Reappearance of the minor alpha -sarcomeric actins in postnatal muscle

Teresa Collins1, Josephine E. Joya1, Ruth M. Arkell1, Vicki Ferguson2, and Edna C. Hardeman1

1 Muscle Development Unit and 2 Cell Biology Unit, The Children's Medical Research Institute, Westmead, New South Wales 2145, Australia

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The postnatal expression profiles of alpha -sarcomeric actin transcripts and protein are quantified in mouse striated muscles from birth to postnatal day 56 by Northern and Western blot analyses. alpha -Cardiac actin (alpha -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 alpha -CA is the alpha -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. alpha -Skeletal actin (alpha -SA) transcripts accumulate to ~32% of the total alpha -sarcomeric actin transcripts in the adult heart. Our study shows that 1) there is a simultaneous reappearance of alpha -CA and alpha -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 alpha -sarcomeric actins.

alpha -cardiac actin; alpha -skeletal actin; contractile protein gene families; heart and skeletal muscle; mouse; development

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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, beta /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 alpha -sarcomeric actin gene family consists of two isoforms, alpha -cardiac actin (alpha -CA) and alpha -skeletal actin (alpha -SA), which are coexpressed in striated muscle (9, 19, 29). alpha -CA is the predominant isoform during early myogenesis but is replaced by alpha -SA as the predominant isoform after birth. In cardiac muscle, alpha -CA is the predominant isoform throughout development in the rodent. For the alpha -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 alpha -sarcomeric actins, alpha -CA and alpha -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 alpha -sarcomeric actin isoforms postnatally in both striated muscle types. By postnatal day 21 in the quadriceps muscles, alpha -CA transcript levels are ~90% that found in the heart. By postnatal day 42, alpha -SA transcripts comprise ~30% of the alpha -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 alpha -CA in skeletal muscle and alpha -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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. alpha -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 · ml-1 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 alpha -sarcomeric actin mRNAs in the adult skeletal muscle is ~95% alpha -SA and 5% alpha -CA, and the composition of the newborn heart is ~95% alpha -CA and 5% alpha -SA (12). With the use of the alpha -sarcomeric actin probe (described in alpha -Sarcomeric actins), the amount of alpha -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 alpha -CA and alpha -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 alpha -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

alpha -CA. A 97-base pair (bp) oligonucleotide corresponding to the 3'-UTR of the mouse alpha -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.

alpha -SA. A 132-bp oligonucleotide corresponding to the 3'-UTR of the mouse alpha -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).

alpha -Sarcomeric actins. Total alpha -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 alpha -sarcomeric actin genes. This probe contains only two nucleotide mismatches between itself and each of the mouse alpha -CA and alpha -SA mRNAs. This probe was washed under low-stringency conditions. To confirm that this probe was detecting both alpha -sarcomeric actin transcripts, the alpha -CA and alpha -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 alpha -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 beta -tropomyosin gene and to exon 9b of the rat fast alpha -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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Abstract
Introduction
Materials & Methods
Results
Discussion
References

alpha -CA Transcript Levels Increase in Postnatal Skeletal Muscle

alpha -Sarcomeric actin gene expression was determined by Northern blot analysis using alpha -CA and alpha -SA isoform-specific probes. Representative autoradiographs from the time course, presented in Fig. 1, show that both alpha -CA and alpha -SA transcripts are coexpressed in postnatal quadriceps muscles. In Fig. 1A, the level of alpha -CA mRNA from postnatal day 12 to day 42 remains higher than that detected in the newborn hindlimb. This postnatal increase in alpha -CA expression is unexpected as the replacement of alpha -CA by alpha -SA has been reported to be complete within 4 days after birth (9). Indeed, alpha -CA levels between 5 and 11 days after birth and in the adult are equivalent. alpha -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 alpha -CA and alpha -SA mRNA values in postnatal skeletal muscle are represented relative to total alpha -sarcomeric actin mRNA in adult cardiac or skeletal muscle, respectively (Fig. 1, C and D). alpha -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 alpha -sarcomeric actin mRNA in the adult heart. The very low levels of alpha -CA, characteristic of the adult phenotype, are not achieved until after postnatal day 42 (3.5 ± 0.2%). The alpha -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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Fig. 1.   alpha -Sarcomeric actin expression in postnatal skeletal muscle. Total RNA (5 µg) was analyzed from quadriceps muscles from the time points shown and from adult heart and quadriceps muscles (>12 wk). mRNA was detected using alpha -cardiac actin (alpha -CA; A) and alpha -skeletal actin (alpha -SA; B) isoform-specific probes. Corrections were made for loading and transfer errors by 18S hybridization. Quantifications of alpha -CA (C) and alpha -SA (D) isoform expression in postnatal skeletal muscle are shown. Values are expressed as a percent of alpha -sarcomeric actin transcript level (%SarcActin) in the adult muscle where the isoform is maximally expressed. Adult value was set at 100%. alpha -CA expression in day 56 quadriceps muscles is equivalent to that of adult (data not shown). Data points represent averages of 3 Northern blots with SE shown. Error bars are not shown if they fall within symbols. Quad, quadriceps muscles.

