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-sarcomeric actins in postnatal
muscle
1 Muscle Development Unit and 2 Cell Biology Unit, The Children's Medical Research Institute, Westmead, New South Wales 2145, Australia
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The postnatal
expression profiles of 
-sarcomeric actin transcripts and protein are
quantified in mouse striated muscles from birth to postnatal
day 56 by Northern and Western blot
analyses. -Cardiac actin (-CA) transcripts transiently increase
between 12 and 21 days after birth in the quadriceps muscle, reaching ~90% that found in the adult mouse heart. Although -CA is the -sarcomeric actin isoform expressed in the immature fiber, the expression profiles of other contractile protein isoforms indicate that
this postnatal period is not reflective of an immature phenotype. -Skeletal actin (-SA) transcripts accumulate to ~32% of the total -sarcomeric actin transcripts in the adult heart. Our study shows that 1) there is a
simultaneous reappearance of -CA and -SA in postnatal skeletal
and heart muscles, respectively, and 2) the contractile protein gene
expression profile characteristic of adult skeletal muscle is not
achieved until after 42 days postnatal in the mouse. We propose there
is a previously uncharacterized period of postnatal striated muscle
maturation marked by the reappearance of the minor -sarcomeric
actins.
-cardiac actin; -skeletal actin; contractile protein gene
families; heart and skeletal muscle; mouse; development
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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THE PROTEINS OF THE contractile apparatus of striated muscle, the sarcomere, are encoded by multigene families. Isoforms are expressed from the gene families characteristic of the embryonic/fetal and adult stages of muscle development (reviewed in Ref. 30). A study from our laboratory showed that immature skeletal myofibers initially express a common isoform profile encompassing a large number of contractile protein gene families that is not reflective of future adult myofiber types (34). As myofibers mature, this embryonic or immature isoform profile is replaced by the adult isoform pattern. This replacement occurs presumably as a result of genetic programming and in response to environmental cues and functional demands. The changes in expression of many of these contractile protein genes during early muscle development and differentiation have been well documented (Ref. 19, reviewed in Refs. 30 and 33). However, the subsequent maturation-based patterns of gene expression in the postnatal period have not been so well established.
The postnatal period represents a phase of development that results in a spectrum of specialized muscle phenotypes with functional differences. Physiological changes that take place during postnatal maturation of skeletal muscle include significant fiber growth, motoneuron synapse elimination, the establishment of adult fiber type, and changes in hormonal environments. The postnatal growth of rodent muscles is due primarily to the growth of existing fibers (24). Mononucleated muscle precursor cells, or satellite cells, are responsible for providing additional myonuclei to enlarging fibers. During periods of muscle growth, the relative number of satellite cells decreases (10). There is a tremendous increase in the number of myofilaments per fiber postnatally. This is due to increases in both fiber diameter and the relative contribution of the contractile filaments to the total fiber volume (reviewed in Ref. 31).
The relationship between motoneuron and muscle fiber is established postnatally. The adult muscle has a single axon per fiber; however, at birth, muscles exhibit polyaxonal innervation. Therefore, maturation involves a mechanism whereby the number of axons per fiber is reduced. In the mouse, this occurs in the first 2 wk of postnatal life (8). The activity imposed by the motoneuron plays a significant role in determining the physiological and biochemical properties of the muscle (25). Innervation can play an instructive role in determining the muscle fiber types. Adult skeletal muscle fibers are broadly divided into fast-twitch (types 2A, 2B, or 2X) or slow-twitch (type 1) fibers, which are established in the perinatal period (reviewed in Ref. 30). These fibers are characterized by their speed of contraction and their metabolic activities. The nerve also influences the transitions of muscle-specific proteins during postnatal maturation (reviewed in Ref. 30).
The myogenic potential of an individual muscle fiber is affected by the
hormonal status. Thyroid hormone influences the acquisition of the
mature muscle phenotype and appears to accelerate the normal maturation
process. Thyroid hormone affects the expression of many contractile
protein genes postnatally, including the adult fast myosin heavy chain
(MHC) isoforms in skeletal muscle (Ref. 6, reviewed in
Ref. 30) and -sarcomeric actin in both striated muscle types (38).
