1Institute of Biochemistry, Faculty of Medicine, University of Szeged, H-6701 Szeged, Hungary; and 2Laboratorium Voor Fysiologie, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Submitted 18 December 2002 ; accepted in final form 27 May 2003
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ABSTRACT |
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slow type sarcoplasmic reticulum Ca2+ pump; MyHCI; nerve influence
In the present study, we followed the mRNA and protein levels of SERCA2a in regenerating rat soleus muscles that were denervated by transecting either the sciatic nerve or the soleus nerve. Our results demonstrate that the SERCA2a mRNA and protein are highly expressed in sciatic-denervated (whole limb denervated) regenerating soleus fibers, whereas the My-HCI mRNA and the protein are absent. The same absolute dependence on innervation of MyHCI expression, but not of SERCA2a expression, was inferred from regeneration experiments after the soleus nerve was cut (selective denervation). In this latter model, the weight of soleus was not decreased after 10 days, unlike in sciatic-denervated regeneration. Furthermore, we show that in vivo transfection of innervated regenerating muscle with a dominant negative Ras mutant or transfection of denervated regenerating muscles with a Ras mutant that selectively and constitutively activates the MAPK pathway only affects My-HCI expression (19) in the fibers but not SERCA2a expression.
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MATERIALS AND METHODS |
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Animals and treatments. Experiments with animals were approved by the Ethics Committee of Animal Treatment of the Medical Faculty of the University of Szeged.
The soleus muscles of the left hindlimb of male Wistar rats 3 mo of age and weighing 300-360 g were treated with notexin as described previously (34). For transfection experiments, the muscles were reinjected 4 days later with 50 µg of plasmid DNA in 50 µl of 20% sucrose (19). Eight days after plasmid transfection (12 days after notexin injection), the muscles were dissected and frozen in isopentane cooled in boiling liquid nitrogen. The muscles were kept at -70°C until use.
Dissecting a 1-cm-long part of the sciatic nerve high in the thigh was used to denervate the hindlimb. In some experiments, a selective disruption of the innervation of the soleus muscle was obtained by removing a 2- to 3-mm-long piece from the soleus nerve leading to the proximal end of the muscle.
RT-PCR. RNA extraction and reverse transcription were carried out
as described previously
(34-35).
Primers and PCR conditions are shown in
Table 1. All the PCR cycles
were carefully adjusted to the log/linear phase of amplification. The ratio RT
PCR of SERCA1/SERCA2 mRNA was done as described previously
(33). Shortly, primers
hybridizing to both SERCA1 and SERCA2 simultaneously amplified two fragments
of the same size, and a restriction enzyme digest distinguished between the
fragments. To radiolabel the PCR fragments for quantification, 5 µl (i.e.,
one-tenth of the total volume) of the primary PCR mixture was transferred to a
new tube containing 50 µl of the same amplification buffer, except that
[-32P]dCTP was added. Two additional PCR cycles were
executed with the same cycle parameters used in the primary PCR. The fragment
of SERCA1 was cut by Mse I, which left SERCA2 intact, whereas
NcoI, which did not cut SERCA1, digested the fragment of SERCA2. The
ratio of SERCA2/SERCA1 mRNAs was calculated from the level of uncut fragments.
The amplification products were analyzed on a 6% (wt/wt) polyacrylamide gels,
which were then either stained with Vistra Green and the bands quantified by
means of a model 840 Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA)
or air-dried and the 32P spots quantified by means of the Typhoon
9400 Variable Mode Imager program (Molecular Dynamics). When
[
-32P]dCTP was used for labeling, the band intensities were
corrected for the CG (cytosine and guanine base) content of the amplified
fragment.
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Immunoblotting. The SERCA2a and MyHCI were measured on extracts from the same muscles. When denervated regenerating muscles were used, the volume of extracting buffer was decreased in proportion to their lower fresh weight. SERCA2a immunoblots were made from the mitochondrial-microsomal fraction as in Ref. 36. The pellets of the first centrifugation (1,000 g for 10 min) were used to extract myosin according to Ref. 9. Equal parts of the muscles were loaded on each lane of the gel for both SERCA and myosin analysis. The SERCA2a antiserum R-15 (rabbit, 1:500; Ref. 32) and MyHCI antibody (BA-D5, mouse, 1:100, Ref. 25) were used as primary antibodies in the immunoblot analysis. The bands were either visualized by Ni-DAB staining (33) and then quantified by densitometry on Gel Doc (Bio-Rad Laboratories, Hercules, CA) or stained with the Vistra ECF system (Amersham Biosciences), following the protocol of the manufacturer, and quantified by model 840 Storm Chemifluorescence Imager (Molecular Dynamics).
