The sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCA) can be expressed from three genes in mammals . The SERCA1 gene is encoding the fast muscle specific SERCA1a and the neonatal SERCA1b. The mRNAs of these isoforms differ in a 42bp exon missing from the SERCA1b. As a result of this the C-terminal glycin of the SERCA1a protein is replaced in SERCA1b by a highly-charged eigth-aminoacid-long tail . The transcript of the SERCA2 gene is spliced into 4 types of mRNA. The class 1 is typical in slow and heart muscles, the class 2 is ubiquitous in most cells, the class 3 is co-expressed with class 2 in nonmuscle cells and the class 4 is confined to neurons . The SERCA3 gene is expressed in blood cells, endothelial cells, mast cells and glandular tissue, etc. and its transcript can be spliced into three variants: SERCA3a, SERCA3b and SERCA3c . The SERCA1 and SERCA2 proteins are essential for muscle relaxation; when they remove the Ca2+ from the sarcoplasm the muscle relaxes. The homology between the SERCA transcripts offers the possibility to asses their relative levels by ratio RT PCR .
Also, isoform-specific antibodies are available for most of the SERCA isoforms. Using these advantages we aimed to detect the expression of the SERCA transcripts and proteins in muscles regenerating from necrosis induced by the venom of the Australian tiger snake (Notechis scutatus scutatus) . This snake venom contains notexin, a toxin which probably provokes hypercontraction of the muscle fibers and finaly results in necrosis of the muscle, but leaves the nerves and the connective tissue relatively intact . After the necrosis the muscle regeneneration takes about 28 days. The regeneration starts with the activation of satellite cells (these are latent cells positioned between the sarcolemma and the lamina basalis) into myoblasts which first proliferate then fuse into myotubes . The myotubes develop into myofibers meanwhile they receive reinnervation  This regeneration system have been useful to describe the expression of several factors important for muscle development and function. Here we summarize our work on the SERCA isoforms in the regenerating fast twitch extensor digitorum longus (EDL) and the slow twitch soleus muscles. We also studied how the fiber type specific expression of the SERCA isoforms changed during the regeneration.
Materials and Methods
The methods are described in more sufficient details in [10-13]. Shortly, 300-360 g male Wistar rats were used for the experiments. The soleus muscles were injected with snake venom as it is described in , but for the EDL three times more venom (60 µg) was administered in 100 µl volume. The muscles were isolated at 1,3,5,7,10,21 and 28 days of regeneration. At least three muscles were used for each time point. The passive stretch of the soleus was achieved by immobilizing the left leg in tarsus flexed position with plaster of Paris. After dissection the muscles were frozen and used for preparation of RNA and RT PCR as it has been described previously . The PCR conditions are also described in that reference .
For Western blotting of the SERCA proteins combined mitochondrial and microsomal fractions were used. The quantification of SERCAs was done by gel scanning of immunoblots using SERCA1, SERCA2a and SERCA2b specific antibodies and peroxidase coupled secondary antibodies . The fiber specific expression was demonstrated with the same antibodies and also using MHC1, MHC2a and MHC2b antibodies in four muscles. Statistics: T-test was used to find the differences at least p<0.05.
The tissue-specific sarcoplasmic/endoplasmic reticulum Ca2+ ATPases were gradually disappeared from the muscles after the injection of notexin. The time course of abolision was depended on the muscle's resistance to the toxin; the slow twitch soleus was less and the fast twitch EDL was more toxin-resistant. The soleus was completely necrotised after the first day while it took 3 days for the EDL on the third day. This was documented not just by the morphology of the fibers but also by the expression of desmin and the level of alpha-actin mRNA, a marker of muscle specific gene expression. The general markers of gene expression, the "housekeeping" SERCA2b and the GAPDH mRNA levels were not significantly changed during the necrosis and the subsequent regeneration.
After the necrosis the neonatal SERCA1b mRNA level was the first to increase in both muscles then it was gradually replaced by the adult fast SERCA1a. In the regenerating soleus a switch occured between the SERCA1a and the slow muscle specific SERCA2a Ca2+ pump at the time of reinnervation. In the EDL, the level of SERCA2a transcript was low and variable and hardly any SERCA3 mRNA was detectable in both types of muscles. After 28 days the levels of all SERCA transcripts were similar to those of the controls and so was the level of SERCA proteins which during the regeneration followed their mRNA levels within two-three days. The early increase of SERCA1 probably also reflected the expression of rather the SERCA1b than SERCA1a. We also studied the fiber specific expression of SERCA proteins in relation to the MHC isoforms. The SERCA1 was coexpressed with the fast type MHC IIa and the MHC IIb, while the SERCA2a was coexpressed with the slow type MHC I isoform. However, after the regeneration the situation did not completely revert to the prenecrosis state. Several changes at the level of fiber specific expression of the SERCA proteins remained. In particular in the soleus, where 98% of the regenerated fibers expressed SERCA2a compared to 81% in the controls.
Also, in the regenerated soleus, 12% of SERCA1 positive fibers were formed compared to 26% in the controls, but these fibers, since they also expressed the slow SERCA isoform could be determined as hybrid fibers. In the regenerated EDL the SERCA2a positive fibers occured in cluster whereas in the controls it was observed in single fibres.
After three days of muscle streching we observed in soleus an increased level of the SERCA1b and a decreased level of the SERCA1a transcripts. The number of SERCA1 positive fibers was also decreased reflecting the changes at the protein level.
Discussion and Conclusion
In regenerating muscles the expression of SERCA isoforms change in a characteristic time dependent pattern. This pattern is similar to that described in the embryonic myogenesis [1,10]. The morphological markers of regeneration provided a background against wich the appearance of the these isoforms could be observed and their developmental importance interpreted. First the myoblasts were proliferating and then the myotubes were formed when the neonatal SERCA1b isoform was dominatly present. Later the myotubes developed into primitive fibers and the neonatal SERCA1b was gradually replaced by the adult fast isoform SERCA1a. This change also coincided with initial reinnervation of the newly formed muscle fibers .
As the reinnervation became established, the adult fast isoform remained permanent in the fast twitch EDL muscles but it started to decline in the soleus. Three weeks after the administration of the toxin the slow type SERCA2a is completely replaced the fast type SERCA1a in the regenerating soleus. This shows that the type of innervation helped to reestablish the former pattern of SERCA isoforms, similar to that reported for the MHC isoforms . During regeneration the level of SERCA proteins followed the level of their mRNAs.
This suggested that the regulation of SERCAs in the regenerating muscles took place primarily at the transcriptional level. In the regenerated muscles differences remained in the fiber specific expressions of SERCAs compared to the control muscles. This suggested that although the expression pattern in the regeneration was similar to that of development the result of the two processes cannot be the same. One possible reason for this is that development and regeneration take place in different enviroments. An other reason might be that the regeneration occurs from satellite cells (latent myoblasts), whereas the embryonic myogenesis occurs from active myoblasts. In the regenerated soleus more fibers expressed the slow SERCA2a and slow MHC than in the normal muscle. That might reflect the stronger effect of slow innervation on the regenerating soleus compared to the developing soleus. Also, the grouped pattern of slow fibers in the regenerated EDL muscle suggested that might have been easier for the fewer slow nerve endings to find neighbouring target fibers instead of distant ones.
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|Zador, E.; Mendler, L.; Szakonyi, G.; Dux, L.; Wuytack, F.; (1998). The Sarco(endo)plasmic Reticulum Ca2+-ATPases in Regenerating and Stretched Muscles. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/|
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