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F Wuytack

Publications and source records attributed to F Wuytack.

At least 19 recordsLinked to original sources
PubMed2000

The Golgi PMR1 P-type ATPase of Caenorhabditis elegans. Identification of the gene and demonstration of calcium and manganese transport.

In recent years, it has been well established that the Ca(2+) concentration in the lumen of intracellular organelles is a key determinant of cell function. Despite the fact that essential functions of the Golgi apparatus depend on the Ca(2+) and Mn(2+) concentration in its lumen, little is known on the transport system responsible for ion accumulation. The Golgi ion pump PMR1 has been functionally studied only in yeast. In humans, mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. We report here the identification of the PMR1 homologue in the model organism Caenorhabditis elegans and after ectopic expression the direct study of its ion transport in permeabilized COS-1 cells. The C. elegans genome is predicted to contain a single PMR1 orthologue on chromosome I. We found evidence for alternative splicing in the 5'-untranslated region, but no indication for the generation of different protein isoforms. C. elegans PMR1 overexpressed in COS-1 cells transports Ca(2+) and Mn(2+) with high affinity into the Golgi apparatus in a thapsigargin-insensitive manner. Part of the accumulated Ca(2+) can be released by inositol 1,4,5-trisphosphate, in agreement with the idea that the Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca(2+) store.

Adenosine Triphosphatases↗
PubMed2000

Low temperature molecular adaptation of the skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA 1) in the wood frog (Rana sylvatica).

We have compared the primary sequence and enzymatic properties of the sarcoplasmic reticulum Ca(2+)-ATPases from a cold-tolerant frog Rana sylvatica with those of a closely related cold-intolerant frog, Rana clamitans. Sarcoplasmic reticulum isolated from leg muscles of both species contains a major protein ( approximately 100 kDa) that reacts with a monoclonal antibody against sarco(endo)plasmic reticulum Ca(2+)-ATPase type 1 (SERCA1). The apparent molecular mass of R. sylvatica SERCA1 is 115 kDa, whereas that of R. clamitans is 105 kDa. However, the deduced amino acid sequences obtained from cDNAs do not indicate a difference in molecular weight, thus suggesting post-translational protein modification of R. sylvatica SERCA1. Comparison of the temperature dependence of both ATP hydrolysis and Ca(2+) transport indicates that R. sylvatica SERCA1 exhibits significantly lower activation energy below 20 degrees C and an approximately 2-fold greater Ca(2+)-ATPase activity near 0 degrees C. Furthermore, R. sylvatica SERCA1 exhibits simple Michaelis-Menten kinetics with ATP and Ca(2+) as opposed to the two-site ATP kinetics and positive cooperativity with Ca(2+) observed for R. clamitans and mammalian SERCA1s. Cooperativity has been linked to protein-protein interaction in SERCA1, and this property may be altered in R. sylvatica SERCA1. Primary sequence comparison shows that R. sylvatica SERCA1 exhibits seven unique amino acid substitutions, three of which are in the ATP binding domain. We also report for the first time the presence of alternative splicing in the frog, resulting in isoforms SERCA1a and SERCA1b. Thus, it appears that the low temperature muscle contractility of R. sylvatica can be explained partially by significant functional and structural differences in SERCA1.

Adaptation, Physiological↗
PubMed2000

Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium.

During development fast-contracting atrial and ventricular chambers develop from a peristaltic-contracting heart tube. This study addresses the question of whether chamber formation is paralleled by a matching expression of the sarcoplasmic reticulum (SR) Ca(2+) pump. We studied indo-1 Ca(2+) transients elicited by field stimulation of linear heart tube stages and of explants from atria and outflow tracts of the prototypical preseptational E13 rat heart. Ca(2+) transients of H/H 11+ chicken hearts, which constitute the prototypic linear heart tube stage, were sensitive to verapamil only, indicating a minor contribution of Ca(2+)-triggered SR Ca(2+) release. Outflow tract transients displayed sensitivity to the inhibitors similar to that of the linear heart tube stages. Atrial Ca(2+) transients disappeared upon addition of ryanodine, tetracaine, or verapamil, indicating the presence of Ca(2+)-triggered SR Ca(2+) release. Quantitative radioactive in situ hybridization on sections of E13 rat hearts showed approximately 10-fold higher SERCA2a mRNA levels in the atria compared to nonmyocardial tissue and approximately 5-fold higher expression in compact ventricular myocardium. The myocardium of atrioventricular canal, outflow tract, inner curvature, and ventricular trabecules displayed weak expression. Immunohistochemistry on sections of rat and human embryos showed a similar pattern. The significance of these findings is threefold. (i) A functional SR is present long before birth. (ii) SR development is concomitant with cardiac chamber development, explaining regional differences in cardiac function. (iii) The pattern of SERCA2a expression underscores a manner of chamber development by differentiation at the outer curvature, rather than by segmentation of the linear heart tube.

