Follistatin treatment suppresses SERCA1b levels independently of other players of calcium homeostasis in C2C12 myotubes.

Follistatin (FS) is a high affinity activin-binding protein, neutralizing the effects of the Transforming Growth Factor-beta (TGF-ß) superfamily members, as myostatin (MSTN). Since MSTN emerged as a negative regulator, FS has been considered as a stimulator of skeletal muscle growth and differentiation. Here, we studied the effect of FS administration on the Ca2+-homeostasis of differentiating C2C12 skeletal muscle cells. FS-treatment increased the fusion index, the size of terminally differentiated myotubes, and transiently elevated the expression of the calcium-dependent protein phosphatase, calcineurin, at the beginning of differentiation. Functional experiments did not detect any alterations in the Ca2+ transients following the stimulation by KCl or caffeine in myotubes. On the other hand, decreased Ca2+-uptake capability was determined by calculating the maximal pump rate (332 ± 17 vs. 279 ± 11 µM/s, in control and FS-treated myotubes, respectively; p < 0.05). In the same way, the expression and ATPase activity of the neonatal sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA1b) were decreased (0.59 ± 0.01 vs. 0.19 ± 0.01 mM ATP/min, in control and FS-treated myotubes, respectively; p < 0.05). However, the expression level of other proteins involved in Ca2+-homeostasis and differentiation (calsequestrin, STIM1, MyoD) were not affected. Our results suggest that the FS controlled myotube growth is paralleled with the tight regulation of cytosolic calcium concentration, and the decline of SERCA1b appears to be one of the key components in this process.

The neonatal sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA1b): a neglected pump in scope.

The neonatal isoform of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA1b) is formed by developmental splicing and expressed fully only in developing muscle. As a major Ca(2+) pump in myotubes, SERCA1b must be detected in excitation contraction coupling or in store-operated calcium entry. The available pan SERCA1 antibodies also recognise SERCA1b but these are more frequently used to detect SERCA1a, the adult muscle-specific isoform characteristically expressed in fast fibres of skeletal muscle. In such applications, the pan SERCA1 antibodies are frequently claimed to be SERCA1a antibodies without proving it. Realistically, such an antibody cannot be made since it should recognise a single glycine at the C-terminal, the only part of SERCA1a that is different from SERCA1b. The false interpretation of the antibody specificity created inconsistence in the literature and led to false conclusions attributing features only to SERCA1a although those at least are also shared by SERCA1b. In contrast, a SERCA1b antibody has been made against the eight amino acid peptide tail that replaces the glycine of SERCA1a at the C-terminal. Therefore, the expression of SERCA1b can be specifically demonstrated, unlike that of SERCA1a, in various stages and conditions of skeletal muscle. This review argues against misbeliefs related to the distinction, expressions and functions of the two muscle-specific SERCA1 isoforms.

The neonatal sarcoplasmic reticulum Ca2+-ATPase gives a clue to development and pathology in human muscles.

The sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1) has two muscle specific splice isoforms; SERCA1a in fast-type adult and SERCA1b in neonatal and regenerating skeletal muscles. At the protein level the only difference between these two isoforms is that SERCA1a has C-terminal glycine while SERCA1b has an octapeptide tail instead. This makes the generation of a SERCA1a specific antibody not feasible. The switch between the two isoforms is a hallmark of differentiation so we describe here a method based on the signal ratios of the SERCA1b specific and pan SERCA1 antibodies to estimate the SERCA1b/SERCA1a dominance on immunoblot of human muscles. Using this method we showed that unlike in mouse and rat, SERCA1b was only expressed in pre-matured infant leg and arm muscles; it was replaced by SERCA1a in more matured neonatal muscles and was completely absent in human foetal and neonatal diaphragms. Interestingly, only SERCA1a and no SERCA1b were detected in muscles of 7-12 years old boys with Duchenne, a degenerative-regenerative muscular dystrophy. However, in adult patients with myotonic dystrophy type 2 (DM2), the SERCA1b dominated over SERCA1a. Thus the human SERCA1b has a different expression pattern from that of rodents and it is associated with DM2.

Characterization of Skeletal Muscle Regeneration Revealed a Novel Growth Network Induced by Molecular Acupuncture-like Transfection.