The responsiveness of the contractile protein genes to thyroid hormone
also changes with age. Hence, muscle maturation involves the complex
interaction of environmental influences on the newly forming fibers.
Of the gene families that encode the thick and thin filaments of the
sarcomere, MHC has been studied in the most detail during postnatal
maturation. In rodent hindlimb muscles, the embryonic and neonatal MHC
isoforms in fetal fibers are replaced by one of the four adult MHC
isoforms, 
/slow, 2A, 2X, or 2B (reviewed in Ref. 30). The age when
the adult fast myosin phenotype is achieved varies from 30 days for the
mouse longissimus muscle (6) to 115 days for the rat diaphragm (17). In
contrast, the expression of the mature isoforms of other contractile
gene families appears to be relatively simple and rapid after birth. For example, the -sarcomeric actin gene family consists of two isoforms, -cardiac actin (-CA) and -skeletal actin (-SA), which are coexpressed in striated muscle (9, 19, 29). -CA is the
predominant isoform during early myogenesis but is replaced by -SA
as the predominant isoform after birth. In cardiac muscle, -CA is
the predominant isoform throughout development in the rodent. For the
-sarcomeric actin genes, the mature phenotype appears to be achieved
by the first few days after birth.
In this study, we establish the detailed expression pattern of the
-sarcomeric actins, -CA and -SA, in the quadriceps muscle and
the heart during postnatal development of the B6D2 strain of mouse. We
report novel increases in the transcripts of the minor -sarcomeric
actin isoforms postnatally in both striated muscle types. By postnatal
day 21 in the quadriceps muscles,
-CA transcript levels are ~90% that found in the heart. By
postnatal day 42, -SA transcripts
comprise ~30% of the -sarcomeric actin transcripts in the heart.
We compare the postnatal expression profiles of the early developmental
isoforms from the myosin light chain
(MLC), troponin I
(TnI), and troponin T
(TnT) gene families, the adult fast
isoforms from MHC,
MLC,
TnI,
TnT, troponin C
(TnC), and tropomyosin gene
families, and desmin. We propose that the transient
increases in -CA in skeletal muscle and -SA in the heart may
represent a previously uncharacterized period of muscle maturation that
is likely to be regulated at least in part, by posttranscriptional
mechanisms.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Materials
The TRIzol reagent was obtained from Life Technologies (Mt. Waverley, Vic, Australia). Hybond N nylon membranes were from Amersham (North Ryde, NSW, Australia), and Immobilon membranes were from Millipore (Lane Cove, NSW). Radionucleotides were supplied by DuPont (North Ryde, NSW) and Gigaprime oligonucleotide labeling kits were from Bresatec (Adelaide, SA, Australia). Enzymes, acrylamide stocks, and disodium 3-phenyl phosphate (CSPD) were obtained from Boehringer Mannheim (Castle Hill, NSW). Monoclonal antibody (MAb) clone 5C5 was supplied by Sigma (Castle Hill, NSW).RNA Isolation
Postnatal samples were collected from B6D2 males (F1 progeny of C57BL/6J female × DBA/2J male matings) from day 0 (<24 h postbirth) until day 56 postpartum. The primary time course entailed collections on days 0, 5, 12, 15, 16/17, 21, 28, 35, 42, 56 and was utilized in the hybridizations of all probes.-Sarcomeric actin
expression was analyzed in a more detailed time course, including daily
time points between days 12 and
21. The final adult time point was taken at >12 wk of age and represents the adult phenotype. All litters were housed separately in light- and temperature-controlled quarters and provided with standard chow and water ad libitum. Litters
were weaned at approximately day 21.
Body weights were recorded when animals were killed. Postnatal skeletal
muscle samples consisted of entire quadriceps muscles except for the
day 0 sample, which included all the
hindlimb proximal to the hock with skin removed. Left and right
quadriceps were collected separately, frozen directly in liquid
N2, and stored at 
80°C.
