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J Appl Physiol 101: 1546-1555, 2006. First published July 27, 2006; doi:10.1152/japplphysiol.00221.2006
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Mechanisms underlying myosin heavy chain expression during development of the rat diaphragm muscle

Paige C. Geiger,1 Jeffrey P. Bailey,1 Carlos B. Mantilla,1,2 Wen-Zhi Zhan,1 and Gary C. Sieck1,2

Departments of 1Physiology and Biomedical Engineering and 2Anesthesiology, Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 21 February 2006 ; accepted in final form 1 July 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
During early postnatal development in rat diaphragm muscle (Diam), significant transitions in myosin heavy chain (MHC) isoform expression occur that are associated with fiber growth and increased MHC protein. At present, there is no direct information regarding the transcriptional regulation of MHC isoform expression during postnatal Diam development. We hypothesized postnatal changes in MHC isoform mRNA expression are followed by concomitant changes in MHC protein expression. The Diam was removed at postnatal days 0, 14, 28, and 84 (adult). MHC mRNA expression was determined by real-time RT-PCR. MHC protein expression was determined by SDS-PAGE. There was a significant effect of postnatal age on MHC isoform mRNA and protein expression. At birth, the MHCNeo isoform accounted for 28% of MHC mRNA and 54% of total MHC protein. By postnatal day 14, MHCNeo mRNA and protein increased significantly, and both decreased significantly by day 28, consistent with transcriptional control of the expression of this developmental isoform. By postnatal day 28, there were minimal changes in mRNA expression for MHCSlow and MHC2X, yet protein expression increased significantly. MHC2A mRNA and protein expression did not change during this time. Thus changes in MHC protein expression did not follow (or parallel) changes in MHC mRNA for the adult MHC isoforms. The present findings indicate that changes in MHC expression in the developing rat Diam are not driven solely by changes in mRNA expression. Knowledge of isoform-specific MHC mRNA expression only yields predictive information on MHC protein expression for the MHCNeo isoform.

real-time reverse transcriptase-polymerase chain reaction; electrophoresis; myosin heavy chain gene regulation; muscle plasticity

IN THE RAT DIAPHRAGM MUSCLE (Diam), significant changes in myosin heavy chain (MHC) isoform expression occur during the first 4 wk of postnatal development (22–24, 38, 39, 41). Most fibers express protein for the MHCNeo isoform at birth (P-0), but the relative expression of this isoform gradually decreases until it disappears by postnatal day 28 (P-28). In contrast, MHC2X and MHC2B protein isoforms are not expressed in the rat Diam until at least the second postnatal week. Postnatal development of the rat Diam is also characterized by changes in muscle contractile properties and by significant growth of muscle fibers, most notably those expressing MHC2X and MHC2B (15, 22, 36, 38, 42, 43, 45, 47). Thus the relative MHC isoform composition changes during postnatal development of the Diam, and ultimately it determines Diam function. At the present time, very little is known about the underlying mechanisms regulating MHC phenotype expression.

Previous studies of postnatal development, both in limb muscles and the Diam, have focused exclusively on changes in the relative protein expression of the different MHC isoforms in the whole muscle (9, 11, 22–24, 30, 31, 33, 38, 39, 45). However, during postnatal development of the rat Diam, MHC content (measured by MHC mass per half-sarcomere volume and fiber cross-sectional area) increases markedly (15). This increase varies across fiber types (classified based on MHC isoform expression). At single Diam fibers, the developmental increase in MHC content is greater in fibers expressing MHC2X and/or MHC2B isoforms than in those expressing MHCSlow or MHC2A. Thus, during postnatal development, not only do Diam fibers increase in size but also they increase their MHC protein content. This substantial increase in total MHC protein expression across Diam fiber types is not evident if only the relative expression of MHC isoform is examined.

