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Departments of 1Physiology and Biophysics and 2Anesthesiology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905
Submitted 19 September 2002 ; accepted in final form 26 March 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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innervation patterns; electrophoresis; myosin heavy chain gene regulation; neuromuscular control
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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300 g). The animals were assigned to either control (Ctl; n = 6)
or one of four Dnv groups (n = 6 each). Animals were kept under a
12:12-h light-dark cycle, fed with Purina rat chow, and provided with water ad
libitum while housed in separate cages. Body weights were monitored daily in
all groups. Aseptic conditions were maintained during all surgical procedures,
and recovery from surgery was carefully monitored. The Institutional Animal
Care and Use Committee of the Mayo Clinic approved all procedures. Unilateral Denervation
The procedure for unilateral Dnv has been previously described in detail
(27,
40,
63–65).
Briefly, an intramuscular injection of ketamine (60 mg/kg) and xylazine (2.5
mg/kg) was used to anesthetize the animals. At a point beneath the
sternomastoid muscle in the lower neck, the right phrenic nerve was exposed
and transected. To prevent reinnervation of the Diam and to
minimize neurotrophic effects emanating from the remaining nerve stump, a
portion (10–20 mm) of the distal end was removed. The wound was
subsequently sutured and treated with topical antibiotics. Periods of Dnv were
maintained for 1, 3, 7, or 14 days. At the end of the assigned Dnv period,
inactivity of the right Diam was verified by the absence of
electromyogram activity before muscle extraction.
Protein and mRNA Extraction
The right costal Diam was excised from the animal and rapidly frozen in isopentane cooled in liquid nitrogen. Myosin protein was extracted from the muscle segments by scissorsmincing in a high-salt solution (in mM: 300 NaCl, 100 NaH2PO4, 50 Na2HPO4, 1 Na4P2O7, 10 EDTA; pH 6.5) and incubated at 4°C for 30 min (8). Extracts were centrifuged and supernatants recovered. Ten microliters of supernatant were 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 dilution 1:200 in sodium dodecyl sulfate (SDS) sample buffer [62.5 mM Tris (hydroxymethyl) aminomethane hydrochloride, 2% (wt/vol) SDS, 10% glycerol, 5% (vol/vol) 2-mercaptoethanol, and 0.001% (wt/vol) bromophenol blue at pH 6.8]. Protein samples were then boiled for 2 min and stored at -80°C until further processing.
The right costal Diam was rapidly excised from the animal, and total RNA was extracted by using the TRIzol reagent (Invitrogen, Carlsbad, CA) following the company protocol. Total RNA concentration was determined spectrophotometrically at a wavelength of 260 nm.
Gel Electrophoresis
MHC isoforms were separated by SDS-PAGE gel electrophoresis. Gel
preparation was based on a modification of the procedure by Sugiura and
Murakami (55). 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% (vol/vol) glycerol. 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. (44). The
relative expression of different MHC isoforms was then quantified by
densitometry measurements on a high-resolution (300 dpi) digital scan of each
gel. The MHC content of the Diam sample was normalized to the
weight of the tissue extracted.
Identification of MHC isoforms by migration patterns was confirmed by Western blot analysis as previously described (20, 21). Briefly, rat Diam bundles were run on SDS-PAGE and transferred to nitrocellulose overnight at 1 Amp. The nitrocellulose sheet was divided into five sections, and one segment was stained with colloidal gold to ensure adequate protein transfer and visualize protein band migration. One of the following mouse monoclonal or polyclonal antibodies was used to stain the four additional segments: NCL (IgG, Novocastra, Newcastle, UK), which reacts with MHCSlow; SC.71 (IgG, ATCC, Manassas, VA), which reacts with MHC2A; BF-F3 (Schiaffino, IgM), which reacts with MHC2B; and BF-35 (Schiaffino, IgG), which reacts with all but the MHC2X isoform. Isoform specificity of these antibodies was previously determined (35, 49). The nitrocellulose segments were stained with a biotinylated secondary antibody specific to IgG (NCL, SC.71, BF-35) or IgM (BF-F3) and visualized with alkaline-phosphatase (Vectastain ABC kit, Vector Laboratories, Burlingame, CA).