Total heart samples included both atria and ventricles. All samples
were pooled, using 10 individuals at days
0 and 5, 4 individuals
at days 10-17, and 3 individuals
for all samples after day 17. Total
cellular RNA was isolated from hearts and right quadriceps muscle
homogenates using TRIzol reagent following the protocol supplied. RNA
was quantified by measuring absorbance at 260 nm. All left quadriceps samples were designated for protein estimation.
Northern Analysis
Total RNA (2-5 µg) was denatured and subjected to electrophoresis on 1% agarose gels containing 2.2 M formaldehyde except blots probed for total actin, which were run on 1.4% gels to allow the separation of muscle from nonmuscle transcripts. RNA was transferred to Hybond N nylon membrane as described by Sambrook et al. (28). DNA probes were labeled specifically or by the random-priming method using Gigaprime labeling kit with [32P]dCTP. Probes were then hybridized to RNA blots at 106 counts · min
1 · ml1
in a solution of 4× sodium chloride sodium citrate
(SSC), 50 mM
NaH2PO4
(pH 7.0), 5× Denhardt's solution, and 10% dextran sulfate (wt/vol) at 65°C for 16 h. All blots were washed
three times at 65°C in 0.5× SSC-0.1% sodium dodecyl sulfate
(SDS) for 20 min unless otherwise stated. Filters were exposed to
DuPont NEN reflection film for 1-14 days. To verify that
equivalent amounts of RNA were transferred, the blots were stripped
according to manufacturer's specifications and reprobed with an
end-labeled 18S rRNA probe under conditions of probe excess and then
washed with 4× SSC-0.1% SDS at 55°C. Autoradiographic
signals were quantitated with the Molecular Dynamics model 300 series
computing densitometer and the analysis program Imagequant. The
composition of the -sarcomeric actin mRNAs in the adult skeletal
muscle is ~95% -SA and 5% -CA, and the composition of the
newborn heart is ~95% -CA and 5% -SA (12). With the use of
the -sarcomeric actin probe (described in
-Sarcomeric actins),
the amount of -sarcomeric actin mRNA in the adult heart was
determined to be 13.2% of that found in the adult skeletal muscle in
this strain of mouse (data not shown). Using the above calculations, we
were able to compare the relative levels of -CA and -SA mRNAs in
both cardiac and skeletal muscle, respectively.
Protein Determination
Muscle samples were collected as described for the RNA assay. Left quadriceps were weighed and then solubilized by sonification in dithiothreitol (DTT) buffer [10 mM tris(hydroxymethyl)aminomethane (pH 7.6), 2% SDS, and 2 mM DTT]. Total protein levels for each sample were determined (20). Equivalent amounts of samples from the same time point were pooled, size fractionated by SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membrane (Immobilon). Total-sarcomeric
actin protein was detected by immunoblotting with MAb clone 5C5 (Sigma)
and CSPD. The Western blots were scanned by densitometry, and levels
were expressed as a percent of adult heart, which was set at 100%.
Silver-stained gels were run concurrently to check for equal loading.
DNA Probes
-CA.
A 97-base pair (bp) oligonucleotide corresponding to the 3'-UTR
of the mouse -CA actin sequence (supplied by J. Lessard, Univ.
Cincinnati) was synthesized. This was specifically labeled with a
primer corresponding to the last 20 bp of 3'-UTR and hybridized at 48°C in a 50% formamide solution and washed at 50°C in
0.1× SSC.
-SA.
A 132-bp oligonucleotide corresponding to the 3'-UTR of the mouse
-SA gene was synthesized and
specifically primed with a 20-bp oligonucleotide corresponding to the
final UTR sequences. This probe was shown to be mouse specific (data
not shown).
-Sarcomeric actins.