Ras plasmids. A number of vectors expressing mutant Ras proteins were kindly provided by Dr. A. Serrano (Padua, Italy). The constitutively active Ras mutants H-RasV12 and H-RasV12S35 in the pDCR expression vector under the control of the CMV promoter were used (23, 24). These Ras mutants had a hemagglutinin (HA) epitope added at their COOH termini. An additional mutant used in this experiment was the dominant negative H-RasN17, which had no HA tag.
Immunostaining. Cryosections of 20 µm thickness obtained from regenerating muscles were stained by peroxidase immunohistochemistry (as in Ref. 36) by using the BA-D5 antibody (mouse 1:50) for MyHCI and the R-15 antiserum (rabbit, 1:400) for SERCA2a. The expression from the HA-tagged Ras plasmids was identified by HA antibody (mouse 1:200; Calbiochem) and expression of the dominant negative Ras by the pan Ras (Ab-1) antibody (mouse 1:20; Santa Cruz).
Fiber cross-sectional area. Cross-sectional area (CSA) was measured on hematoxilin/eosin-stained sections by the Olympos DP-soft, version 3.2 program (Olympus, Hamburg, Germany).
Statistics. The Newman-Keuls test was used to test for significant differences. The numbers of SERCA2a- and My-HCI-expressing fibers were compared by using a t-test.
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RESULTS |
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SERCA2a and SERCA1 mRNAs in denervated regenerating soleus muscle. Figure 2 shows that in denervated regenerating soleus, MyHCI mRNA was absent, but the SERCA2a mRNA was clearly expressed on days 7 and 10 and elevated on day 21. In the innervated regenerating control muscles, both MyHCI and SERCA2a mRNAs increased gradually from days 7 to 21. Denervation apparently decreased the levels of SERCA2a mRNA 3- and 0.25-fold, respectively, on days 10 and 21 compared with the innervated controls (Fig. 2A). The ratio of SERCA2/SERCA1 mRNAs was determined by a ratio RT-PCR technique (33, 35) (Fig. 2B). We found that denervation suppressed SERCA1 mRNA nearly completely by day 7 and, therefore, strongly increased the SERCA2a/SERCA1 mRNA ratio. However, at later stages of regeneration the level of SERCA1 mRNA increased again. These observations are in line with earlier ones showing that denervation of a slow muscle promotes the fast phenotype (7, 31).
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It should be remarked that the PCR primers used in all of these assays would coamplify SERCA2a and SERCA2b cDNAs. However, under our experimental conditions, the SERCA2a isoform accounted for practically all SERCA2. Tests with SERCA2b-specific primers showed the near absence of this isoform, so SERCA2a accounted for nearly all SERCA2.
In denervated regenerating soleus, a pronounced increase of SERCA2a protein is followed by a gradual decrease. Next, we compared the time course of SERCA2a, SERCA1, and MyHCI proteins during regeneration. In innervated control muscles, SERCA2a proteins gradually increased during regeneration and followed an expression pattern similar to that of the corresponding mRNA. Remarkably, denervation led to a three-fold higher level of SERCA2a compared with the innervated control on day 7 of regeneration (Fig. 3). Subsequently, SERCA2a expression gradually declined at days 10 and 21. This shows that although innervation is clearly not a prerequisite for the expression of SERCA2a, it is needed for maintaining a long-term expression of the Ca2+ pump.
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The expression of the fast-type sarcoplasmic reticulum pump protein SERCA1 was less affected by denervation than that of SERCA2a; it showed a transient increase by 20% on day 10 in the denervated regenerating soleus compared with the innervated control, but it declined back to the normal level afterward (Fig. 3).