The low efficiency of in vivo transfection of a few fibres revealed a novel tissue network that temporally amplified growth stimulation in the entire regenerating rat soleus muscle. This acupuncture-like effect was demonstrated when the fibres began to grow after complete fibre degradation, synchronous inflammation, myoblast and myotube formation. Neonatal sarcoplasmic/endoplasmic reticulum ATPase (SERCA1b) was first detected in this system. The neonatal, fast and slow SERCA isoforms displayed consequent changes with innervation and differentiation, recapitulating events in muscle development. In vivo transfection of myotubes with plasmids expressing dominant negative Ras or a calcineurin inhibitor peptide (Cain/cabin) proved that expression of the slow myosin heavy chain and the slow muscle type SERCA2a are differentially regulated. In vivo transfection of a few nuclei of myotubes with dnRas or SERCA1b shRNA stimulated fibre size growth in the whole regenerating muscle but only until the full size had been reached. Growth stimulation by Ras and SERCA1b antisense was abolished by co-transfection of Cain or with perimuscular injection of IL4 antibody. This revealed a novel signalling network resembling scale-free networks which, starting from transfected fibre myonuclei as "hubs", can amplify growth stimulation uniformly in the entire regenerating muscle.

SERCA1 protein expression in muscle of patients with Brody disease and Brody syndrome and in cultured human muscle fibers.

Brody disease is an inherited myopathy associated with a defective function of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) protein. Mutations in the ATP2A1 gene have been reported only in some patients. Therefore it has been proposed to distinguish patients with ATP2A1 mutations, Brody disease (BD), from patients without mutations, Brody syndrome (BS). We performed a detailed study of SERCA1 protein expression in muscle of patients with BD and BS, and evaluated the alternative splicing of SERCA1 in primary cultures of normal human muscle and in infant muscle. SERCA1 reactivity was observed in type 2 muscle fibers of patients with and without ATP2A1 mutations and staining intensity was similar in patients and controls. Immunoblot analysis showed a significant reduction of SERCA1 band in muscle of BD patients. In addition we demonstrated that the wild type and mutated protein exhibits similar solubility properties and that RIPA buffer improves the recovery of the wild type and mutated SERCA1 protein. We found that SERCA1b, the SERCA1 neonatal form, is the main protein isoform expressed in cultured human muscle fibers and infant muscle. Finally, we identified two novel heterozygous mutations within exon 3 of the ATP2A1 gene from a previously described patient with BD.

The Meeting of Micropeptides with Major Ca2+ Pumps in Inner Membranes-Consideration of a New Player, SERCA1b.

Calcium is a major signalling bivalent cation within the cell. Compartmentalization is essential for regulation of calcium mediated processes. A number of players contribute to intracellular handling of calcium, among them are the sarco/endoplasmic reticulum calcium ATP-ases (SERCAs). These molecules function in the membrane of ER/SR pumping Ca2+ from cytoplasm into the lumen of the internal store. Removal of calcium from the cytoplasm is essential for signalling and for relaxation of skeletal muscle and heart. There are three genes and over a dozen isoforms of SERCA in mammals. These can be potentially influenced by small membrane peptides, also called regulins. The discovery of micropeptides has increased in recent years, mostly because of the small ORFs found in long RNAs, annotated formerly as noncoding (lncRNAs). Several excellent works have analysed the mechanism of interaction of micropeptides with each other and with the best known SERCA1a (fast muscle) and SERCA2a (heart, slow muscle) isoforms. However, the array of tissue and developmental expressions of these potential regulators raises the question of interaction with other SERCAs. For example, the most abundant calcium pump in neonatal and regenerating skeletal muscle, SERCA1b has never been looked at with scrutiny to determine whether it is influenced by micropeptides. Further details might be interesting on the interaction of these peptides with the less studied SERCA1b isoform.

Silencing SERCA1b in a few fibers stimulates growth in the entire regenerating soleus muscle.

The neonatal isoform of the sarcoplasmic/endoplasmic reticulum Ca²(+) ATPase 1 (SERCA1b) is a dominant Ca²(+) pump in the young fibers of regenerating muscle. In vivo transfection of about 1% of the fibers with SERCA1b RNAi plasmid resulted in no apparent change in the transfected fibers, but enhanced the increase of fresh weight and fiber size in the whole regenerating rat soleus muscle, until the normal size was reached. Co-transfection of calcineurin inhibitor cain/cabin-1 with SERCA1b RNAi was sufficient to cut down the widespread growth stimulation, but the subsequent transfection of cain into the SERCA1b RNAi transfected muscle did not inhibit muscle growth. The SERCA1b RNAi preferably upregulated the expression of the NFAT reporter lacZ compared to controls when co-transfected into the fibers. Notably, perimuscular injection of interleukin-4 (IL-4) antibody but not that of an unrelevant antibody completely abolished the growth-promoting effect of SERCA1b RNAi. This indicates that silencing SERCA1b in a few fibers stimulates the calcineurin-NFAT-IL-4 pathway and fiber growth in the whole regenerating soleus. These results suggest the presence of an autocrine-paracrine coordination of growing muscle fibers, and put forward a new method to stimulate skeletal muscle regeneration.