Total -sarcomeric actin mRNA levels were measured by using a 95-bp
oligonucleotide probe corresponding to the second exon of the coding
region of the human -sarcomeric actin genes. This probe contains
only two nucleotide mismatches between itself and each of the mouse
-CA and -SA mRNAs. This probe was washed under low-stringency
conditions. To confirm that this probe was detecting both
-sarcomeric actin transcripts, the -CA and -SA transcript values were summed at each time point and the values were compared directly with those obtained with this probe. Both sets of values reflected similar levels of -sarcomeric transcripts (data not shown).
Desmin. A probe was generated by polymerase chain reaction (PCR) amplification of rat soleus muscle cDNA to produce a 166-bp fragment corresponding to exons 7, 8, and 9 (32).
MHC. A 2.3-kilobase Hind III-BamH I restriction fragment from a human fast-twitch skeletal muscle MHC clone was used under low-stringency wash conditions to detect all fast isoforms. This clone was first described as MHC fast 2A (36), but subsequent findings indicate it encodes the human MHC fast 2X isoform (P. Gunning, personal communication).
MLC. A Bal I-Taq I restriction fragment was used to detect MLC 1 slow a (MLC-1sa). A 155-bp PCR product from the 3'-UTR was used for MLC 1/3 fast (MLC-1/3f) (33).
Tropomyosin.
A probe corresponding to exon 9a of the rat -tropomyosin gene and to
exon 9b of the rat fast -tropomyosin (11) was used under standard
conditions.
Troponins. TROPONIN I. For fast TnI (TnIf), a 200-bp Bgl I-Rsa I restriction fragment was used that contains amino acids 11-78 of the protein-coding region of the human cDNA. For slow TnI (TnIs), a 250-bp Pst I restriction fragment of the human cDNA was used that consists of the 5'-UTR and a short region of proximal protein-coding sequence. Troponin I probes have been described in Sutherland et al. (33).
TROPONIN T. For cardiac TnT (TnTc), a 252-bp Pvu II-Sma I restriction fragment from the human cDNA was used. Fast TnT (TnTf) was detected with a Pvu II fragment containing the coding and 3'-UTR regions. TnT probes were described in Sutherland et al. (33). TROPONIN C. For fast TnC (TnCf), a 177-bp Msa I restriction fragment containing coding and 3'-UTR sequences was used. Slow TnC (TnCs) was detected with a 260-bp Pst I-Bgl II fragment containing coding and 3'-UTR sequences. TnC probes were described by Sutherland et al. (33).| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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-CA Transcript Levels Increase in Postnatal
Skeletal Muscle
-Sarcomeric actin gene expression was determined by
Northern blot analysis using -CA and -SA isoform-specific probes.
Representative autoradiographs from the time course, presented in Fig.
1, show that both -CA and
-SA transcripts are coexpressed in postnatal quadriceps muscles. In
Fig. 1A, the level of -CA mRNA
from postnatal day 12 to
day 42 remains higher than that
detected in the newborn hindlimb. This postnatal increase in -CA
expression is unexpected as the replacement of -CA by -SA has
been reported to be complete within 4 days after birth (9). Indeed,
-CA levels between 5 and 11 days after birth and in the adult are
equivalent. -SA transcripts increased steadily after birth (Fig.
1B), in accordance with other
studies (9, 19). Quantitative densitometry was performed on the
autoradiographs and normalized to 18S ribosomal RNA levels as described
in MATERIALS AND METHODS. Both -CA
and -SA mRNA values in postnatal skeletal muscle are represented relative to total -sarcomeric actin mRNA in adult cardiac or skeletal muscle, respectively (Fig. 1,
C and
D). -CA expression initially
declines after birth and then rises rapidly at day
12 with a postnatal peak in transcript accumulation
occurring at day 21, reaching 88 ± 7% that of the level of -sarcomeric actin mRNA in the adult heart.
The very low levels of -CA, characteristic of the adult phenotype,
are not achieved until after postnatal day
42 (3.5 ± 0.2%). The -SA profile
shows a marked induction of expression by postnatal
day 5, reaching an adult maximum by day 17 (105 ± 5%) followed by an
~50% decrease in mRNA level by day
18 (62 ± 4%). The adult level is reached by
day 21 (111 ± 5%).
