene by thyroid hormone has been
extensively studied. Thyroid responsive elements have
been characterized in the a-MHC promoter (Izumo and
Mahdavi, 1988; Tsika et al., 1990; Flink and Morkin,
1990; Subramaniam et al., 1991; Rindt et al., 1995) and
thyroid hormones increased the expression of a-MHC
both at the levels of mRNA and protein in the ventricular myocardium of rats and mice (Butler-Browne et al.,
1987; D’Albis et al., 1987; Lompré et al., 1984). Since a
significant increase in thyroxin (T4) has been reported
to occur in the pig during the last days of gestation
(Berthon et al., 1993), and very high levels of triiodothyronine (T3) and T4 have been measured during the first
week following birth in the pig (Slebodzinski et al.,
1981), thyroid hormones could therefore be the factor
that triggers the transitory expression of a-MHC in
both the heart (Lompré et al., 1981) and skeletal muscle
(present study).
Why thyroid hormones might act selectively on a
subpopulation of secondary fibers, close to the primary
The combined use of antibodies and riboprobes on
serial sections confirmed the occurrence of a-MHC in
developing pig ST muscle both at the levels of the
transcript and corresponding protein. The red portion
of mature porcine ST muscle is characterized by a
typical arrangement of fibers in rosettes, with islets of
type I fibers occupying the central core position surrounded by a ring of type II fibers (Fig. 4a). This ordered
pattern of fiber distribution is unique to the pig and
contrasts with the checkerboard pattern of fiber types
seen in skeletal muscles of most other mammals. The
expression of a- and b-MHC within a rosette of fibers
from 90-day gestation to 6 months of age is schematized
in Figure 6. It is noteworthy that the most important
expression of a-MHC occurs during the first postnatal
weeks, a period when the greatest changes occur in the
maturation of pig muscle fibers (Suzuki and Cassens,
1980; Lefaucheur and Vigneron, 1986). Our results
show that the expressions of a- and b-MHC isoforms
are differentially regulated in a spatial and temporal
manner within the pig ST muscle. The close correlation
observed between the presence of the a- and b-MHC
mRNAs and corresponding isozymes during development suggests that transcriptional mechanisms are
involved in the regulation of MHC phenotype, as suggested by Medford et al. (1983), Lompr[41d] ae et al.
(1984), Cox and Buckingham (1992), Boheler et al.
(1992).
The biological significance of the transitory expression of a-MHC in pig ST muscle is unknown but one
could speculate that it influences the contractile performance of skeletal muscle. Indeed, the a-MHC has been
shown to have an ATPase activity three times that of
b-MHC (Pope et al., 1980), and the velocity of contraction of a particular fiber is directly proportional to its
112
LEFAUCHEUR ET AL.
Fig. 4. Serial sections of the deep medial portion of semitendinosus
muscle at 6 days of age. Serial sections were processed for histochemical
demonstration of Ca21 activated acto-myosin ATPase after preincubation
at pH 4.6 (a) and 10.4 (b), reacted with NOQ7-5-4D (b-MHC) (c) and
F8812F8 (a-MHC) (d) mAbs, and processed for in situ hybridization with b
(e) and a- (f) cRNA probes. Phase contrast pictures corresponding to (e)
and (f) were used to identify the fibers. Three primary fibers are labelled P
on each serial section. Scale bar 5 50 µm.