The period from birth to adulthood provides a unique and significant opportunity to examine the mechanisms underlying rapid muscle growth and phenotype transitions. In rat limb muscles, postnatal muscle phenotype is regulated by MHC gene expression either as a result from mechanical stretch induced by skeletal growth and/or increased muscle activity (18). At present, there is no direct information regarding the transcriptional regulation of MHC isoform expression during postnatal development of the rat Diam.

An increase in MHC protein of the rat Diam during postnatal development could be due to transcriptional, translational, and/or posttranslational mechanisms, which may vary depending on fiber type (10). Assuming transcriptional rate remains constant, an increase in mRNA expression would result in increased protein expression. For example, patterns of MHC isoform-specific mRNA and protein expression are very similar during postnatal development in pigs (25). We hypothesized that postnatal transitions in MHC isoform mRNA expression in the rat Diam are followed by concordant changes in MHC protein expression. To address this question, measurements of MHC mRNA and protein were performed using quantitative real-time RT-PCR and SDS-PAGE, respectively, at postnatal days 0, 14 (P-14), and 28 and in the adult [postnatal day 84 (P-84)] rat Diam.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Experiments were performed on male Sprague-Dawley rats at P-0, P-14, P-28, and P-84 adults. Pregnant mothers were obtained at 14 days of gestation, and pups were randomly assigned to one of the three developmental time points at birth. Up until postnatal day 21, pups were housed with their lactating mothers at which time they were weaned and placed in separate cages under a 12:12-h light-dark cycle. Adult rats and postweaning pups were fed with Purina rat chow and provided with water ad libitum. Body weights were monitored daily in all groups. Different animals were used for mRNA and protein measurements at each developmental time point. The Institutional Animal Care and Use Committee of the Mayo Clinic approved all procedures.

MHC Isoform mRNA Expression

RNA extraction.   The whole Diam was excised in all groups except in the adult where only the right costal Diam was obtained (n = 6 for each age group). The Diam samples were rapidly frozen in liquid nitrogen and stored at –80°C until analyzed. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. RNA concentration was determined spectrophotometrically at a wavelength of 260 nm. To assess RNA integrity, the ratio of 260- and 280-nm measurements was obtained and verified to be in the range of 1.8–2.1. Before real-time RT-PCR, microfluidic technology using the BioAnalyzer 2100 (Agilent Technologies, Palo Alto, CA) was used to confirm quantitation and integrity results, in accordance with the manufacturer's protocol.

Quantitation of MHC mRNA.   To quantify mRNA amounts across Diam samples and MHC isoforms, a real-time RT-PCR technique was developed using a synthetic mimic as an internal control where the mimic spans a fragment of the gene target and contains the same primer pair for real-time PCR.

CONSTRUCTION OF REAL-TIME RT-PCR INTERNAL CONTROL.   PCR primers were designed for real-time RT-PCR using LightCycler Probe Design software (version 1.0, Roche Molecular). It was not possible, however, to design reliable primer pairs for the MHC2B isoform suitable for real-time RT-PCR using a mimic internal control. This isoform is usually expressed at low levels and rarely is singly expressed (15, 37), and thus it is unlikely to produce a measurable impact on conclusions regarding MHC transitions during postnatal development. To document any relative change in MHC2B mRNA expression across developmental time points, Northern blot analyses of MHC2B mRNA were conducted, as previously described (13).

Mimic internal control DNA was created for each MHC isoform by oligonucleotide overlap extension and amplification by PCR with the respective mimic or MHC composite primer pairs (Table 1). The PCR product was then agar purified to eliminate any nonspecific PCR product. The purified mimic product was incubated with 5 units of Taq polymerase and 1 pmol of dATP at 72°C for 15 min to create a 3'(A) overhang, cloned into the pCR-4 TOPO vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen), and transformed into bacteria. Correct insertion of the mimic DNA was confirmed by sequencing performed at the Molecular Biology Core Facility, Mayo Clinic. Mimic internal control cRNA was generated by in vitro transcription using the MAXIscript T3/T7 transcription kit (Ambion) with linearized vector DNA as template. Either T7 RNA polymerase (for the MHC2A isoform) or T3 RNA polymerase (for the other MHC isoforms) was used to drive in vitro transcription as appropriate. Isoform-specific mimic cRNA was then treated with 10 units of RNase-free DNase I (Roche), purified through a NucAway spin column (Ambion), and quantitated spectrophotometrically. Tenfold serial dilutions of mimic cRNA were then used in real-time RT-PCR.