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What Are the Relative Levels of the -Sarcomeric
Actin Transcripts Postnatally?
-SA and -CA mRNAs to total
-sarcomeric actin transcript levels in skeletal muscle. Using a
probe that detects both -CA and -SA transcripts equally (see MATERIALS AND METHODS), we
determined the relative levels of total -sarcomeric actin
transcripts in adult skeletal muscle (data not shown). The
quadriceps-to-heart ratio was determined to be 7.6:1.0 in the adult
B6D2 mouse. In Fig. 2, the amount of -CA and -SA in skeletal
muscle is expressed as a percent of total -sarcomeric actin
transcripts in adult skeletal muscle. Figure 2 clearly shows that, although -CA
increases, -SA remains the predominant isoform in skeletal muscle
throughout the postnatal period. Relative to -SA, the maximal
contribution of -CA is 12 ± 0.9% that of total -sarcomeric
actin transcripts in skeletal muscle.
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-SA Transcript Levels Increase in Postnatal
Cardiac Muscle
-sarcomeric
actin transcript levels in the heart in concert with skeletal muscle.
The expression patterns of both -CA and -SA in postnatal heart
were analyzed over the same time period. Representative Northern blots
are shown in Fig. 3,
A and
B. The transcript levels are expressed
as a percent of the adult maximum. At all postnatal time points
analyzed, -CA remains the predominant isoform (Fig. 3C). -CA mRNA levels peak in the
heart at day 15 (140 ± 11% of total -sarcomeric actin transcripts in the adult heart) and then decline at day 17 (83 ± 15%) and
rise again by day 21 (114 ± 21%). This is in contrast to skeletal muscle, in which the peak of -CA transcript accumulation occurs at day
21 (Fig. 1).
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Unexpectedly, -SA transiently increases in the postnatal heart (Fig.
3C). -SA mRNA levels remain
<9.0% that of total -sarcomeric actin transcripts in the adult
heart until day 21 (13 ± 4%).
There is a significant rise in the -SA mRNA level detected from
day 28 to day
42 (25 ± 3 to 32 ± 9% that of the total
-sarcomeric actin transcripts in the adult heart). The true adult
phenotype is also not achieved in cardiac muscle until after
day 42, when -SA has declined (9.0 ± 0.1%). Thus there is a concomitant increase in the minor
-sarcomeric actin isoform in each of the striated muscles commencing
around postnatal day 21.
Expression of the -Sarcomeric Actins in Postnatal
Skeletal Muscle Is Regulated Primarily by Transcriptional Mechanisms
-sarcomeric actin probe to determine the postnatal profile of the
total -sarcomeric actin transcript pool (Fig.
4A). Transcript levels were calculated as a percent of those detected in adult skeletal
muscle and presented graphically in Fig.
4C. The results show that the
-sarcomeric actin transcripts accumulate rapidly from the newborn
time point to day 17, after which the
adult level is maintained. The sarcomeric actin mRNA level directly
reflects the level of the predominant -SA transcript in skeletal
muscle.
Total -sarcomeric actin protein was determined by
Western blot analysis on the skeletal muscle samples using an antibody that recognizes both -CA and -SA (Fig.
4B). Total -sarcomeric actin
protein accumulates steadily from postnatal day
0 to day 17, after
which the maximal adult output is maintained (Fig.
4C). There is remarkable similarity
between the mRNA and protein accumulation profiles, with the exception
of the peak in day 17 transcript levels, which is not reflected in the protein data (Fig.
4C). The data suggest that this gene
family is regulated primarily by transcriptional
mechanisms.
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Increase in -CA Transcripts in Postnatal Skeletal
Muscle Does Not Reflect the Reemergence of an Immature Phenotype
-CA is the major
-sarcomeric actin isoform expressed in the immature fiber (3, 29).
It is reasonable to propose that the increase in the -CA transcript
level postnatally in skeletal muscle may reflect the reemergence of the
immature phenotype. To test this hypothesis, we assayed for the
expression of three other contractile protein genes that are
characteristic of the immature expression profile: MLC-1sa, TnIs, and
TnTc (Fig.