To determine the MHC concentration in rat Diam samples, increasing volumes of a known concentration of purified rabbit myosin [Sigma Chemical, St. Louis, MO; concentrations verified with the Bradford method (5)] were loaded on polyacrylamide gels and separated by SDS-PAGE as described. Gels were silver stained, and a high-resolution scanner (Microtek ScanMaker 5) was used to image the gels. 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, or densitometric measurement of electrophoretic bands, and myosin content in the rabbit myosin samples was used to determine the myosin content in rat Diam samples. This method has been validated and previously described (19, 20).
Northern Blot Analysis
A total of 2.0 µg of total RNA was electrophoresed on a 1.0% agarose gel containing formaldehyde, transferred to a positively charged nylon membrane (Roche Molecular Biochemicals, Indianapolis, IN) in 20x 3 M sodium chloride, 0.3 M sodium citrate (SSC) and ultraviolet (UV) cross-linked. The membrane was hybridized with DIG-dUTP tailed oligonucleotide specific for each isoform by using the DIG Easy Hyb hybridization buffer (Roche Molecular Biochemicals) at 35°C overnight. The probes used to hybridize to the adult MHC isoforms were previously described (14), as was the probe used to detect 18S rRNA (4). The probes were labeled by using the DIG oligonucleotide tailing kit (Roche Molecular Biochemicals). The 18S probe was random prime labeled by using the DIG DNA Labeling and Detection (Roche Molecular Biochemicals). The membrane was twice washed in 2x SSC/0.1% SDS at the hybridization temperature for 15 min and then washed twice in 1x SSC/0.1% SDS at the hybridization temperature for 15 min. The signal was detected after the addition of the chemiluminescence substrate (CDP-Star, Roche Molecular Biochemicals) and exposure to Lumi-Film chemiluminescent detection film (Roche Molecular Biochemicals) at room temperature. The exposed film was scanned, and the digitized signal was quantified by using MetaMorph (Universal Imaging, Downingtown, PA) software after background subtraction. The MHC isoform signal was normalized to the 18S rRNA signal for each sample. To assess RNA integrity, before transfer the gels were visualized by use of UV transillumination of ethidium bromide-stained samples. The relative intensity of 28S to 18S bands and the lack of obvious smearing of rRNA bands were used as gross indicators of RNA integrity (12). In addition, any smearing or additional banding of MHC evident after Northern detection with the DIG-labeled oligo probes was used as an indicator of RNA degradation.
Statistical Analysis
MHC protein concentration and MHC mRNA concentration were compared across
different MHC isoforms and between control and Dnv fibers by two-way ANOVA
according to MHC isoform and Dnv group. A Student's t-test with
Bonferroni correction was used as a post hoc analysis to compare between fiber
types when appropriate. A power analysis was performed for each parameter to
determine the minimal change from Ctl values that could be detected by use of
the number of animals per experimental group (n = 6) at a 
level of 0.8. Statistical significance was indicated by a P value
< 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Relative MHC isoform composition. MHC isoform protein expression
was determined after separation by SDS-PAGE as shown in
Fig. 1A. The relative
expression of different MHC isoforms in the rat Diam changed
dramatically after unilateral Dnv (Table
1). On the basis of electrophoretic analysis, the relative
expression of MHCSlow (20%), MHC2A (25%), and
MHC2X (40%) after 1 day of Dnv did not change, whereas the
relative expression of MHC2B protein decreased significantly (to 15
± 1% of total MHC; P < 0.05). Between 1 and 3 days of Dnv,
the relative expression of all MHC isoforms remained constant. The relative
expression of MHCSlow increased between 3 and 7 days of Dnv,
whereas the relative expression of MHC2B decreased slightly. The
relative expression of MHC2A, MHC2X, and
MHC2B did not change between 7 and 14 days of Dnv. The greatest
increase in relative expression of MHCSlow occurred by 14 days of
Dnv when MHCSlow accounted for 31 ± 2% of the total MHC
protein (P < 0.05 relative to Ctl). On the other hand, the
relative expression of MHC2A (28 ± 1% of total MHC;
P < 0.05) and MHC2X (32 ± 2% of total MHC;
P < 0.05) was unchanged, whereas MHC2B decreased (to 9
± 1% of total MHC; P < 0.05) after 14 days of Dnv of the
rat Diam.