What Are the Relative Levels of the alpha -Sarcomeric Actin Transcripts Postnatally?

We determined the contributions of alpha -SA and alpha -CA mRNAs to total alpha -sarcomeric actin transcript levels in skeletal muscle. Using a probe that detects both alpha -CA and alpha -SA transcripts equally (see MATERIALS AND METHODS), we determined the relative levels of total alpha -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 alpha -CA and alpha -SA in skeletal muscle is expressed as a percent of total alpha -sarcomeric actin transcripts in adult skeletal muscle. Figure 2 clearly shows that, although alpha -CA increases, alpha -SA remains the predominant isoform in skeletal muscle throughout the postnatal period. Relative to alpha -SA, the maximal contribution of alpha -CA is 12 ± 0.9% that of total alpha -sarcomeric actin transcripts in skeletal muscle.


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Fig. 2.   Relative contribution of alpha -CA and alpha -SA isoform mRNAs to total alpha -sarcomeric actin transcripts in skeletal muscle. Levels of alpha -CA and alpha -SA transcripts are expressed as a percent of alpha -sarcomeric actin transcript level in the adult quadriceps muscle (which was set at 100% as described in MATERIALS AND METHODS). Values represent means ± SE. Error bars are not shown if they fall within symbols.

alpha -SA Transcript Levels Increase in Postnatal Cardiac Muscle

We investigated whether there was any modulation in alpha -sarcomeric actin transcript levels in the heart in concert with skeletal muscle. The expression patterns of both alpha -CA and alpha -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, alpha -CA remains the predominant isoform (Fig. 3C). alpha -CA mRNA levels peak in the heart at day 15 (140 ± 11% of total alpha -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 alpha -CA transcript accumulation occurs at day 21 (Fig. 1).


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Fig. 3.   alpha -Sarcomeric actin expression in postnatal cardiac muscle. Total RNA (5 µg) was analyzed from whole hearts from the time points shown and from controls, adult heart, and quadriceps muscles. Northern blots were probed with alpha -CA (A) and alpha -SA (B) isoform-specific probes. Corrections were made for loading and transfer errors by 18S hybridization. C: alpha -CA and alpha -SA mRNA levels expressed as a percent of alpha -sarcomeric actin transcript level in the adult heart, which was set at 100%. Data points represent averages of 2 Northern blots with range shown. Error bars are not shown if they fall within symbols.

Unexpectedly, alpha -SA transiently increases in the postnatal heart (Fig. 3C). alpha -SA mRNA levels remain <9.0% that of total alpha -sarcomeric actin transcripts in the adult heart until day 21 (13 ± 4%). There is a significant rise in the alpha -SA mRNA level detected from day 28 to day 42 (25 ± 3 to 32 ± 9% that of the total alpha -sarcomeric actin transcripts in the adult heart). The true adult phenotype is also not achieved in cardiac muscle until after day 42, when alpha -SA has declined (9.0 ± 0.1%). Thus there is a concomitant increase in the minor alpha -sarcomeric actin isoform in each of the striated muscles commencing around postnatal day 21.

Expression of the alpha -Sarcomeric Actins in Postnatal Skeletal Muscle Is Regulated Primarily by Transcriptional Mechanisms

Quantitative Northern blot analysis was performed using the alpha -sarcomeric actin probe to determine the postnatal profile of the total alpha -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 alpha -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 alpha -SA transcript in skeletal muscle.

Total alpha -sarcomeric actin protein was determined by Western blot analysis on the skeletal muscle samples using an antibody that recognizes both alpha -CA and alpha -SA (Fig. 4B). Total alpha -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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Fig. 4.   Total alpha -sarcomeric actin transcript and protein levels in postnatal skeletal muscle. A: total RNA (2 µg) from quadriceps muscles from postnatal time points shown and from adult heart and quadriceps muscles was analyzed by Northern blot. TAs, total alpha -sarcomeric actin transcripts. Corrections were made for loading and transfer errors by 18S hybridization. B: total alpha -sarcomeric actin protein (1 µg) samples from postnatal and from control heart were analyzed by Western blot. Samples were collected on postnatal days shown and were size-fractionated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and probed with a monoclonal antibody to the alpha -sarcomeric actins (5C5). A silver-stained gel was used to normalize total protein loadings. Size markers (in kDa) run with gels are indicated (left). C: mRNA values are expressed as a percent of alpha -sarcomeric actin transcript level (SarcActin mRNA) in the adult quadriceps (quad), which was set at 100%. alpha -Sarcomeric actin protein values are expressed as a percent of alpha -sarcomeric actin protein (SarcActin protein) in the adult heart, which was set at 100%. mRNA and protein values represent means ± SE for 3 experiments. Error bars are not shown if they fall within symbols.

Increase in alpha -CA Transcripts in Postnatal Skeletal Muscle Does Not Reflect the Reemergence of an Immature Phenotype

Immature myofibers express a particular profile of isoforms from many of the contractile protein gene families (34). alpha -CA is the major alpha -sarcomeric actin isoform expressed in the immature fiber (3, 29). It is reasonable to propose that the increase in the alpha -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 alpha -CA during this postnatal period does not reflect the reemergence of an immature myofiber phenotype.