myotube, is an interesting question. Fiber-specific differences in nuclear T3 receptors, local growth factors and
hormones, and/or neuronally imposed mechanical work
could be involved. Recent results reported by Peuker
and Pette (1995) demonstrate that low-frequency stimulation strongly induced a-MHC mRNA in rabbit tibialis
anterior muscle, which suggests that neural input and
its associated mechanical work are likely to be involved
in the spatial distribution of a-MHC expression. Interestingly, the expression of a-MHC we observed seemed
to precede that of b-MHC in the same fibers, as
suggested by Peuker and Pette (1995) in their stimulation study. The significance of the a-MHC in the maturation of muscle fibers and the mechanisms involved
Fig. 5. Expression of b- and a-MHCs on serial sections of the deep
medial portion of the semitendinosus muscle at 90 days of gestation
(a–d), and 1 day (e–h), 6 days (i–l), 5 weeks (m–p), and 6 months (q–t) of
age. The sections were reacted with NOQ7-5-4D (b-MHC) (a,e,i,m,q) and
F8812F8 (a-MHC) (c,g,k,o,s) mAbs and processed for in situ hybridization with b (b,f,j,n,r) and a (d,h,l,p,t) cRNA probes. White arrowheads
denote corresponding myofibers on serial sections at a given age. Scale
bars 5 50 µm.
114
LEFAUCHEUR ET AL.
Fig. 6. Schematic representation of a- and b-MHC expression within a
rosette of fibers in the red portion of pig semitendinosus muscle from
90-day gestation (dg) to 6 month of age (6 mo). The central fiber of each
rosette is a primary fiber (thick circle) and the islet of slow fibers is formed
by the gradual conversion of some surrounding secondary generation
fibers to type I during the first weeks after birth. During the period of
maturation that we have analyzed, the expression of a-MHC was limited
to a subpopulation of secondary muscle fibers which later mature to
become slow fibers. Within each rosette, a-MHC was only expressed in
those secondary fibers which were in the direct vicinity of the primary
fibers and which were also the first of the secondary generation muscle
fibers to express slow MHC. At 6d, a- and b-MHCs were mostly present in
the same fibers, i.e., in 23% of the secondary fibers. During the
subsequent maturation, the a-MHC was gradually eliminated, while
b-MHC continued to accumulate. These fibers progressively matured to
type I fibers, giving rise to the typical islets of type I fibers surrounded by
type II fibers encountered in the adult pig. It should be noted that the
primary generation type I muscle fibers never expressed a-MHC nor did
the more peripheral type I fibers of each slow islet and the fast secondary
fibers. This demonstrates that even though all the fibers within a slow islet
are type I in mature muscle, they got there by different pathways. Scale
bars 5 50 µm.
remain to be established. On the other hand, it is well
documented that expression of b-MHC would be mostly
neurally maintained by maturation of the type I motor
units and their associated contractile activity and
excitation-contraction coupling (Butler-Browne et al.,
1982). Since the piglet can immediately walk and run
after birth, the dramatic increase in physical activity at
birth, i.e., the increased contractile activity, could be
part of the mechanism leading to myosin transitions
during the perinatal period in the pig.
by sequencing and in situ hybridization. Therefore,
a-MHC can be assigned as an additional element in the
succession of MHC isoforms in skeletal muscle during
development in the pig. Up to now, no transitory
expression of a-MHC in a limb skeletal muscle has been
reported in other mammals, but one could speculate
that one of the ‘‘slow’’ isoforms recognized by antibodies
in human and rat developing muscle in Hughes et al.’s
paper (1993) was actually a-MHC. However, they did
not identify the corresponding cDNAs, and further
studies are needed to know if the pig is unique in that
respect. It is likely that interplay between hormonal
and neuronal factors is involved in the regulation of the
spatial and temporal expression of a-MHC in pig
skeletal muscle, and more research is still needed to
answer the question. Finally, the present study shows
that a-MHC can be considered as a developmental
isoform in pig semitendinosus muscle, joining a grow-
CONCLUSION
This study in the pig is the first unequivocal demonstration of a transitory expression of a-MHC in a
subpopulation of secondary fibers in a limb skeletal
muscle during development in mammals. The presence
of a-MHC has been demonstrated both at the levels of
the protein by immunocytochemistry and its transcript
MYOFIBER DIVERSITY IN PIG DEVELOPMENT
ing amount of other studies that show that expression
of a-MHC is far from being exclusive to the myocardium.