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MHC protein expression. After 1 day of Dnv, total MHC protein decreased significantly from 2.69 ± 0.34 to 1.60 ± 0.19 µg MHC protein/g tissue. Between 1 and 3 days of Dnv, total MHC protein decreased even further to 1.05 ± 0.11 µg MHC protein/g tissue at 3 days of Dnv. From 3 to 14 days of Dnv, total MHC protein remained relatively constant (1.17 ± 0.09 at 7 days and 0.98 ± 0.06 µg MHC protein/g tissue at 14 days). Total MHC protein was significantly lower than Ctl values at all Dnv time points. On the basis of measurements of total MHC protein and the relative expression of each isoform, the amount of protein represented by each isoform was calculated (Fig. 2). After only 1 day of Dnv, the amount of each isoform of MHC protein decreased across all isoforms, with the greatest decrease in MHC2B protein. Between 1 and 3 days of Dnv, MHC protein continued to decrease across all MHC isoforms. The absolute amount of MHCSlow and MHC2A protein increased slightly between 3 and 7 days of Dnv and remained stable through 14 days of Dnv. In contrast, the amount of MHC2X and MHC2B protein did not change between 3 and 7 days of Dnv and decreased slightly by 14 days of Dnv. Overall, the most dramatic changes in MHC protein expression occurred early (1 day after Dnv).
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MHC mRNA Expression After Unilateral Dnv of the Diam
Relative MHC isoform composition. MHC mRNA expression in the rat
Diam was determined from Northern blot analysis
(Fig. 1,
B–E). The relative expression of mRNA for
the different MHC isoforms in the rat Diam also displayed dramatic
changes after unilateral Dnv (Table
2). In contrast to the changes in relative MHC protein expression,
there were no significant changes in relative MHC mRNA expression until 7 days
of Dnv. By 7 days of Diam Dnv, MHC2A relative expression
increased to 30% of MHC mRNA, whereas the relative expression of
MHC2X decreased to 20%, and MHCSlow and
MHC2B remained unchanged. From 7 to 14 days of Dnv,
MHC2A mRNA relative expression decreased to 22 ± 6% of total
MHC mRNA, and although it remained higher than Ctl this difference was no
longer significant (P > 0.05). From 7 to 14 days of Dnv,
MHC2X mRNA relative expression remained relatively unchanged.
Interestingly, the relative expression of MHCSlow and
MHC2B mRNA remained relatively unchanged after Dnv, unlike the
relative protein expression of this isoform. After 14 days of Dnv of the rat
Diam, MHC2X decreased to 16 ± 4% (P <
0.05). In comparison, MHCSlow, MHC2A and
MHC2B relative mRNA expression were not different from Ctl values
(50 ± 8, 22 ± 6, and 11 ± 4% respectively; P
> 0.05) after 14 days of Diam Dnv.
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MHC mRNA expression. To assess changes in MHC mRNA expression from
Northern analysis, we measured the amount of mRNA for each MHC isoform by
normalizing for the amount of 18S rRNA present in each sample. The expression
of 18S rRNA was not altered by Dnv (data not shown). Overall, mRNA expression
changes showed greater variability than those observed for MHC protein levels.
After 1 day of Dnv, the amount of MHCSlow mRNA dramatically
decreased to 50% of Ctl levels (Fig.
3A). Although not significant, MHC2A,
MHC2X, and MHC2B mRNA expression increased slightly
after 1 day of Dnv. The most dramatic changes occurred after 3 days of Dnv,
when there was 75% reduction in mRNA expression for MHCSlow
and MHC2X isoforms, whereas MHC2A and MHC2B
mRNA decreased only slightly. There was very little change in MHC mRNA
expression from 3 to 14 days of Dnv.
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Correlation Between MHC Isoform Protein and mRNA Patterns After Unilateral Dnv of the Diam
MHC isoform protein and mRNA amounts in the denervated Diam did
not necessarily change in the same direction or with the same temporal
pattern. To assess any relative changes in MHC mRNA and protein levels, we
calculated the ratio of normalized amounts of MHC mRNA and protein present in
each sample by using arbitrary units. In Ctl rat Diam, the ratio of
MHC mRNA to protein was 23.4 for MHCSlow, 5.9 for MHC2A,
13.6 for MHC2X, and 2.9 for MHC2B. After 1 day of Dnv,
the ratio of MHCSlow and MHC2A mRNA to protein increased
slightly to 31.6 and 8.3, respectively. At the same time, the ratio of
MHC2X mRNA to protein increased approximately twofold (to 43.7),
and the ratio for MHC2B increased approximately fivefold (to 16.8).