The expressions of SERCA2a and MHCI are also uncoupled in selectively denervated regenerating soleus. In the experiments described above, denervation was done by removing a piece of the sciatic nerve. A drawback of this method is that it paralyzes the hindlimb and results in a decrease of both the fresh weight and the fiber diameter of regenerating soleus muscles. This side effect can be partially circumvented by selectively disrupting the soleus nerve instead of the main branch sciatic nerve. In such selectively denervated regenerating muscles, the denervation atrophy was somewhat delayed. After 10 days, the fresh weight, RNA, and protein content were similar to those in innervated regeneration (Table 2). However, the fiber mean CSA was smaller (1,010 ± 308 µm2 vs. 1,944 ± 534 µm2) and similar to that of hindlimb denervated (1,031 ± 156 µm2) regenerating muscles. In selectively denervated regeneration, the slow myosin was nearly abolished, whereas SERCA2a was still present in most fibers (Fig. 4A, a and b). During innervated regeneration, MyHCI and SERCA2a were coexpressed, but some fibers expressed more MyHCI and SERCA2a than others (Ref. 33; Fig. 4A, c and d). By day 12 of regeneration, these isoforms were expressed in most fibers (see Fig. 1, A and B). In selectively denervated regenerating soleus, the MyHCI mRNA were nearly absent and the extracted MyHCI protein levels were extremely low (Fig. 4B); nonetheless, the SERCA2a mRNA level was less decreased than in sciatic denervation (87 ± 17.7% vs. 33.8 ± 13.7% of normal, P < 0.05). The levels of SERCA2a protein did not change significantly compared with those of the innervated regenerating muscle. This confirms the view that the denervated soleus muscle maintains SERCA2a expression but loses MyHCI expression. Furthermore, it shows that the uncoupled expression of MyHCI and SERCA2a is not linked to the degenerative changes like, e.g., the loss of fresh weight, RNA, and protein content of the muscle.
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Ras mutants do not affect SERCA2a expression. Murgia et al. (19) showed that the in vivo expression of a dominant negative Ras mutant, RasN17, is able to mimic the effect of denervation on MyHCI expression in regenerating soleus muscle. Fibers constitutively expressing this mutant are deprived of Ras pathways transmitting nerve influence to the slow myosin transcription. We transfected regenerating soleus with RasN17 to investigate its effect on SERCA2a expression. Our hypothesis was that if SERCA2a and MyHCI were coregulated by the Ras pathway, they would both follow similar kinetics. However, we found that My-HCI-negative fibers (Fig. 5Aa) still expressed SERCA2a with the same intensity as the MyHCI-positive fibers (Fig. 5Ab). This showed that whereas Ras affects MyHCI expression, it apparently does not affect that of the SERCA2a isoform.
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The effect of denervation on MyHCI in regenerating soleus can also be reversed by the ectopic expression of RasS35, a constitutively active form of Ras that specifically stimulates the MAPK pathway (19). In vivo expression of this RasS35 mutant in regenerating soleus (Fig. 5Bc) did not stimulate SERCA2a expression above the normal level (Fig. 5Bb) in the MyHCI-expressing regenerating fibers (Fig. 5Ba). A similar result was obtained with the overexpression of the Ras12V mutant (data not shown). Ras12V is also a constitutively active Ras mutant known to stimulate MyHCI expression but which, in contrast to RasS35 besides the MAPK pathway, also targets the phosphatidyl inositol-3-OH kinase pathway and ralGDS, the guanine dissociation stimulator for the GTPase Ral (19). The above observations confirm that the expression of SERCA2a is independent from Ras.
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DISCUSSION |
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The three-fold increase of SERCA2a level after 1 wk of regeneration in denervated muscles compared with the innervated controls can clearly not be ascribed to a direct neuronal effect on SERCA2a expression. This is an indirect effect, which might rather be the result of apoptotic changes (14) causing the dramatic loss of fresh weight of denervated regenerating muscles. Remarkably, the time-dependent changes in the ratio of SERCA2/SERCA1 mRNA levels paralleled changes in the level of the SERCA2a protein rather than that of the SERCA2a mRNA level. The ratio of SERCA2/SERCA1 mRNAs is a possible indicator of the progress of soleus muscle regeneration: it is lower than 1 shortly after the onset of regeneration (day 5) but increases to values >3 when regeneration is accomplished (35).