ACKNOWLEDGMENTS
We are grateful to Dr. R.B. Fitzsimons for her generous gift of the NOQ7-5-4D monoclonal antibody. We
thank Zsuzsanna Paltzmann and Tom Moran for their
expert technical assistance. This work was financially
supported by a grant from the Muscular Dystrophy
Association of America to A.M. Kelly.
REFERENCES
Barany M. ATPase activity of myosin correlated with speed of muscle
shortening. J. Genet. Physiol. 1967;50:197–218.
Berthon D, Herpin P, Duchamp C, Dauncey MJ, Le Dividich J.
Modification of thermogenic capacity in neonatal pigs by changes in
thyroid status during late gestation. J. Dev. Physiol. 1993;19:253–
261.
Boheler KR, Chassagne C, Martin X, Wisnewsky C, Schwartz K.
Cardiac expressions of a- and b-myosin heavy chains and sarcomeric a-actins are regulated through transcriptional mechanisms.
J. Biol. Chem. 1992;267:12979–12985.
Bredman JJ, Wessels A, Weijs WA, Korfage JAM, Soffers CAS,
Moorman AFM. Demonstration of ‘‘cardiac-specific’’ myosin heavy
chain in masticatory muscles of human and rabbit. Histochem. J.
1991;23:160–170.
Bredman JJ, Weijs WA, Korfage HAM, Brugman P, Moorman AFM.
Myosin heavy chain expression in rabbit masseter muscle during
postnatal development. J. Anat. 1992a;180:263–274.
Bredman JJ, Weijs WA, Moorman AFM. Presence of cardiac a-myosin
correlates with histochemical myosin Ca21 ATPase activity in rabbit
masseter muscle. Histochem. J. 1992b;24:260–265.
Brooke MH, Kaiser KK. Muscle fibre types: How many and what kind?
Arch. Neurol. 1970;23:369–379.
Buckingham ME, Alonso S, Barton P, Bugaiski G, Cohen A, Daubas A,
Minty A, Robert B, Weydert A. Actin and myosin multigene families:
Their expression during the formation of skeletal muscle. Am. J.
Med. Genet. 1986;25:623–634.
Butler-Browne G, Bugaisky L, Cuenoud S, Schwartz K, Whalen R.
Denervation of newborn rat muscles does not block the appearance
of adult fast myosin heavy chain. Nature 1982;299:830–833.
Butler-Browne GS, Prulière G, Cambon N, Whalen RG. Influence of
the dwarf mouse mutation on skeletal and cardiac myosin isoforms.
Effect of one injection of thyroxine on skeletal and cardiac muscle
phenotype. J. Biol. Chem. 1987;262:15188–15193.
Chomczynski P, Sacchi N. Single step method of RNA isolation by acid
guanidinium thiocyanate-phenol chloroform extraction. Anal. Biochem. 1987;162:156–159.
Cox RD, Buckingham ME. Actin and myosin genes are transcriptionally regulated during mouse skeletal muscle development. Dev. Biol.
1992;149:228–234.
D’Albis A, Weinman J, Mira J-C, Janmot C, Couteaux R. Regulatory
rôle of thyroid hormones in myogenesis. Analysis of myosin isoforms
during muscle regeneration. C.R. Acad. Sci. Paris 1987;305:697–
702.
D’Albis A, Janmot C, Mira JC, Couteaux R. Characterization of a
ventricular V1 myosin isoform in rabbit masticatory muscles.
Developmental and neural regulation. BAM. 1991;1:23–34.
D’Albis A, Anger M, Lompre AM. Rabbit masseter expresses the
cardiac alpha myosin heavy chain gene evidence from mRNA
sequence analysis. Febs Letts. 1993;324:178–180.
Dechesne CA, Léger JOC, Bouvagnet P, Claviez M, Leger JJ. Fractionation and characterization of two molecular variants of myosin from
adult human atrium. J. Mol. Cell. Cardiol. 1985;17:753–757.