Between 1 and 3 days of Dnv, there was a generalized reduction in the
mRNA-to-protein ratio for all MHC isoforms. The MHCSlow
mRNA-to-protein ratio decreased slightly to 21.07 (90% of Ctl), and
although the mRNA-to-protein ratio for MHC2A also decreased
slightly to 7.9, it remained 35% above the Ctl value. During this same
period, the mRNA-to-protein ratio also decreased for MHC2X and
MHC2B (to 26.9 and 5.4, respectively) but remained at levels
approximately twofold higher than Ctl. Between 3 and 7 days of Dnv, the
mRNA-to-protein ratio remained relatively constant for MHCSlow (at
22.7) and MHC2A (at 13.1, approximately twofold higher than Ctl),
decreased for MHC2X (to 9.3; 70% of Ctl), and increased for
MHC2B (to 10.9; approximately fourfold higher than Ctl). Between 7
and 14 days of Dnv, the mRNA-to-protein ratios for
MHCSlow,MHC2A, MHC2X, and MHC2B
remained relatively unchanged (at 22.7, 17.1, 8.8, and 18.2, respectively).
Thus, by 14 days of Dnv, the mRNA-to-protein ratio was near Ctl levels for
MHCSlow, and the ratio for MHC2X was only reduced
slightly. In contrast, the ratio of MHC mRNA to protein increased for
MHC2A and MHC2B compared with Ctl (approximately
threefold and sixfold, respectively). Considering the significant reductions
in MHC protein expression observed in MHC2A, MHC2X, and
MHC2B and the relatively unchanged expression of MHCSlow
protein, the ratio of MHC isoform-specific mRNA to protein suggests that mRNA
availability was not limiting to the continued synthesis of MHC proteins.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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In a previous study from our laboratory
(19), we reported similar
changes in MHC isoform expression after 14 days of Dnv. We found considerable
reductions in the maximum specific force across all isoforms. Specifically,
the maximum specific force of fibers expressing MHC2X was reduced
by 40% and in fibers expressing MHC2A and MHCSlow
was decreased by 20%. Dnv also reduced the MHC protein content in fibers
expressing MHC2X, with no effect on fibers expressing
MHC2A and MHCSlow. When normalized for MHC content per
half-sarcomere, force generated by Dnv fibers expressing MHC2X and
MHC2A was decreased compared with control fibers. These results
suggest the force per cross bridge is also affected by Dnv. Therefore, loss of
MHC protein leads to a decrease in muscle function.
Transitions in fiber type and corresponding alterations in MHC isoform
expression have been observed in response to altered activity levels. In
contrast to the intermittent activation pattern of limb muscles, the
Diam is continuously activated with a high duty cycle of
40–50% to support ventilation
(50). A number of studies have
suggested that activation history (total activity and activity pattern) exerts
a pronounced effect on muscle contractile and metabolic properties
(1,
45,
46,
54,
56,
58). For example, chronic
low-frequency stimulation of fast-twitch muscle results in a fast-to-slow
fiber-type transition (1,
45,
46,
56), whereas unloading of
slow-twitch muscle results in slow-to-fast fiber-type transitions
(45,
54,
58). The unique activation
pattern of the Diam may result in heightened susceptibility to
conditions of imposed inactivity. In this regard, the observed decrease in
MHCSlow and MHC2X mRNA after Dnv of Diam,
with little to no change in MHC2A and MHC2B mRNA, is
interesting compared with other studies in limb muscles. After Dnv of hindlimb
muscles, expression of MHC2A increases in the soleus, and
MHC2X and MHC2B mRNA increases in the soleus, adductor
longus, and gastrocnemius muscles
(33,
34,
39). In addition,
interpretation of the results of hindlimb studies is obscured by other factors
such as unloading and uncontrolled length changes, rather than Dnv and/or
inactivity per se.
Our results are consistent with a previous report of MHC mRNA expression
changes after Dnv of the Diam. Yang et al.