5A).
Figure 5B shows the transcript levels
of these isoforms expressed as a percent of the level in the adult
muscle in which each is expressed maximally. Clearly, the levels of
these isoforms decrease as skeletal muscle matures. MLC-1sa transcripts
can still be detected up to postnatal day
17 (14 ± 6.3%); however, the level declines
steadily from birth. The lack of increase in expression of these
isoforms between days 12 and
21 indicates that the increase in
-CA during this postnatal period does not reflect the reemergence of
an immature myofiber phenotype.
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Contractile Protein Adult Fast Isoforms Are Upregulated
Concomitantly With the Increase of -CA in the
Quadriceps Muscles
-CA transcript level in postnatal skeletal muscle
may signal a phase of maturation in which physiological demands require
a general increase in protein output. To address this possibility, we
assayed the induction of the adult isoforms from four other
myofibrillar gene families (tropomyosin, troponin, MLC, and
MHC). The portion of the quadriceps
muscles used in this study consists predominately of fast-twitch fibers
in the adult. Thus, during the postnatal period, there is an
upregulation of the fast isoform from each contractile protein gene
family. Figure 6 shows the fast isoform
expression pattern from these gene families. The mean mRNA level for
each isoform is graphed as a percent of the level detected in the adult
quadriceps, which was set at 100%. All the adult fast isoforms of each
contractile gene family examined accumulate after birth. However, there
are differences in the rate of transcript accumulation between gene
families. The fast isoforms of the MLC
families, MLC-1/3f and MLC-2f, rapidly increase and exceed their adult
maximum by postnatal day 12. Fast
-Tropomyosin and -tropomyosin levels reach their adult maximum
around days 15-17. In contrast,
the profiles of the three troponin transcripts, TnTf, TnIf, and TnCf,
show a more gradual increase from days
0 to 56, with the
level at day 17 equal to ~90, 80, and 60% of that of the adult maximum, respectively. All adult isoforms
are expressed at approximately 
50% that of their adult maximum when
-CA increases at day 12.
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A Maturation Cue Initiates a Common Response in Three Gene Families
The notable exceptions to the above maturation expression profiles are the MHC gene family and desmin (Fig. 6). Fast MHC transcripts accumulate rapidly by postnatal day 12 (57 ± 6% that of the adult quadriceps), after which there is a peak in expression at day 17 (93 ± 4%) followed by a decline at day 18 (53 ± 10%). The adult maximum is achieved after day 42. The expression profile of the intermediate filament protein, desmin, also shows a significant peak at day 17 (85 ± 5%), followed by a decline at day 18 (41 ± 2%). Likewise, the adult maximum is not achieved until after day 42. This expression profile notably is similar to that of the-SA isoform (Fig. 1D).
Hence, the -SA, fast
MHC, and desmin genes appear to
respond in a similar manner to a maturation agent(s) between postnatal
days 17 and
21.
Increase in -CA Signals a Period of Muscle Growth
-CA. We measured total body and quadriceps muscle weight of the B6D2
mice during maturation. Total body weight increased over 20-fold during
the 56-day experimental period (data not shown). This is in agreement
with growth studies in the postnatal rat (4). A period of rapid growth
between days 14 and
21 has been identified in the rat
quadriceps muscles. To determine if there was a similar phase of muscle
hypertrophy in the mouse quadriceps, we determined the
quadriceps-to-body weight ratio over the maturation period (Fig.
7). Interestingly, the quadriceps-to-body
weight ratio changes as these muscles mature and shows its most rapid increase between days 12 and
15 in the mouse. These data show that
the quadriceps muscles undergo the most rapid growth during the period
when the -CA transcript level increases.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We describe a novel expression profile of the -sarcomeric actin
genes during postnatal striated muscle maturation in the mouse. The
transcript level for -CA, the predominant isoform in embryonic
skeletal and cardiac muscle, transiently increases in the quadriceps
muscle between days 12 and
42 after birth. -SA, the
predominant isoform in adult skeletal muscle, increases in the heart
between days 21 and
42. Contrary to published findings, the adult levels of these isoforms in the striated muscles are not
achieved until after 42 days postbirth. We propose that this postnatal
period indicated by the induction of the minor -sarcomeric actin
isoforms may represent an as yet uncharacterized period of muscle
maturation.