Animals↗
PubMed2000

Corticosteroids decrease mRNA levels of SERCA pumps, whereas they increase sarcolipin mRNA in the rat diaphragm.

1. In order to explore the potential role of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA)-type pumps and of their modulators phospholamban (PLB) and sarcolipin (SLN) in the functional alterations of the diaphragm induced by corticosteroid treatment, expression of SERCA, PLB and SLN was assessed by RT-PCR in the diaphragm of rats treated daily for 5 days either with triamcinolone (80 mg kg-1, n = 8) or with saline (control; 0.6 ml, n = 8). 2. Triamcinolone treatment reduced the normalised overall amount of all SERCA mRNA in diaphragm by 70 % compared to controls (P < 0.05). This reduction was accounted for by a relatively larger decrease in the SERCA1 mRNA (-69 %, P < 0.05) whilst the decrease in SERCA2 mRNA (-49 %, P = 0.09) did not reach statistical significance. As a result the relative proportion of SERCA2 mRNA was increased from 43 +/- 7 % in control diaphragm to 52 +/- 4 % after triamcinolone treatment (P < 0.05). 3. Only the adult isoform of SERCA1 (i.e. SERCA1a) mRNA was found in the diaphragm of the 15-week-old control rats. Furthermore, triamcinolone treatment resulted in reduced levels of SERCA2a (-40 %, P < 0.05) and increased levels of SLN mRNA (+100 %, P < 0.05), while the decrease in PLB mRNA (-31 %, P = 0.277) did not reach statistical significance. SERCA1b, SERCA2b and SERCA3 mRNA levels fell below the detection limit in the diaphragm of both control and triamcinolone-treated rats. 4. Compared to control diaphragm, control rat heart showed a relatively high PLB/(SERCA1 + SERCA2) mRNA ratio of 7.88 while this ratio amounted only to 0.16 in control extensor digitorum longus (EDL) muscle. Remarkably, the SLN/(SERCA1 + SERCA2) mRNA ratio in normal cardiac muscle (0.96) was nearly the same as in diaphragm, but in EDL it amounted to only 0.05 that in diaphragm. This indicates the very low expression of SLN in rat EDL. 5. These data reveal that considerable alterations in SERCA mRNA levels accompany the functional changes seen in diaphragm after corticosteroid treatment. The relatively larger decrease in SERCA1 mRNA is in agreement with the selective type II fibre atrophy previously observed in the diaphragm of triamcinolone-treated rats, but the magnitude of SERCA alterations is more pronounced than expected on the basis of the structural changes in the diaphragm. The increase in SLN mRNA levels may represent a compensatory mechanism.

Adrenal Cortex Hormones↗
PubMed2000

The expression of SR calcium transport ATPase and the Na(+)/Ca(2+)Exchanger are antithetically regulated during mouse cardiac development and in Hypo/hyperthyroidism.

The mouse has been used extensively for generating transgenic animal models to study cardiovascular disease. Recently, a number of transgenic mouse models have been created to investigate the importance of sarcoplasmic reticulum (SR) Ca(2+)transport proteins in cardiac pathophysiology. However, the expression and regulation of cardiac SR Ca(2+)ATPase and other Ca(2+)transport proteins have not been studied in detail in the mouse. In this study, we used multiplex RNase mapping analysis to determine SERCA2, phospholamban (PLB), and Na(+)/Ca(2+)-exchanger (NCX-1) gene expression throughout mouse heart development and in hypo/hyperthyroid animals. Our results demonstrate that the expression of SERCA2 and PLB mRNA increase eight-fold from fetal to adult stages, indicating that SR function increases with heart development. In contrast, the expression of the Na(+)/Ca(2+)-exchanger gene is two-fold higher in fetal heart compared to adult. Our study also makes the important observation that in hypothyroidic hearts the NCX-1 mRNA and protein levels were upregulated, whereas the SERCA2 mRNA/protein levels were downregulated. In hyperthyroidic hearts, however, an opposite response was identified. These findings are important and point out that the expression of NCX-1 is regulated antithetically to that of SERCA2 during heart development and in response to alterations in thyroid hormone levels.