(61) reported relative
reductions in MHC mRNA across all MHC isoforms, except MHC2B, after
1 wk of Diam Dnv. When expressed as MHC isoform mRNA relative
to mean Ctl values, our results differed. We found larger decreases in
MHCSlow (70 vs. 50%), MHC2X (80 vs.
60%) and MHC2B (40 vs. 30%) mRNA, and a smaller
decrease in MHC2A (4 vs. 70%) mRNA. Despite these
relative differences, the trends were similar except for MHC2A
mRNA. We did not find any significant change in MHC isoform mRNA between 7 and
14 days of Dnv. Although Yang et al. studied Diam 8 days post-Dnv,
the different time point would not explain the different results. It is
possible that some of the discrepancy can be attributed to differences in
hybridization probes, conditions, and detection methodology. In agreement with
their findings, the reported changes in MHC mRNA cannot solely account for the
changes in MHC protein expression. Our results are also similar to those
reported in another study investigating the relative proportion of MHC mRNA
expressed in several muscle groups in adult rats, including Diam,
by use of RT-PCR (36).
Although we found similar relative isoform expression for MHC2X
(45%) and MHC2B (5%), a higher relative isoform
expression was observed for MHCSlow (40 vs. 20%) whereas
a lower relative isoform expression (10 vs. 30%) was found for
MHC2A.
Several studies have begun to examine the transcriptional, translational,
and posttranslational regulation of MHC expression
(2). Most of the information
currently available on MHC transcription concerns the transcriptional control
of type I () MHC of cardiac muscle (equivalent to MHCSlow in
skeletal muscle). The 5' flanking region of the MHCSlow gene
has been well described and is known to include at least three
cis-regulatory elements
(17,
29,
41). Regulatory
transcriptional activators or repressors can bind to these DNA sequences and
thus control the expression of MHC genes. The regulatory regions for
MHCSlow are conserved across genes for other myofilament proteins,
including troponin T and 
-actin, suggesting the existence of common
regulatory pathways in the expression of different muscle phenotypes. The
regulatory elements for other MHC isoforms are currently unknown, although it
is likely that common pathways also contribute to the expression of other MHC
isoforms. In the present study, although we did not specifically examine
transcriptional rates for the different MHC isoforms, we found time-dependent
and isoform-specific changes in MHC mRNA. However, the changes in MHC mRNA did
not parallel the changes in MHC protein. Interestingly, the slow-to-fast
fiber-type transition reported for rat soleus after hindlimb suspension (and
muscle unloading) was associated with a concomitant decrease in promoter
activity within a specific regulatory region of the MHCSlow gene
(22) and an increase in
MHC2B mRNA (30). In
addition, studies in the rat plantaris muscle (predominantly expressing
MHC2B and MHC2X) demonstrated an increase in the
MHCSlow promoter activity after muscle overload (by removal of the
antagonist muscles) (23). The
isoform-specific MHC promoter activity after Dnv has not been examined. It is
possible that examination of mixed muscles will provide differing results
compared with muscles of predominantly homogeneous fiber type composition and
different activation history.
There are currently no data available directly regarding MHC
isoform-specific protein synthesis or degradation rates after Dnv. However, it
is generally believed that Dnv-induced atrophy is the result of decreased
protein synthesis, as well as increased protein degradation
(24,
25,
32,
60). Key regulatory points in
MHC synthesis include initiation of translation mediated by eukaryotic
initiation factor 2 and ribosomal S6 kinase and protein elongation mediated by
eukaryotic elongation factor 2
(32,
43). In fact, increased
phosphorylation of p70 has been shown in conditions associated with muscle
hypertrophy and increased translational activity. The phosphorylation state of
p70 and elongation factors after Diam Dnv is unknown, but altered
phosphorylation could lead to decreased translational efficiency. In addition,
polyribosomal clustering and disruption of the translational machinery have
been implicated in conditions of muscle atrophy
(57). Turner and Garlick
(59) showed that, after
unilateral Dnv of the Diam, the rate of protein synthesis in the
Dnv hemidiaphragm increased by 3 days of Dnv and remained elevated by 10 days
of Dnv. However, the calculated rate of protein degradation increased to twice
that of Ctl during the same period, reflecting an overall shift in the balance
toward increased degradation rates after Dnv. Of note, the half-life of
myofibrillar myosin has been reported to be 7 days
(47), possibly providing a
means for a rapid shift in overall protein composition during conditions
associated with muscle atrophy. In a recent study, Bodine et al.
