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.

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.

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.

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 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.

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.

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.

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 compact mutation of myostatin causes a glycolytic shift in the phenotype of fast skeletal muscles.

Myostatin is an important negative regulator of skeletal muscle growth. The hypermuscular Compact (Cmpt) mice carry a 12-bp natural mutation in the myostatin propeptide, with additional modifier genes being responsible for the phenotype. Muscle cellularity of the fast-type tibialis anterior (TA) and extensor digitorum longus (EDL) as well as the mixed-type soleus (SOL) muscles of Cmpt and wild-type mice was examined by immunohistochemical staining of the myosin heavy chain (MHC) proteins. In addition, transcript levels of MHC isoforms were quantified by qPCR. Based on our results, all investigated muscles of Cmpt mice were significantly larger compared with that of wild-type mice, as characterized by fiber hyperplasia of different grades. Fiber hypertrophy was not present in TA; however, EDL muscles showed specific IIB fiber hypertrophy while the (I and IIA) fibers of SOL muscles were generally hypertrophied. Both the fast TA and EDL muscles of Cmpt mice contained significantly more glycolytic IIB fibers accompanied by a decreased number of IIX and IIA fibers; however, this was not the case for SOL muscles. In summary, despite the variances found in muscle cellularity between the different myostatin mutant mice, similar glycolytic shifts were observed in Cmpt fast muscles as in muscles from myostatin knockout mice.

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.

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.

Phytoecdysteroids and vitamin D analogues--similarities in structure and mode of action.

Phytoecdysteroids are plant steroids with identical or analogue structures to the molting hormone in arthropods. The ecdysteroids exert several beneficial effects on mammals, from which the most cited and deeply examined one is the increase of muscle size and strength. This shows similarities with the mode of action of the androgenic steroids but the ecdysteroids do not bind to the cytoplasmic/nuclear receptor of the mammalian steroids. These findings led to the hypothesis that ecdysteroids possibly bind to membrane bound receptors and they are likely to influence signal transduction pathways. Probably because of their closely related chemical structures, ecdysteroids exert some similar effects in vertebrates to those of the hormone 1 alpha,25-dihydroxyvitamin D3 (1,25D) which is produced in the kidney from 25-hydroxyvitamin D3, after being converted in the liver from Vitamin D3. 1,25D generates biological responses via both genomic and rapid, nongenomic mechanisms. Structure-activity relationship studies with different Vitamin D analogues could open the possibility to show that the two ways of action (genomic and nongenomic) can be influenced separately. The connection between the Vitamin D status and muscle function is already well-described in clinical studies, and several efforts have been made to evaluate the effect of Vitamin D deficiency or supplementation on muscle morphological changes and the underlying molecular mechanisms. This paper aims to summarize the main structural commonalities between the ecdysteroids, 1,25D and other Vitamin D analogues. The similarities in their effects and pathways that might be involved in the mechanism of action of these compounds will also be discussed.

The slow sarco/endoplasmic reticulum Ca2+ -ATPase declines independently of slow myosin in soleus muscle of diabetic rats.

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) isoforms are normally expressed in coordination with the corresponding myosin heavy chain (MyHC) isoforms in the fibers of skeletal muscle but this coordination is often disrupted in pathological conditions. In the streptozotocin-induced diabetes of rats (stz-rats), the soleus muscle showed peripheral neuropathy and the SERCA2a level decreased in type I (slow-oxidative) fibers compared to the control muscles, whereas the expression of the corresponding slow MyHC1 did not change. No difference was found at the mRNA and protein levels of SERCA and MyHC isoforms in the whole soleus, except that the level of the SERCA2a protein specifically declined in stz-rats compared to the controls. This shows that the coordinated expression of SERCA2a and MyHC1 is disrupted at the SERCA2a protein level in the diabetic soleus. The results are in line with previous observations that regulators of the Ca-homeostasis may adapt faster to type I diabetes than the contractile elements.

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.

20-Hydroxyecdysone increases fiber size in a muscle-specific fashion in rat.

20-Hydroxyecdysone (20E) is an ecdysteroid hormone that regulates moulting in insects. Interestingly, 20E is also found most abundantly in plant species and has anabolic effects in vertebrates, i.e. increasing muscle size without androgen influence. The effect of 20E on slow and fast fiber types of skeletal muscle has not been reported yet. Here we present that 20E affects the size (cross-sectional area, CSA) of the different fiber types in a muscle-specific manner. The effect on fiber size was modified by the distance from the site of the treatment and the presence of a regenerating soleus muscle in the animal. Besides the fiber size, 20E also increased the myonuclear number in the fibers of normal and regenerating muscles, suggesting the activation of satellite cells. According to our results 20E may provide an alternative for substitution of anabolic-androgenic steroids in therapeutic treatments against muscle atrophy.

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.

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.

Intermittent spontaneous breathing protects the rat diaphragm from mechanical ventilation effects.

OBJECTIVE: Short-term mechanical ventilation has been proven to reduce diaphragm force and fiber dimensions. We hypothesized that intermittent spontaneous breathing during the course of mechanical ventilation would minimize the effects of mechanical ventilation on diaphragm force and expression levels of transcription factors (MyoD and myogenin). DESIGN: Randomized, controlled experiment. SETTING: Animal basic science laboratory. SUBJECTS: Male Wistar rats, weighing 350-500 g. INTERVENTIONS: Anesthetized and tracheotomized rats were submitted to either 24 hrs of spontaneous breathing (SB, n = 5), 24 hrs of continuous controlled mechanical ventilation (CMV, n = 7), or controlled mechanical ventilation with intermittent spontaneous breathing: 60 mins every 5 hrs of mechanical ventilation repeated four times (ISB60, n = 8) or 5 mins every 5 hrs 55 mins of mechanical ventilation repeated four times (SB5, n = 9). They were compared with control animals free from intervention (C, n = 5). MEASUREMENTS AND MAIN RESULTS: The profile of the diaphragm force-frequency curve of the controls and SB group was significantly different from that of the ISB and CMV groups; especially, the mean asymptotic force was less in the ISB and CMV compared with controls and SB. CMV resulted in a significant decrease in the diaphragm type I (-26%, p < .05 vs. C) and type IIx/b (-39%, p < .005 vs. C and SB) cross-sectional area, whereas this was not observed in the ISB groups. Diaphragm MyoD protein expression was significantly decreased after ISB60 (-35%, p < .0001 vs. C and SB) and even more after CMV (-73%, p < .0001 vs. others). The same pattern was observed with myogenin protein levels. Positive relationships between diaphragm MyoD and myogenin protein levels and diaphragm force were observed. CONCLUSIONS: The data demonstrated that intermittent spontaneous breathing during the course of mechanical ventilation may minimize the deleterious effect of controlled mechanical ventilation on diaphragm force, fiber dimensions, and expression of transcription factors.