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  		Table 1. Primers used for amplification of MHC isoform mRNA in rat diaphragm muscle and synthetic internal control construction

 
REVERSE TRANSCRIPTION.   Total Diam RNA (1.0 µg) and titrated amounts of mimic cRNA were reverse transcribed using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen) following the kit protocol. Briefly, sample RNA, 0.5 µl of oligo(dT) (0.5 µg/µl), 0.5 µl of random primers (50 ng/µl), 1 µl of 10 mM dNTP, and RNase-free sterile water to 10 µl were incubated at 65°C for 5 min and chilled on ice for 1 min to denature RNA. A mix with 2 µl of 10x RT buffer, 4 µl of 25 mM MgCl2, 1 µl of RNaseOUT RNase inhibitor, and 2 µl of 0.1 M dithiothreitol was then added to each sample and incubated at room temperature for 2 min. After this, 50 units of SuperScript II RT were added to each sample. Samples were then incubated 10 min at room temperature, 50 min at 42°C, 15 min at 70°C, and placed on ice. All reverse transcription reactions were done in triplicate.

REAL-TIME PCR.   For gene expression analysis, 2 µl of the reverse transcription reaction was added to a reaction mix containing 1x SYBRgreen I Master (Roche Molecular), 2.0 mM MgCl2, and 0.5 µM of the respective primer pair (5' and 3' primers in Table 1). Amplification and quantitation of MHC isoform mRNA were performed on a LightCycler (Roche Molecular) with the following parameters: 10 min at 95°C followed by 33–37 cycles of 15 s at 95°C, 10 s at 58°C, and 24 s at 72°C. Fluorescence measurements were taken at the end of each cycle (product extension period). After amplification, a melting curve analysis was performed to verify amplification product specificity. Briefly, amplification products were denatured at 95°C, and then they were quickly cooled to 65°C for 15 s. Products were then heated at a rate of 0.1°C/s to a final temperature of 95°C while continuously measuring fluorescence. The derivative of the melting curve fluorescence was plotted vs. temperature, thus yielding melting peaks indicative of products generated during amplification (Fig. 1A). The lack of primer-dimer complexes and the presence of an appropriate single peak in the resultant melting curve were indicators of correct amplification products. These products were subsequently sequenced to confirm the nature of the amplified product. All PCR reactions were performed in duplicate for each reverse transcription product.


Figure 1
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  		Fig. 1. Representative real-time PCR melting and amplification curves for the different myosin heavy chain (MHC) isoforms demonstrating the method used for mRNA quantitation. A: melting curves of mimic internal control and diaphragm (Diam) MHC mRNA. Vertical lines, melting peak temperature for the MHC mimic (red) and Diam (green) amplification products. Note lack of nonspecific amplification and clear separation between the mimic and Diam MHC mRNA products. B: SYBRgreen I fluorescence during PCR amplification of mimic internal control and Diam MHC mRNA. The crossing point with the baseline fluorescence (threshold cycle) for each amplification product was calculated using the second-derivative method. A plot of mimic internal control concentration vs. crossing point (inset) was then used to determine the amount of unknown MHC in each sample. P-14, postnatal day 14; P-84, postnatal day 84 (adult).

 
QUANTITATIVE ANALYSES.   An amplification fluorescence plot was generated for each sample with LightCycler software (version 3.5.3, Roche Molecular) using the second-derivative method (Fig. 1B). From this plot, a threshold cycle value was calculated that corresponds to the cycle number at which fluorescence was detected above threshold. This threshold cycle value was used to calculate the initial amount of target cDNA. A linear relationship was obtained by plotting the threshold cycle and the logarithm of the mimic cDNA dilution used as an internal control for each MHC isoform. Comparison to the respective standard permitted determination of the MHC isoform mRNA concentration for each Diam sample.