Dechesne CA, Léger JOC, Léger JJ. Distribution of a- and b-myosin
heavy chains in the ventricular fibers of the post-natal developing
rat. Dev. Biol. 1987;123:169–173.
115
DeNardi C, Ausoni S, Moretti P, Gorza L, Velleca M, Buckingham M,
Schiaffino S. Type 2X-myosin heavy chain is coded by a muscle fiber
type-specific and developmentally regulated gene. J. Cell Biol.
1993;123:823–835.
Eller M, Stedman HH, Sylvester JE, Fertels SH, Wu Q, Raychowdhury MK, Rubinstein NA, Kelly AM, Sarkar S. Human
embryonic myosin heavy chain cDNA. Interspecies sequence conservation of the myosin rod, chromosomal locus and isoform specific
transcription of the gene. FEBS Lett. 1989;256:21–28.
Emerson CP, Bernstein SI. Molecular genetics of myosin. Ann. Rev.
Biochem. 1987;56:695–726.
Flink IL, Morkin E. Interaction of thyroid hormone receptors with
strong and weak cis-acting elements in the human a-myosin heavy
chain gene promoter. J Biol. Chem. 1990;265:11233–11237.
Gambke B, Lyons GE, Haselgrove J, Kelly AM, Rubinstein NA.
Thyroidal and neural control of myosin transitions during development of rat fast and slow muscles. FEBS Lett. 1983;156:335–339.
Gulick J, Subramaniam A, Neumann J, Robbins J. Isolation and
characterization of the mouse cardiac myosin heavy chain genes. J.
Biol. Chem. 1991;266:9180–9185.
Guth L, Samaha FJ. Procedure for the histochemical demonstration of
actomyosin ATPase. Exp. Neurol. 1970;28:365–367.
Hämäläinen N, Pette D. The expression of an additional slow myosin
heavy chain isoform in transforming skeletal muscle of the rabbit. J.
Muscle Res. Cell Motil. 1994;15:189.
Hughes SM, Cho M, Karsch-Mizrachi I, Travis M, Silberstein L,
Leinwand LA, Blau HM. Three slow myosin heavy chains sequencially expressed in developing mammalian skeletal muscle. Dev.
Biol. 1993;158:183–199.
Ianuzzo D, Patel P, Chen V, O’Brian P, Williams P. Thyroidal trophic
influence on skeletal muscle protein. Nature 1977;270:74–76.
Izumo S, Mahdavi V. Thyroid hormone receptor a-isoforms generated
by alternative splicing differentially activate myosin HC gene
transcription. Nature 1988;334:539–541.
Izumo S, Nadal-Ginard B, Madahavi V. All members of the MHC
multigene family respond to thyroid hormone in a highly tissuespecific manner. Science 1986;231:597–600.
Jaenicke T, Diederich KW, Haas W, Schleich J, Lichter P, Pfordt M,
Bach A, Vosberg HP. The complete sequence of the human b-myosin
heavy chain gene and a comparative analysis of its product.
Genomics 1990;8:194–206.
Jolesz F, Sreter FA. Development, innervation and activity pattern
induced changes in skeletal muscle. Annu. Rev. Physiol. 1981;43:531–
552.
Kucera J, Walro JM, Gorza L, Expression of type-specific MHC
isoforms in rat intrafusal muscle fibers. J. Histochem. Cytochem.
1992;40:293–307.
Lefaucheur L, Vigneron P. Post-natal changes in some histochemical
and enzymatic characteristics of three pig muscles. Meat Sci.
1986;16:199–216.
Lefaucheur L, Edom F, Ecolan P, Butler-Browne GS. Pattern of muscle
fiber type formation in the pig. Dev. Dyn. 1995;203:27–41.
Léger JOC, Bouvagnet P, Pau B, Roncucci R, Léger JJ. Levels of
ventricular myosin fragments in human sera after myocardial
infarction determined with monoclonal antibodies to myosin heavy
chains. Eur. J. Clin. Invest. 1985;15:422–429.