Poststerone increases muscle fibre size partly similar to its metabolically parent compound, 20-hydroxyecdysone.

In mice, poststerone is a major in vivo metabolite of the worldwide popular anabolic food supplement 20-hydroxyecdysone (20E). Here we present the first study on this ecdysteroid in view of the in vivo anabolic effect of its parent compound, 20E in mammals. We have monitored muscle fibre type cross sectional areas (CSA) of developing rats after treatment with poststerone as we did in a previous study with 20E. The muscle mass and fibre CSAs of soleus and EDL were increased by poststerone in a muscle specific manner as by 20E but there were some differences. Notably, the CSAs of type I and type IIa fibres in the soleus were less elevated by poststerone than by 20E. However poststerone increased the CSA of each four fibre types (I, IIa, IIx, IIb) in the EDL more effectively than 20E did. Poststerone, like 20E, also increased the number of myonuclei in the EDL of both hind limbs. Overall, this shows for the first time that poststerone having steroid nucleus and no side chain of 20E has a partly overlapping effect with that of 20E.

Phytoecdysteroids and anabolic-androgenic steroids--structure and effects on humans.

Phytoecdysteroids are structural analogs of the insect molting hormone ecdysone. Plants comprise rich sources of ecdysteroids in high concentration and with broad structural diversity. Ecdysteroids have a number of proven beneficial effects on mammals but the hormonal effects of ecdysteroids have been proven only in arthropods. Their structures are somewhat similar to those of the vertebrate steroid hormones but there are several structural differences between the two steroid groups. Despite of these essential structural differences, ecdysteroids exert numerous effects in vertebrates that are similar to those of vertebrate hormonal steroids, and they may serve as effective anabolic, hepatoprotective, immunoprotective, antioxidant and hypoglycemic agents. Ecdysteroids do not bind to the cytosolic steroid receptors, instead, they are likely to influence signal transduction pathways, like the anabolic steroids, possibly via membrane bound receptors. The application of phytoecdysteroids is a promising alternative to the use of anabolic-androgenic steroids because of the apparent lack of adverse effects. The prospective use of phytoecdysteroids may extend to treatments of pathological conditions where anabolic steroids are routinely applied. One of the most cited aspects of phytoecdysteroid application (on the Internet) is the increase of muscle size. However in this field too stringent research is needed as an adequate cytological explanation is not yet available for the anabolic. This paper reports on the most important structural differences between androgenic hormones, their synthetic analogs and ecdysteroids. The anabolic/hormonal effects and the possible mechanisms of action of these compounds are also discussed as concerns the skeletal muscle.

dnRas stimulates autocrine-paracrine growth of regenerating muscle via calcineurin-NFAT-IL-4 pathway.

Ras and calcineurin are members of two independent pathways in muscle growth but their interaction is not known. This work shows that the transfection of about 1% of the muscle fibers with dominant negative Ras (dnRas) shows a wilder effect; it stimulates the fiber growth in the entire regenerating soleus muscle, including the nontransfected fibers. Co-transfection with the calcineurin inhibitor cain/cabin prevented the growth stimulation. Injection of antibody for interleukin-4 (IL-4) also abolished the growth ameliorating effect. These results suggest that the inactivation of Ras in 1% of the fibers upregulates the calcineurin-NFAT-IL-4 pathway and the secreted IL-4 triggers fiber growth stimulation in the whole regenerating soleus muscle of the rat. The results highlight the importance of the autocrine-paracrine regulation in muscle regeneration and hint to a novel method of gene theraphy of degenerative-regenerative muscle dystrophies.

The effect of passive movement on denervated soleus highlights a differential nerve control on SERCA and MyHC isoforms.

The sarco-endoplasmic reticulum Ca2+ ATP-ase (SERCA) and myosin heavy chain (MyHC) levels were measured in hindlimb-denervated and selectively denervated rat soleus muscles. Selective denervation allowed passive movement of the soleus, whereas hindlimb denervation rendered it to passivity. To minimize chronic effects, we followed the changes only for 2 weeks. Selective denervation resulted in less muscle atrophy, a faster slow-to-fast transition of MyHC isoforms, and less coordinated expressions of the slow vs fast isoforms of MyHC and SERCA. Generally, expression of the slow-twitch type SERCA2a was found to be less dependent, whereas the slow-twitch type MyHC1 was the most dependent on innervation. Our study shows that passive movement is able to ameliorate denervation-induced atrophy of the soleus and that it also accentuates the dyscoordination in the expression of the corresponding slow and fast isoforms of MyHC and SERCA.