The significant increase in the size of the quadriceps muscles during
the postnatal period could signify myofiber growth by several
mechanisms. The fusion of muscle precursor cells to existing myofibers
and/or a general increase in transcriptional output from the
contractile protein gene families to support the need for rapid
sarcomere accumulation could result in the increase in -CA. Newly
formed and immature myofibers display a distinct expression profile of
many gene families that comprise the thick and thin filaments of the
sarcomere (34). -CA is the predominant -sarcomeric actin isoform
expressed in immature myofibers (3, 29). Therefore, if the immature
phenotype is being recapitulated, one might expect the concomitant
induction of signature isoforms such as MLC-1sa, TnIs, and TnTc.
However, these transcripts do not increase postnatally, suggesting that
the postnatal requirement for -CA is separate from its role during
muscle differentiation.
Modulations in -sarcomeric actin gene expression during maturation
may reflect alterations in muscle activity. The predominant behavior of
the neonate in the first 2 wk of life is quiescent, consisting of
activities such as sleeping and nursing. By 21 days after birth, the
pups are highly coordinated, exhibiting rapid motile and propulsive
movements such as scurrying and jumping. This highly active phase
declines with further maturity. Electromyographic studies indicate that
the masseter muscle phenotype changes concomitantly with the
disappearance of the neonatal MHC transcripts, when patterns of
activity change from sucking to biting around 21 days after birth in
the rat (21). Contraction and stretch have been correlated with
increased rates of desmin transcription in cardiac muscle (37). The
significant increase in desmin mRNA accumulation in postnatal
quadriceps muscles may reflect this increased period of activity.
Contractile-responsive DNA elements such as those identified recently
in -MHC (23) and in -SA (5) may play a role in the regulation of
these genes during this postnatal period.
The quadriceps muscles of the mouse are predominantly fast-twitch
muscles with a small contribution of slow-twitch fibers provided by the
vastus intermedius portion (14). The increased expression of the
-CA gene may occur equally in all
fibers within the muscles or it could occur in a selective subset of
fibers such as the slow-twitch fiber component. Alternatively,
-CA expression may be restricted to
a specific region within the myofiber where myofibrillogenesis occurs.
During growth, new myofibrils assemble in the myotendinous regions at
the ends of the myofiber. Slow MHC is preferentially expressed in this
region during the addition of sarcomeres in response to stretch (7). It
is possible that mRNA accumulation localized to the site of
myofibrillogenesis may allow for rapid fiber growth during the
maturation phase.
There is a synchronous and rapid increase in -SA, adult fast MHC,
and desmin transcripts around postnatal day
17 in skeletal muscle. In addition, we find that -CA
transcripts decline in the heart at day
17 after birth. A maturation signal that is capable of
inducing a response in both striated muscle types is likely to be
provided systemically. Modulations in circulating hormones may provide
the postnatal trigger for the induction of genes required for the
maturation phase in striated muscle. Thyroid hormone is known to
influence the acquisition of the adult phenotype within the first few
weeks after birth. The transition from neonatal to adult MHC isoform
expression in rodent fast-twitch fibers is influenced by thyroid
hormone (Ref. 18, reviewed in Ref. 30). The same
MHC gene can be regulated
differentially by thyroid hormone in different muscles (13, 16). In
postnatal skeletal muscle, hypothyroidism results in the delayed
repression of the -CA gene and the
augmentation of -SA transcript levels (2).