MHC Isoform Protein Expression

Protein extraction.   In additional animals (n = 6 for each age group), the Diam was rapidly excised as described above. The Diam samples were rapidly frozen in isopentane cooled in liquid nitrogen and stored at –80°C until analyzed. Myosin protein was extracted as previously reported (13, 15). Briefly, muscle segments were minced in a high-salt solution (300 mM NaCl, 100 mM NaH2PO4, 50 mM Na2HPO4, 1 mM Na4P2O7, 10 mM EDTA, pH 6.5), and extracts were incubated at 4°C for 30 min (7). Extracts were then centrifuged and supernatants recovered. A 10-µl aliquot of supernatant was diluted (1:10) in a low-salt buffer consisting of 1 mM EDTA and 0.1% 2-mercaptoethanol (vol/vol) and stored overnight at 4°C to allow precipitation of myosin filaments. The filament solution was subsequently centrifuged to form a pellet, which was then dissolved in myosin sample buffer (500 mM CaCl2, 10 mM NaH2PO4), followed by a 1:200 dilution in SDS sample buffer [62.5 mM Tris, 2% SDS (wt/vol), 10% glycerol, 5% 2-mercaptoethanol (vol/vol), and 0.001% bromophenol blue (wt/vol at pH 6.8)]. The samples were boiled for 2 min and stored at –80°C.

Gel electrophoresis.   Different MHC protein isoforms were electrophoretically separated by SDS-PAGE. A modification of the procedure by Sugiura and Murakami (40) was used to prepare the gel. A 3.5% acrylamide concentration (pH 6.8) was used in the stacking gel, and the resolving gel (8 x 10 cm in size, 0.75 mm thick) consisted of a gradient of 5–8% acrylamide (pH 8.8) with 25% glycerol (vol/vol). Increasing volumes of a known concentration of purified rabbit myosin (Sigma, St. Louis, MO) were loaded on the gels [standard myosin concentrations verified with the Bradford method (5)]. All samples were run at a constant current of 20 mA/gel until the tracking dye reached the bottom of the gel (~1.75 h). After completion of the gel run, the gels were removed from the plates and silver-stained according to the procedure of Oakley et al. (29). All MHC isoforms were identified based on their migration pattern, and their relative expression was quantified by densitometry measurements on a high-resolution (300 dpi) digital scan (Microtek ScanMaker 5) of each gel. The brightness-area product of each rabbit myosin sample was determined from the area and average brightness of each densitometric band after subtraction of local background. A linear relationship between the brightness-area product and the myosin content across rabbit myosin samples was used to determine the myosin content in the individual rat Diam samples. This method has been previously described and validated (13, 14, 16). For each Diam sample, MHC content was normalized to the tissue weight.

Identification of MHC isoforms by migration patterns was verified by Western blot analysis as previously described (16, 17). Briefly, rat Diam bundles were run on SDS-PAGE and transferred to nitrocellulose overnight. The nitrocellulose sheet was divided into six sections, and one segment was stained with colloidal gold to ensure adequate protein transfer and visualize protein migration. One of the following mouse monoclonal or polyclonal antibodies was used to stain the five additional segments: anti-MHCNeo (IgG, Novocastra, Newcastle, UK); NCL (IgG, Novocastra), which reacts with MHCSlow; SC.71 (IgG, ATCC, Manassas, VA), which reacts with MHC2A; BF-F3 (IgM, Schiaffino), which reacts with MHC2B; and BF-35 (IgG, Schiaffino), which reacts with all isoforms but MHC2X. Isoform specificity of these antibodies has been previously determined (20, 34). The nitrocellulose segments were stained with a biotinylated secondary antibody specific to IgG or IgM as appropriate, and they were visualized with alkaline phosphatase (Vectastain ABC kit, Vector Laboratories, Burlingame, CA).