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Fig. 5.   Myosin light chain 1 slow a (MLC-1sa), slow Troponin I (TnIs), and cardiac troponin T (TnTc) mRNA expression profiles in postnatal skeletal muscle. A: total RNA (2-5 µg) samples from postnatal and control adult muscle were run in the lanes indicated. Adult muscle used as a control was that in which isoform was expressed maximally; MLC-1sa and TnIs were compared with adult soleus, and TnTc was compared with adult heart. Northern blots were probed with MLC-1sa, TnIs, and TnTc isoform-specific probes shown. Each blot was stripped and hybridized to an 18S rRNA probe to correct for loading and transfer errors, an example of which is shown. B: transcript level for each isoform is expressed as a percent of the level in the adult muscle in which it is expressed maximally. Adult level was set at 100%. Values represent means ± SE of 3 Northern blots for each gene family. Error bars are not shown if they fall within symbols.

Contractile Protein Adult Fast Isoforms Are Upregulated Concomitantly With the Increase of alpha -CA in the Quadriceps Muscles

The increase in alpha -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 alpha -Tropomyosin and beta -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 alpha -CA increases at day 12.


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Fig. 6.   Accumulation of adult fast isoforms of tropomyosin (alpha -Tmf and beta -Tm), troponin (TnT fast, TnC fast, and TnI fast), myosin light chain (MLC-1/3 fast and MLC-2 fast), and myosin heavy chain (MHC) gene families and desmin in postnatal skeletal muscle. Values for each plot are calculated from Northern blot analysis using isoform-specific probes as described in MATERIALS AND METHODS. Values are expressed as a percent of transcript level in adult quadriceps, which was set at 100%, and represent means of 3-4 Northern blots. Samples from postnatal days 0, 5, 12, 15, 16/17, 21, 28, 35, and 56 were analyzed with the following exceptions: day 42 was included for TnI fast; days 17, 18, 42 were included for MHC and desmin. Error bars are not shown if they fall within symbols.

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 alpha -SA isoform (Fig. 1D). Hence, the alpha -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 alpha -CA Signals a Period of Muscle Growth

Muscle hypertrophy has been correlated with an increased output of some myofibrillar protein genes (5). Therefore, we wanted to determine if there is a period of muscle growth associated with the reappearance of alpha -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 alpha -CA transcript level increases.


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Fig. 7.   Quadriceps-to-body weight ratio of postnatal skeletal muscle. Quadriceps muscles and total body weight (BW) were determined at postnatal days 0-56 as described in MATERIALS AND METHODS. Values are calculated from mean weights of 3-10 individuals at each time point. Vertical bars represent SE. Error bars are not shown if they fall within symbols.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We describe a novel expression profile of the alpha -sarcomeric actin genes during postnatal striated muscle maturation in the mouse. The transcript level for alpha -CA, the predominant isoform in embryonic skeletal and cardiac muscle, transiently increases in the quadriceps muscle between days 12 and 42 after birth. alpha -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 alpha -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 alpha -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). alpha -CA is the predominant alpha -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 alpha -CA is separate from its role during muscle differentiation.

Modulations in alpha -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 alpha -MHC (23) and in alpha -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 alpha -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, alpha -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 alpha -SA, adult fast MHC, and desmin transcripts around postnatal day 17 in skeletal muscle. In addition, we find that alpha -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 alpha -CA gene and the augmentation of alpha -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 alpha -SA gene (22) and in the first intron of the rodent alpha -SA gene (2). The alpha -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 alpha -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 alpha -SA and alpha -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).

alpha -SA induction in the postnatal heart constitutes a significant portion of the total alpha -sarcomeric actin mRNA content (32% that of alpha -sarcomeric actin mRNA in the adult heart). The inclusion of alpha -SA in the cardiac sarcomere may provide a functional adaptation that better suits a period of growth and increased activity. Increased levels of alpha -SA mRNA in the hearts of the BALB/c strain of mice are associated with increased contractility of the myocardium (15). Alternatively, alpha -SA may be expressed because the alpha -CA output alone may be insufficient in meeting the increased demand for alpha -sarcomeric protein during this phase of heart growth.

Our studies indicate that transcriptional mechanisms are primarily involved in the regulation of the alpha -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, alpha -SA, MHC, and desmin. Posttranscriptional mechanisms have been demonstrated in the regulation of the alpha -sarcomeric actins in other studies (1, 9). Variation in alpha -CA transcript levels in the adult heart between several mouse lines is not reflected at the protein level. Similarly, normal levels of alpha -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 alpha -tropomyosin, and beta -tropomyosin) rapidly achieve mRNA levels that are greater than that of the adult quadriceps muscles by approximately postnatal day 15. In contrast, alpha -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.

    ACKNOWLEDGEMENTS

We thank Dr. Jim Lessard for providing the mouse alpha -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.

    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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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