Thyroid hormone acts through nuclear receptors that repress or activate
the transcription of genes by binding to thyroid-responsive elements
(TREs). Putative TREs have been identified and characterized in the
proximal promoter of the human -SA
gene (22) and in the first intron of the rodent
-SA gene (2). The
-CA gene also has a less
well-conserved consensus sequence within the second intron (2). In
addition, thyroid hormone responses can be mediated by other, as yet
unidentified, regulatory mechanisms in addition to the TREs (27).
Increases in thyroid hormone plasma concentration, which occur
postnatally at days 16 and
22 in the mouse (6), correlate well
with the peaks in -SA transcript accumulation (days
17 and 21). Because
thyroid hormone is capable of inducing antithetical responses, thyroid
hormone may in part be the trigger for the concomitant up- and
downregulation of the -SA and
-CA genes, respectively, at
day 17. Alternatively, thyroid hormone
effects may be mediated indirectly. Growth hormone is a major regulator of proliferative aspects of muscle growth. Serum levels of growth hormone are high in the neonatal rodent and decline with age (26). Growth hormone promoter activity is subject to thyroidal control (reviewed in Ref. 35).
-SA induction in the postnatal heart constitutes a significant
portion of the total -sarcomeric actin mRNA content (32% that of
-sarcomeric actin mRNA in the adult heart). The inclusion of -SA
in the cardiac sarcomere may provide a functional adaptation that
better suits a period of growth and increased activity. Increased levels of -SA mRNA in the hearts of the BALB/c strain of mice are
associated with increased contractility of the myocardium (15).
Alternatively, -SA may be expressed because the -CA output alone
may be insufficient in meeting the increased demand for -sarcomeric
protein during this phase of heart growth.
Our studies indicate that transcriptional mechanisms are primarily
involved in the regulation of the -sarcomeric actins after birth. In
addition, there may be mechanisms in play around day 17 that alter mRNA stability and/or rates of
decay that could account for the sharp decline in transcript pool size
detected in three genes, -SA,
MHC, and desmin. Posttranscriptional
mechanisms have been demonstrated in the regulation of the
-sarcomeric actins in other studies (1, 9). Variation in -CA
transcript levels in the adult heart between several mouse lines is not
reflected at the protein level. Similarly, normal levels of
-sarcomeric actin protein are present in the BALB/c heart despite a
fivefold reduction in transcript levels.
We demonstrate that the timing for each individual contractile protein
gene family to achieve its final adult phenotype varies between gene
families. For example, four adult fast isoforms (MLC-1/3f, MLC-2f, fast
-tropomyosin, and -tropomyosin) rapidly achieve mRNA levels that
are greater than that of the adult quadriceps muscles by approximately
postnatal day 15. In contrast, -CA, desmin, and the adult fast isoforms from four other gene families (TnT,
TnC,
TnI, and
MHC) do not achieve their adult
transcript levels until after day 42.
This suggests that the acquisition of the adult phenotype in the
quadriceps muscles is uncoordinated with respect to contractile protein
gene expression. The mature expression profile of these gene families
is not achieved in the mouse until after 42 days postbirth. This
maturation period may represent the fine tuning of the contractile
protein genes in response to functional demand.
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ACKNOWLEDGEMENTS |
|---|
We thank Dr. Jim Lessard for providing the mouse -CA sequence data.
We thank Drs. Peter Gunning and Ron Weinberger for their helpful
discussions and for critical reading of this manuscript. We thank Prof.
Peter Rowe and members of the Hardeman laboratory for their
encouragement. We also thank the Animal Facility under the direction of
Dr. Luana Ferrara.
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FOOTNOTES |
|---|
This work was supported by grants from the National Health and Medical Research Council of Australia to E. C. Hardeman and by the Children's Medical Research Institute.
Present addresses: R. M. Arkell, MRC Clinical Sciences Centre, Royal Postgraduate Medical School, Hammersmith Hospital, London, W12 0NN, UK; T. Collins, Division of Veterinary and Biomedical Sciences, Murdoch University, Perth, WA 6150, Australia.
Address for reprint requests: E. C. Hardeman, The Children's Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia.
Received 2 April 1997; accepted in final form 11 August 1997.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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