Statistical Analysis

All statistical analyses were performed using standard statistical software (JMP 6.0.0, SAS Institute, Cary, NC). MHC protein concentration and MHC mRNA concentration were compared across different MHC isoforms and across developmental and adult samples by two-way ANOVA with MHC isoform and age as grouping variables (F ratio values are reported for each comparison). When appropriate, least squares mean differences across MHC isoforms and age were compared post hoc using a Student's t-test (with Bonferroni correction), and specific t values are reported. Statistical significance was considered at a P value <0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
MHC mRNA Expression During Postnatal Development

On average, 0.57 ± 0.07 µg of total RNA/mg tissue was extracted from Diam across all developmental time points (Fig. 2). There was a nonstatistically significant trend toward a decrease in the amount of total RNA extracted from Diam samples with increasing postnatal age (F = 1.91; P = 0.16).


Figure 2
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  		Fig. 2. Total RNA (solid bars) and total MHC protein (open bars) in the postnatal rat Diam (amounts normalized to tissue weight). *Significant difference from adult values, P < 0.05. #Significant difference from P-14 values, P < 0.05.

 
Quantitation of MHC mRNA isoform expression.   The amount of MHCNeo, MHCSlow, MHC2A, and MHC2X mRNA was measured using real-time RT-PCR at P-0, P-14, and P-28 and in the adult. There were significant changes in the expression of isoform-specific MHC mRNA during the postnatal development of the Diam (F = 19.3; P < 0.0001). Figure 3 shows the amount of isoform-specific MHC mRNA throughout development, and Table 2 presents the relative amounts of each MHC isoform at each postnatal time point. Expression of MHC2B mRNA was determined from Northern blots (data not shown). Importantly, there was no significant change in the relative expression of this isoform throughout postnatal development.


Figure 3
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  		Fig. 3. MHC isoform-specific mRNA in the postnatal Diam determined by real-time RT-PCR at postnatal day 0 (P-0), P-14, postnatal day 28 (P-28), and adult. A: MHCNeo. B: MHCSlow. C: MHC2A. D: MHC2X. Note scale difference on y-axis for MHCNeo and MHC2A isoforms. Values are means ± SE. *Significant difference from adult values, P < 0.05. {dagger}Significant difference from P-28 values, P < 0.05. Significant difference from P-14 values, P < 0.05.

 

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  		Table 2. Postnatal changes in relative MHC mRNA expression in the rat diaphragm muscle

 
At P-0, the predominant mRNA isoform expressed was MHCSlow, accounting for ~40% of all MHC mRNA. Between P-0 and P-14, mRNA expression for the MHCNeo isoform increased significantly and at P-14, MHCNeo accounted for ~80% of all MHC mRNA. There was no significant change in the amount of MHCSlow, MHC2A or MHC2X isoform mRNA expressed. However, the expression of MHCSlow and MHC2A mRNA decreased relative to all MHC mRNA, with each of these adult isoforms, accounting for <10% of all MHC mRNA.

From P-14 to P-28, the amount of MHCNeo mRNA decreased significantly returning to near P-0 levels accounting for ~30% of all MHC mRNA. The amount of MHCSlow and MHC2X mRNA increased slightly, with each accounting for ~30% of all MHC mRNA expressed. The amount of MHC2A mRNA did not change during this period, even when expressed relative to all MHC mRNA expressed.

Between P-28 and adulthood, MHCNeo mRNA continued to decrease accounting for ~1% of all MHC mRNA expressed. The amount of mRNA for MHCSlow and MHC2X isoforms increased further (P < 0.05), with each isoform accounting for ~45% of all MHC mRNA in the adult Diam. The amount of MHC2A mRNA was again unchanged, accounting for 7% of all MHC mRNA.

MHC Protein Expression During Postnatal Development

Total MHC protein normalized by tissue weight changed during development (Fig. 2). The amount of total MHC protein peaked at P-14 (F = 5.38; P = 0.005). Total MHC protein levels in the adult (~3.7 µg MHC/g tissue) were consistent with previous results (13) and were similar to P-0 levels.