The expression of the neonatal sarcoplasmic reticulum Ca2+ pump (SERCA1b) hints to a role in muscle growth and development.

The neonatal isoform of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 1 (SERCA1b) is a Ca2+ pump with a well-known developmentally regulated transcript level but an undefined protein expression and function. Specific antibodies were generated to show that SERCA1b is exclusively expressed in myoblasts and myotubes of cultured and regenerating muscle. However, the SERCA1b protein was not detectable in normal adult fast and slow muscles. Studies of the in vitro differentiating myogenic cell lines C2C12 and sol8 showed that SERCA1b is the main SERCA1 protein isoform induced during differentiation and that it is found in the myotubes. Remarkably in BC3H1 cells, which show incomplete differentiation and are reluctant to form myotubes, express the SERCA1b mRNA but not the corresponding protein. SERCA1b protein was also absent from stretched or denervated adult soleus, in spite of the fact that its mRNA level was upregulated. SERCA1b accounts for nearly the total of SERCA1 expression in the diaphragm of newborn mice, which suggests that the insufficient function and development of the diaphragm in the SERCA1 null mutant mice may be due to the lack of SERCA1b. Our studies point to an important regulation of SERCA1b expression at the protein level and hints to a role in the growth of the developing muscle.

Sarcolipin and phospholamban mRNA and protein expression in cardiac and skeletal muscle of different species.

The widely held view that SLN (sarcolipin) would be the natural inhibitor of SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1), and PLB (phospholamban) its counterpart for SERCA2 inhibition is oversimplified and partially wrong. The expression of SLN and PLB mRNA and protein relative to SERCA1 or SERCA2 was assessed in ventricle, atrium, soleus and EDL (extensor digitorum longus) of mouse, rat, rabbit and pig. SLN protein levels were quantified by means of Western blotting using what appears to be the first successfully generated antibody directed against SLN. Our data confirm the co-expression of PLB and SERCA2a in cardiac muscle and the very low levels (in pig and rabbit) or the absence (in rat and mouse) of PLB protein in the slow skeletal muscle. In larger animals, the SLN mRNA and protein expression in the soleus and EDL correlates with SERCA1a expression, but, in rodents, SLN mRNA and protein show the highest abundance in the atria, which are devoid of SERCA1. In the rodent atria, SLN could therefore potentially interact with PLB and SERCA2a. No SLN was found in the ventricles of the different species studied, and there was no compensatory SLN up-regulation for the loss of PLB in PLB(-/-) mouse. In addition, we found that SLN expression was down-regulated at the mRNA and protein level in the atria of hypertrophic hearts of SERCA2(b/b) mice. These data suggest that superinhibition of SERCA by PLB-SLN complexes could occur in the atria of the smaller rodents, but not in those of larger animals.

Antisense inhibition of myoD expression in regenerating rat soleus muscle is followed by an increase in the mRNA levels of myoD, myf-5 and myogenin and by a retarded regeneration.

It has been reported that muscles of myoD-/- mice present a lower potential to regenerate, but there are no reports on the effect of acute interference with myoD expression limited in space and time to only a particular regenerating muscle. Here we relied on antisense inhibition of this factor. Four different oligos were tested. The suppression of regeneration indices (the expression of desmin, the formation of myotubes and the initiation of endplates) was the most pronounced, with the oligomer targeting a region encompassing the translation start site of myoD. A mixed backbone phosphorothioate-phosphate diester oligo (200 microl at 20 microM) was still detectable in the muscles 1 h after its administration and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the level of the targeted 5' end of the myoD mRNA was selectively decreased. The level of myoD protein was also lowered. Four hours after the antisense treatment, when the oligos were no longer detectable, the myoD mRNA level was restored and 24 h later it exceeded controls together with that of myf-5 and myogenin. After 4 weeks, the antisense-treated soleus muscles were similar to the control-treated and the untreated regenerated soleus with respect to fiber types and motor endplates, however, they contained smaller fibers which reflected the asynchronity of regeneration. This shows that successfully targeted simple antisense oligonucleotides can be used as selective tools for inhibition of individual factors in studying the process of muscle regeneration.

Expression of SERCA2a is not regulated by calcineurin or upon mechanical unloading in skeletal muscle regeneration.