Leinwand LA, Fournier REK, Nadal-Ginard B, Shows TB. Multigene
family for sarcomeric myosin heavy chain in mouse and human
DNA: Localization on a single chromosome. Science 1983;221:766–
768.
Liew CC, Sole MJ, Yamauchi-Takihara K, Kellam B, Anderson DH,
Lin L, Liew JC. Complete sequence and organization of the human
cardiac b-myosin heavy chain gene. Nucleic Acids Res. 1990;18:3647–
3651.
Lompré AM, Mercadier JJ, Wisnewsky C, Bouveret P, Pantaloni C,
D’Albis A, Schwartz K. Species- and age-dependent changes in the
relative amounts of cardiac myosin isoenzymes in mammals. Dev.
Biol. 1981;84:286–290.
Lompré AM, Nadal-Ginard B, Mahdavi V. Expression of the cardiac
ventricular a- and b-myosin heavy chain genes is developmentally
and hormonally regulated. J. Biol. Chem. 1984;259:6437–6446.
116
LEFAUCHEUR ET AL.
Lyons GE, Schiaffino S, Sassoon D, Barton D, Buckingham M.
Developmental regulation of myosin gene expression in mouse
cardiac muscle. J. Cell Biol. 1990;111:24270–2436.
Mahdavi V, Periasamy M, Nadal-Ginard B. Molecular characterization of two myosin heavy chain genes expressed in the adult heart.
Nature 1982;297:659–664.
Mahdavi V, Chambers AP, Nadal-Ginard B. Cardiac a- and b-myosin
heavy chain genes are organized in tandem. Proc. Natl. Acad. Sci.
U.S.A. 1984;81:2626–2630.
Mahdavi V, Izumo S, Nadal-Ginard B. Development and hormonal
regulation of sarcomeric myosin heavy chain gene family. Circ. Res.
1987;60:804–814.
Matsuoka R, Beisel KW, Furutani M, Arai S, Takao A. Complete
sequence of human cardiac a-myosin heavy chain gene and amino
acid comparison to other myosins based on structural and functional
differences. Am. J. Med. Gen. 1991;41:537–547.
Medford RM, Nguyen HT, Nadal-Ginard B. Transcriptional and
cell-cycle-mediated regulation of myosin heavy chain gene expression during muscle cell differentiation. J. Biol. Chem. 1983;258:
11063–11073.
Narusawa M, Fitzsimons RB, Izumo S, Nadal-Ginard B, Rubinstein
NA, Kelly AM. Slow myosin in developing rat skeletal muscle. J. Cell
Biol. 1987;104:447–459.
Pedrosa F, Soukup T, Thornell LE. Expression of an alpha cardiac-like
myosin heavy chain in muscle spindle fibres. Histochemistry 1990;
95:105–113.
Pedrosa-Domellöf F, Thornell L. Expression of myosin heavy chain
isoforms in developing human muscle spindles. J. Histochem.
Cytochem. 1994;42:77–88.
Pedrosa-Domellöf F, Eriksson PO, Butler-Browne GS, Thornell LE.
Expression of alpha-cardiac myosin heavy chain in mammalian
skeletal muscle. Experientia 1992;48:491–494.
Pette D, Staron RS. Cellular and molecular diversities of mammalian
skeletal muscle fibers. Rev. Physiol. Biochem. Pharmacol. 1990;116:
1–76.
Peuker H, Pette D. Reverse transcriptase-polymerase chain reaction
detects induction of cardiac-like a myosin heavy chain mRNA in low
frequency stimulated rabbit fast-twitch muscle. FEBS Lett. 1995;367:
132–136.
Pons F, Léger JOC, Chevallay M, Tomé FMS, Fardeau M, Léger JJ.
Immunocytochemical analysis of myosin heavy chains in human
fetal skeletal muscles. J. Neurol. Sci. 1986;76:151–163.
Pope B, Hoh JFY, Weeds A. The ATPase activities of rat cardiac myosin
isoenzymes. FEBS Lett. 1980;118:205–208.