MHC protein expression.   Based on measurements of total MHC protein and the relative expression of each isoform, the amount of protein represented by each isoform was calculated at each time point. A significant change in MHC isoform expression was evident during postnatal development of the Diam (F = 7.60; P < 0.0001). Figure 4 shows the amount of isoform-specific MHC protein throughout development, and Table 3 presents the relative amounts of each MHC isoform at each postnatal time point.


Figure 4
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  		Fig. 4. MHC isoform-specific protein in the postnatal Diam calculated based on measurements of total MHC protein and the relative expression of each isoform in rat Diam at P-0, P-14, P-28, and adult. A: MHCNeo. B: MHCSlow. C: MHC2A. D: MHC2X. E: MHC2B. Values means ± SE. *Significant difference from adult values, P < 0.05. {dagger}Significant difference from P-28 values, P < 0.05. #Significant difference from P-14 values, P < 0.05.

 

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  		Table 3. Postnatal changes in relative MHC protein expression in the rat diaphragm muscle

 
At P-0, the predominant protein isoform was MHCNeo, accounting for ~50% of all MHC protein; MHC2X and MHC2B isoforms were not detectable. Between P-0 and P-14, MHCNeo showed a significant, but not proportionate, increase in protein expression, decreasing to ~30% of all MHC protein. Expression of the MHCSlow and MHC2A isoforms increased slightly, accounting for just under 20 and 30% of all MHC protein, respectively (a significant change in relative expression for each of these isoforms). Protein expression for MHC2X increased significantly, and accounted for ~20% of all MHC protein. Expression of MHCSlow and MHC2X protein isoforms peaked at P-28, accounting for over 20% and nearly 40% of all MHC protein, respectively. Expression of MHC2A isoform remained relatively unchanged. The P-28 time point was also marked by the disappearance of the MHCNeo protein isoform and by the appearance of the MHC2B protein isoform, which accounted for ~15% of MHC protein expressed.

The period from P-28 to adult was marked by the significant decrease in expression of the MHC2A protein isoform, accounting for ~25% of MHC protein. Expression of MHCSlow, MHC2X and MHC2B proteins decreased slightly, each accounting for 20–30% of MHC protein expressed.

Correlation Between MHC mRNA and Protein Expression During Postnatal Development

To assess any proportionate changes in MHC mRNA and protein levels, we calculated the ratio of MHC protein to mRNA at each developmental time point after normalizing for tissue weight (Fig. 5). At any one time point, there were considerable differences in the ratio of protein to mRNA across isoforms. In general, the protein expression was relatively greater for the MHCNeo and MHC2A isoforms (~50 µg of protein/pg of mRNA) compared with the MHCSlow and MHC2X isoforms (~10 µg of protein/pg of mRNA).


Figure 5
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  		Fig. 5. Spline curves of the ratio of average Diam protein and mRNA expression for each MHC isoform during postnatal development: MHCNeo (bullet), MHCSlow ({blacktriangledown}), MHC2A ({blacksquare}), and MHC2X ({blacklozenge}). The ratio of MHC protein to mRNA was normalized to the peak of each isoform to better visualize temporal patterns in the expression of each isoform. The slope of the spline describes the relative change in protein vs. mRNA such that a line of slope zero indicates a concordant change in protein and mRNA, a line of positive slope indicates a relatively greater increase in protein levels (or decrease in mRNA levels), and a line of negative slope indicates a relatively smaller increase in protein vs. mRNA.

 
Comparing the temporal expression profile of MHC isoform mRNA and protein in the developing rat Diam, it was evident that isoform-specific mRNA and protein levels did not necessarily show corresponding changes. To compare across isoforms, only relative changes in the protein-to-mRNA ratio were considered (normalized to the peak ratio for each isoform). In this analysis, when the change in MHC isoform protein expression parallels the change in mRNA, a line of slope zero is obtained. A greater increase in protein expression relative to the change in mRNA yields a line with a positive slope; conversely, a smaller change in protein expression relative to the change in mRNA yields a line with a negative slope.