This study investigates to what extent the expression of the slow myosin heavy chain (MyHCI) isoform and the slow type sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) isoform are co-regulated in fibers of regenerating skeletal soleus muscle. Both overexpression of cain, a calcineurin inhibitor, or partial tenotomy prevented the expression of MyHCI but left SERCA2a expression unaffected in fibers of regenerating soleus muscles. These data complement those from different experimental models and clearly show that the expression of MyHCI and SERCA2a--the major proteins mediating, respectively, the slow type of contraction and relaxation--are not coregulated in regenerating soleus muscle.

Neocortical c-fos mRNA transcription in repeated, brief, acute seizures: is c-fos a coincidence detector?

The effect of acute brief seizures on neocortical c-fos expression was investigated in rats injected with 5 mg/kg 4-aminopyridine. Electroencephalography in freely moving animals with implanted neocortical electrodes detected an average of 2.67 tonic-clonic convulsions within 1 h following the 4-AP treatment. Tissue samples of the somatosensory neocortex were collected at 30 min, 1 h, 3 h, 5 h and 8 h following the treatment for PCR and immunohistochemistry. The c-fos mRNA displayed the first significant rise at 1 h, and remained significantly higher through 3 h. The number of c-fos protein immunoreactive cells was significantly elevated already at 30 min, peaked at 1 h, and declined by 5 h. We conclude that in repetitive, brief seizures, the first convulsion does not increase c-fos RNA transcription, whilst the second causes a long-lasting gene expression and a large increase of c-fos protein synthesis. The phenomenon may have implications in the pathogenesis of human and animal epilepsies.

The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells.

The sarcoplasmic/endoplasmic reticulum Ca2+ATPases (SERCAs) are the main Ca2+ pumps which decrease the intracellular Ca2+ level by reaccumulating Ca2+ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca2+ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca2+-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca2+]i measured at the 20th and 40th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca2+-uptake capability which was quantified by extracting the maximal pump rate (454±41 µM/s vs. 144±24 µM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 µM/s) and the amount of calcium released (843±75 µM vs. 576±80 µM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca2+]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.

Non-competitive NMDA receptor antagonists moderate seizure-induced c-fos expression in the rat cerebral cortex.

We examined the effects of non-competitive NMDA glutamate receptor antagonists on seizures elicited by 4-aminopyridine (4-AP), and in particular, on the expression of the transcription factor c-fos induced by these seizures. Induction of c-fos mRNA due to 4-AP-elicited seizures was ascertained by reverse transcription polymerase chain reaction in samples of the neocortex. Adult rats were pretreated with the NMDA receptor antagonists amantadine (40 mg/kg), ketamine (3mg/kg), dizocilpine (MK-801; 1mg/kg) or dextrometorphan (40 mg/kg); 4-AP (5mg/kg) was then injected i.p. Controls were treated with either antagonist only or with 4-AP only. Pretreatment with the antagonists (with the exception of amantadine) increased the latency of behavioural seizures, but not all of the antagonists caused symptomatic seizure protection. In the brains which were processed for Fos immunohistochemistry, quantitative evaluation of immunostained cells was performed in the neocortex and hippocampus. Treatment with either antagonist did not induce by itself c-fos expression, with the exception of amantadine, which caused slight Fos induction in the neocortex. Pretreatment with all the antagonists resulted in decrease of seizure-induced Fos immunoreactivity with respect to non-pretreated animals. Decrease of immunostained cells was significant in the neocortex, in the granule cell layer and hilus of the dentate gyrus, in hippocampal areas CA1 and CA2. MK-801, ketamine and dextrometorphan decreased significantly Fos immunoreactivity also in area CA3. The decrease of Fos immunostaining was not directly correlated with a suppression of behavioural seizures. The results support an important role of NMDA receptors in c-fos gene induction in acute 4-AP seizures.

Transfection efficiency along the regenerating soleus muscle of the rat.

We investigated the efficiency of a single plasmid transfection along the longitudinal axis of the regenerating soleus of young rats. This also reflected transfection efficiency along the fibers because the soleus is a nearly fusiform muscle in young animals. The complete regeneration was induced by notexin and the transfection was made by intramuscular injection of enhanced green fluorescent protein- or Discosoma red-coding plasmids after 4 days. One week after transfection the number of transfected fibers was higher at the place of injection (i.e., in the muscle belly) and lower or absent at the ends of the muscle. The inspection of longitudinal sections and neuromuscular endplates indicated that one of the reasons of uneven transfection might be the shortness of transfected myotubes and the other reason might be the limit of diffusion of transgenic proteins from the expressing nuclei. As a result, the efficiency of transfection in the whole regenerating muscle was much lower than it could be estimated from the most successfully transfected part.