(3) identified ubiquitin
ligases implicated in skeletal muscle atrophy, the activity of which increased
concurrently with myofibrillar protein loss after Dnv. The actual contribution
of altered MHC protein synthesis and degradation to Diam atrophy
after Dnv remains to be explored.
Several possible underlying mechanisms might explain the differential effects of Dnv on MHC isoform expression, including altered mechanical load, Diam inactivity, and/or nerve-derived trophic influences. Previous studies have suggested that Diam adaptations after unilateral Dnv are due to the passive strain imposed by continued activation of the intact contralateral side (15, 62). In a previous study from our laboratory (63), sonomicrometry was used to measure passive strain imposed in the sternal and midcostal regions of the Dnv Diam. The orientation of muscle fibers in these two Diam regions is such that continued activation of the intact contralateral side would impose different length changes. Indeed, we found that passive strain imposed was different in these two Diam regions after Dnv. However, the morphological and contractile adaptations in these two Diam regions were similar after Dnv despite differences in passive strain. Furthermore, changes in muscle fiber length never exceeded optimal fiber length in either region; thus unilateral Diam Dnv resulted in minimal passive stress and mechanical load in the ipsilateral Diam. These results indicate that Dnv-induced adaptations of Diam fibers are not the result of passive mechanical strain. This conclusion is further supported by an assessment of the influence of unilateral Diam paralysis induced by cervical spinal cord hemisection at C2 (SH) (40, 64). Although the passive mechanical effects of unilateral Diam paralysis after SH and Dnv were comparable, SH resulted in little, if any, change in Diam fiber cross-sectional area, maximum specific force, or MHC isoform expression. Together, these results indicate that passive length changes and mechanical stress are not the main determinants of the morphological, contractile, or MHC isoform adaptations induced by unilateral Dnv.
Unilateral Dnv may elicit changes in MHC isoforms indirectly through the removal of trophic influences emanating from the motoneuron. For instance, Dnv-induced atrophy in the extensor digitorum longus muscle as well as protein content and fiber cross-sectional area were attenuated by the addition of nerve extracts (13). Specifically, Dnv resulted in greater atrophy of type IIb fibers (expressing the MHC2B isoform) than of type IIa fibers (expressing the MHC2A isoform). Addition of nerve extract attenuated the reduction in type IIb fiber cross-sectional area but had no effect on type IIa fibers (13). A differential trophic effect may explain the isoform-specific changes in MHC mRNA and protein expression after rat Diam Dnv observed in the present study. For example, neurotrophin-3 has been shown to preferentially recover axotomized extensor digitorum longus muscle, a predominantly fast muscle type, with no effect on the predominantly slow soleus muscle (52). Additionally, brain-derived neurotrophic factor and neurotrophin-4 have been shown to convert fast motoneurons into slow motoneurons (26, 42). Removal of these influences after Dnv would thus have a greater effect on MHC2X and MHC2B fiber morphology, contractile properties, and MHC isoform expression than on MHC2A or MHCSlow fibers. These possibilities remain to be explored.
In summary, results of this study reveal a selective effect of Dnv on MHC isoform expression in the rat Diam. The gradual increase in MHCSlow and MHC2A protein after a slight decrease with 1 day of Dnv most likely explains the transient hypertrophy of these isoforms after 2 wk of Dnv. Similarly, the greater decrease in MHC2X mRNA and protein expression could explain previous observations of atrophy and a loss in MHC content per half-sarcomere in fast MHC isoforms after unilateral Dnv. However, the nonconcordant patterns of MHC mRNA and protein expression at early time points after unilateral Dnv of the Diam suggest that MHC mRNA transcription and MHC protein turnover (e.g., synthesis and degradation) are differentially regulated and that MHC mRNA expression does not necessarily determine MHC protein expression.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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E.-K. Pae, J. Wu, D. Nguyen, R. Monti, and R. M. Harper Geniohyoid muscle properties and myosin heavy chain composition are altered after short-term intermittent hypoxic exposure J Appl Physiol, March 1, 2005; 98(3): 889 - 894. [Abstract] [Full Text] [PDF]
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