Between P-0 and P-14, the MHC protein-to-mRNA ratio decreased for the MHCNeo and MHC2A isoforms, reflecting a greater increase in mRNA than in protein expression. In contrast, protein expression of the MHCSlow and MHC2X isoforms increased to a greater extent than mRNA. Beyond P-14, the lack of protein for the MHCNeo isoform causes the slope of the protein-to-mRNA ratio to approach zero. Between P-14 and P-28, the MHC protein-to-mRNA ratio for the MHCSlow and MHC2A isoforms has a positive slope, reflecting the greater increase in protein relative to mRNA levels. Beyond P-14, the slope of the protein to mRNA ratio for the MHC2X isoform was negative, reflecting the increasing mRNA levels despite unchanged protein levels. Between P-28 and adulthood, the slope of the protein-to-mRNA ratio for the MHCSlow and MHC2A isoforms was negative, reflecting an increase in mRNA with stable or decreasing protein levels. These comparisons highlight isoform-specific differences in the temporal pattern of MHC mRNA and protein expression.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The results of the present study demonstrate that during a period of rapid muscle growth and increase in MHC protein, changes in MHC mRNA expression are not concordant, particularly for the adult MHC isoforms. Indeed, the amount of MHC protein does not correspond to the mRNA levels across isoforms or over time. Thus MHC protein expression appears not to be solely under transcriptional control during postnatal development of the rat Diam. These are novel findings, suggesting important developmental differences in MHC isoform mRNA and protein expression.

Contradictory findings exist regarding the correlation between MHC isoform mRNA and protein expression in skeletal muscle. In pigs, the expression patterns of isoform-specific MHC mRNA and protein were very similar during postnatal development in the longissimus and rhomboideus muscles (predominantly composed of fibers expressing MHC2X and/or MHC2B vs. MHCSlow isoforms, respectively) (25). During early stages of development, parallel changes in MHC isoform mRNA and protein expression were also reported in the rabbit Diam (28) and several other skeletal muscles (26). These findings contrast with the present report during development of the rat Diam. Only the postnatal changes in MHCNeo protein expression were matched by changes in mRNA expression. Other studies have shown that the correlation between MHC mRNA and protein expression becomes weaker during development (2, 46) and in conditions associated with rapid transformations in fiber composition (3, 13, 35).

Concordant changes in mRNA and protein expression would suggest a role for transcription in the control of MHC protein expression. Thus it is possible that MHCNeo is under transcriptional control in the postnatal rat Diam. However, this is does not appear to be the case for other (adult) MHC isoforms in the rat. Although the amount of MHC protein increased throughout early development and peaked by P-28 for the MHCSlow and MHC2X isoforms, mRNA expression remained relatively constant up to P-28 and then increased into adulthood. There was little relation between the changes in mRNA and protein expression for the MHC2A isoform. Expression of the MHC2B mRNA isoform was evident early in development despite lack of protein expression, and mRNA expression remained relatively constant throughout postnatal development. The apparent inconsistency in MHC isoform-specific mRNA and protein expression may relate to species-, muscle-, or isoform-specific differences. Nevertheless, knowledge of isoform-specific MHC mRNA levels does not allow for prediction of the levels of protein expression during a period of rapid fiber growth of the rat Diam, emphasizing the need for comprehensive studies that examine MHC isoform-specific mRNA and protein levels.