Rappold GA, Vosberg HP. Chromosomal localization of a human
myosin heavy chain gene by in situ hybridization. Hum. Genet.
1983;65:195–197.
Reiser PJ, Moss RL, Giulian GG, Greaser ML. Shortening velocity in
single fibers from adult rabbit soleus muscles is correlated with
myosin heavy chain composition. J. Biol. Chem. 1985;260:9077–
9080.
Rindt H, Subramaniam A, Robbins J. An in vivo analysis of transcriptional elements in the mouse a-myosin heavy chain gene promoter.
Trans. Res. 1995;4:397–405.
Saez L, Leinwand A. Characterization of diverse forms of myosin
heavy chain expressed in adult human skeletal muscle. Nucleic
Acids Res. 1986;14:2951–2969.
Saez LJ, Granola KM, MacNally EM, Feghali R, Eddy R, Shows TB,
Leinwand LA. Human cardiac myosin heavy chain genes and their
linkage in the genome. Nucleic Acids Res. 1987;15:5443–5459.
Schiaffino S, Gorza L, Sartore S, Saggin L, Ausoni S, Vianello M,
Gundersen K, Lomo T. Three myosin heavy chain isoforms in type 2
skeletal muscle fibers. J. Muscle Res. Cell Motil. 1989;10:197–205.
Schwartz K, Lompré AM, Bouveret P, Wisnewsky C, Whalen RG.
Comparisons of rat ‘‘cardiac’’ myosins at fetal stages in young
animals and in hypothyroid adults. J. Biol. Chem. 1981;257:14412–
14418.
Slebodzinski AB, Nowak G, Zamyslowska H. Sequential observation of
changes in thyroxine, triiodothyronine and reverse triiodothyronine
during the postnatal adaptation of the pig. Biol. Neonate 1981;39:
191–199.
Soussi-Yanicostas N, Whalen RG, Petit C. Five skeletal myosin heavy
chain genes are organized as a multigene complex in the human
genome. Hum. Mol. Genet. 1993;2:563–569.
Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J.
Tissue-specific regulation of the a-myosin heavy chain gene promoter in transgenic mice. J. Biol. Chem. 1991;266:24613–24620.
Suzuki A, Cassens RG. A histochemical study of myofiber types in
muscle of the growing pig. J. Anim. Sci. 1980;51:1449–1461.
Tsika RW, Bahl JJ, Leinwand LA, Morkin E. Thyroid hormone
regulates expression of a transfected human alpha-myosin heavychain fusion gene in fetal rat heart cells. Proc. Natl. Acad. Sci.
U.S.A. 1990;87:379–383.
Weydert A, Daubas P, Lazaridis I, Barton P, Garner I, Leader DP,
Bonhomme F, Catalan J, Simon D, Guénet JL, Gros F, Buckingham
ME. Genes for skeletal muscle myosin heavy chains are clustered
and are not located on the same mouse chromosome as a cardiac
myosin heavy chain gene. Proc. Natl. Acad. Sci. U.S.A. 1985;82:7183–
7187.
Whalen RG, Sell SM, Butler-Browne GS, Schwartz K, Bouveret P,
Pinset-Härstrom I. Three myosin heavy-chain isozymes appear
sequentially in rat muscle development. Nature 1981;292:805–809.
Yamauchi-Takihara K, Sole MJ, Liew J, Ing D, Liew C. Characterization of human cardiac myosin heavy chain genes. Proc. Natl. Acad.
Sci. U.S.A. 1989;86:3504–3508.
Yoon SJ, Seiler SH, Kucherlapati R, Leinwand L. Organization of the
human skeletal myosin heavy chain gene cluster. Proc. Natl. Acad.
Sci. U.S.A. 1992;89:12078–12082.
Документ
Категория
Без категории
Просмотров
3
Размер файла
499 Кб
Теги
402
1/--страниц
Пожаловаться на содержимое документа