The increase of the SERCA2a mRNA on day 21 in the denervated regenerating muscle further indicates that the expression of this Ca2+ pump can occur in the absence of neuronal activity. The lack of correlation between SERCA2a mRNA and its corresponding protein suggests an important posttranscriptional component in the regulation of SERCA2a expression (reviewed in Ref. 18). However, in denervated soleus the SERCA1 mRNA level gradually increased during regeneration and became even higher than that of SERCA2a on days 10 and 21. Hence, the ratio of SERCA2/SERCA1 mRNA declined on days 10 and 21. Also, the SERCA2a protein was lower on days 10 and 21. A similar decrease of SERCA2a protein has been reported in denervated developing muscles (27) and in denervated soleus muscle (16, 20). It has been shown that the expression of fast myosin heavy chains MyHCIIx/d (and MyHCIIa) is upregulated in denervated developing/regenerating muscle and that only after the muscle becomes subject to an electrical stimulation pattern typical for slow-twitch muscle does a switch to the expression of the slow MyHCI form occur (19, 31). Surprisingly, a corresponding upregulation of the SERCA1 mRNA and protein levels was not observed in denervated regenerating muscles on day 7. Instead, the SERCA1 mRNA level was practically abolished on day 7, than increased dramatically on day 10, whereas the SERCA1 protein level did not decrease on day 7 and increased by 20% on day 10 in denervated regenerating muscle.
However, in the innervated soleus, both the SERCA2a and the MyHCI levels gradually increased with the progress of slow muscle regeneration, as could be expected because both are characteristic components of a slow muscle. Calcineurin was suggested as a candidate common regulator of MyHCI and SERCA2a expression because the number of slow fibers coexpressing MyHCI and SERCA2a declines in soleus muscles of rats fed with a cyclosporin A-supplemented diet (2) and because the expression of MyHCI and SERCA2a changed in parallel in fibers of regenerating muscle adapting phenotypically to overload (1). Calcineurin, via transcription factors like NFAT, can mediate excitation-transcription coupling and can therefore regulate slow muscle-type transcription (21). The role of calcineurin in the upregulation of MyHCI expression is well documented as the overexpression of cain, a protein inhibitor of calcineurin, prevents My-HCI expression in the fibers of regenerating soleus (28). However, because denervation does not abolish expression of SERCA2a, it is difficult to perceive a calcineurin-mediated regulatory pathway for this sarcoplasmic reticulum Ca2+ pump as well.
Talmadge et al. (29) demonstrated that the expression of SERCA2a and slow myosin is not always coupled in fibers of muscles phenotypically adapting to spinal cord injury. Such fibers have probably lost nerve influence but remain stretched by the surrounding intact fibers. A similar situation might exist in selective denervation of soleus when the functional innervation of the other hindlimb muscles remains intact. Here, we show that in the regenerating soleus when only the soleus nerve is ablated (selective denervation), the SERCA2a mRNA and protein levels are higher than in the case of ablation of the sciatic nerve (hindlimb denervation), whereas in both types of denervation the MyHCI is abolished. This further points to an uncoupling of expression of these two characteristically slow protein isoforms. Another characteristic of the selective denervation is that the muscle keeps its weight, RNA, and protein content, although the size of its individual fibers is decreased compared with the innervated regenerating control, probably because of the retarded regeneration. Therefore, the expression of SERCA2a is less likely to be stimulated by apoptosis-related changes (14) than in the sciatic-denervated regenerating muscles in which these parameters are lower.
We tested the effect of the Ras oncogene, another reported mediator of neuronal influence, on slow myosin expression. This further supported the view that these contraction and relaxation elements are separately regulated. The overexpression of RasS35, which is able to mimic nerve influence on MyHCI expression in the denervated regenerating muscle (19), left SERCA2a expression unaffected. Similarly, the dominant negative Ras mutant, known to mimic denervation affects on MyHCI expression (19), did not influence the expression of the slow SR Ca2+ pump. The fibers expressing dominant negative Ras are probably stretched by the intact fibers; this situation is somewhat similar to that of the selectively denervated regenerating soleus and that of fibers adapting after spinal cord injury. Apparently, the passive stretch did not compensate for the inhibition of MyHCI expression in fibers expressing dominant negative Ras; however, it might contribute to the maintenance of the level of SERCA2a expression like it did in the selectively denervated regenerating soleus.
It remains at present an open question, what regulates SERCA2a expression in the regenerating soleus muscle. Our results show that this regulation must be more complex than that of MyHCI. SERCA2a expression is regulated at both transcriptional and posttranscriptional levels, unlike that of MyHCI, where the regulation is primarily at the transcriptional level.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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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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