The marked transitions in MHC isoform mRNA and/or protein expression observed during postnatal development of the rat Diam are in general agreement with the reports of several previous studies. However, most previous studies were not comprehensive, precluding analyses of the postnatal control of MHC isoform expression. Kelly and colleagues (23) reported MHC mRNA and protein for MHCNeo, MHC2A, and MHC2B; their results are limited by the fact that MHCSlow and MHC2X isoforms comprise over 50% of the total MHC protein in the adult Diam. The absence of information relating to postnatal changes in expression of these abundantly expressed MHC isoforms is a major deficiency. Changes in MHC isoform expression have been reported by electrophoretic separation of MHC proteins in the whole Diam and single fibers (22, 24, 38, 39) or analysis of Diam cross sections (24, 41). In a previous study on single rat Diam fibers (15), significant fiber growth was evident postnatally. The increase in MHC protein was greatest in fibers expressing MHC2X and/or MHC2B isoforms, in agreement with the results of the present study where MHC2X and MHC2B protein expression increased to a greater extent than MHC2A and MHCSlow in the whole rat Diam.

The increase in relative composition and total MHC protein content of the MHC2X and MHC2B isoforms likely contributes to the differing contractile properties of the postnatal rat Diam (15, 22, 36, 38, 42, 44, 45, 47). The neonatal Diam relies on shallow, rapid breaths, partly because of the highly compliant neonatal chest wall but also due to the inability of the neonatal Diam to generate sufficient force to accommodate deeper breaths. Throughout postnatal development, the chest wall becomes less compliant, and recruitment of fatigable, fast-twitch motor units innervating MHC2X and MHC2B isoforms is possible. These transitions in MHC isoform expression are consistent with the effect of stretch and muscle activity on fiber phenotype of rat limb muscles (18). The increase in relative protein expression of MHC2X and MHC2B isoforms, as well as increased MHC protein content in these fibers, allows for a wider range of functional requirements and motor behaviors in the adult rat Diam compared with the neonate.

The underlying mechanisms for postnatal transitions in MHC isoform expression are currently unknown. Several possibilities include the pattern of innervation or removal of nerve-derived trophic influences, changing hormonal levels (e.g., for thyroid or growth hormones), the degree of weight-bearing activity, and/or genetically determined developmental program(s). Whether innervation of skeletal muscles plays a critical role in the isoform transitions observed during postnatal development is controversial. From embryological day 18 (19) up until P-14, rat Diam innervation is polyneuronal (4, 12), and, therefore, MHC isoform expression in the rat Diam can be influenced by more than one motoneuron during the first 2 postnatal wk. Coincident with the completion of synapse elimination and the withdrawal of polyneuronal innervation, expression of MHC2X and MHC2B protein isoforms occurs, and their relative expression continues to increase through P-28 when the adult pattern of MHC isoform expression is fully established. This suggests a potential role for innervation in regulation of MHC isoform protein expression. However, previous studies have suggested that innervation is not a requirement for the ultimate expression of adult fast MHC protein isoforms (6, 8, 27, 32). Furthermore, during postnatal development of the rat Diam, removal of innervation with unilateral denervation only delayed expression of fast MHC protein isoforms and prolonged expression of the MHCNeo protein isoform (39). Therefore, changes in the pattern of innervation or removal of neural influence may modulate the timing of MHC isoform transitions during postnatal development, but these factors alone do not seem to control postnatal MHC isoform expression in skeletal muscle.

Regulatory processes other than transcriptional control likely contribute significantly to the overall transitions in composition of the rat Diam during early postnatal development, especially for the adult MHC isoforms. Although it appears that a number of factors can modulate postnatal MHC expression, MHC isoform transitions may ultimately depend on preprogrammed fiber type differences. Although changes in the innervation pattern, removal of innervation, and low thyroid levels may alter the timing of postnatal MHC isoform transitions (1, 21, 38), the MHC phenotypes present in the adult are ultimately expressed. Taken together, the results from this and previous studies indicate there are complex differential regulatory mechanisms that control MHC isoform expression in the rat Diam during postnatal development. Future studies will need to focus on the signaling pathways and underlying transcriptional and/or translational mechanisms governing MHC isoform-specific plasticity in skeletal muscle.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grants HL-34817 and HL-37680.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Rebecca Macken, Thomas Keller, and Tucker Johnson for assistance in these studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. C. Sieck, Dept. of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (e-mail: sieck.gary{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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