Animals↗
PubMed2000

Myostatin levels in regenerating rat muscles and in myogenic cell cultures.

Myostatin is a newly described member of the TGF-beta superfamily acting as a secreted negative regulator of skeletal muscle mass in several species, but whose mode of action remains largely unknown. In the present work, we followed the myostatin mRNA and protein levels in rat soleus and extensor digitorum longus (EDL) muscles regenerating in vivo from notexin-induced necrosis, and the myostatin transcript levels in two different in vitro myogenic differentiation models: i.e. in mouse BC3H1 and C2Cl2 cultured cells. The in vivo regenerating rat skeletal muscles showed a characteristic time-dependent expression of myostatin mRNA. After notexin injection, the transcript levels dropped below the detection limit on day 1 in soleus and close to the detection limit on day 3 in EDL, then increased to a maximum on day 7 in soleus and after 28 days finally reached the control values in both types of muscles. In contrast, the myostatin protein levels increased dramatically on the first days of regeneration in both muscles, i.e. at the time when its transcript level was low. Later on the myostatin protein level gradually declined to normal in soleus while in EDL it stayed high some days longer and decreased to normal on days 21-28. In vitro proliferating myoblasts produced low level of myostatin mRNA, which increased upon induction of differentiation suggesting that functional innervation is no prerequisite for myostatin expression. Myostatin production in vitro seems not to be dependent on myocyte fusion either, since it is observed in differentiated BC3H1 cells, which are defective in myofiber formation.

Animals↗
PubMed2000

Abnormal intracellular ca(2+)homeostasis and disease.

A whole range of cell functions are regulated by the free cytosolic Ca(2+)concentration. Activator Ca(2+)from the extracellular space enters the cell through various types of Ca(2+)channels and sometimes the Na(+)/Ca(2+)-exchanger, and is actively extruded from the cell by Ca(2+)pumps and Na(+)/Ca(2+)-exchangers. Activator Ca(2+)can also be released from internal Ca(2+)stores through inositol trisphosphate or ryanodine receptors and is taken up into these organelles by means of Ca(2+)pumps. The resulting Ca(2+)signal is highly organized in space, frequency and amplitude because the localization and the integrated free cytosolic Ca(2+)concentration over time contain specific information. Mutations or functional abnormalities in the various Ca(2+)transporters, which in vitro seem to induce trivial functional alterations, therefore, often lead to a plethora of diseases. Skeletal-muscle pathology can be caused by mutations in ryanodine receptors (malignant hyperthermia, porcine stress syndrome, central-core disease), dihydropyridine receptors (familial hypokalemic periodic paralysis, malignant hyperthermia, muscular dysgenesis) or Ca(2+)pumps (Brody disease). Ca(2+)-pump mutations in cutaneous epidermal keratinocytes and cochlear hair cells lead to, skin diseases (Darier and Hailey-Hailey) and hearing/vestibular problems respectively. Mutated Ca(2+)channels in the photoreceptor plasma membrane cause vision problems. Hemiplegic migraine, spinocerebellar ataxia type-6, one form of episodic ataxia and some forms of epilepsy can be due to mutations in plasma-membrane Ca(2+)channels, while antibodies against these channels play a pathogenic role in all patients with the Lambert-Eaton myasthenic syndrome and may be of significance in sporadic amyotrophic lateral sclerosis. Brain inositol trisphosphate receptors have been hypothesized to contribute to the pathology in opisthotonos mice, manic-depressive illness and perhaps Alzheimer's disease. Various abnormalities in Ca(2+)-handling proteins have been described in heart during aging, hypertrophy, heart failure and during treatment with immunosuppressive drugs and in diabetes mellitus. In some instances, disease-causing mutations or abnormalities provide us with new insights into the cell biology of the various Ca(2+)transporters.

Animals↗
PubMed2000

[Intracellular calcium: physiology and physiopathology].

Many important aspects of our life are regulated by the free cytosolic Ca2+ concentration. The intracellular Ca2+ signal is regulated both in space, frequency and amplitude. Each cell chooses a unique set of Ca2+ signals to control its function. Ca2+ signal transduction is based on rises in free cytosolic Ca2+ concentration. Ca2+ can come from the extracellular space or be released from intracellular stores. Extracellular Ca2+ enters the cell through various types of plasma-membrane Ca2+ channels and leaves the cell using Ca2+ pumps and Na+/Ca(2+)-exchangers. Ca2+ is accumulated in intracellular stores by means of Ca2+ pumps and is released via inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors. Mutations or abnormalities in one of the above mentioned Ca(2+)-transporting proteins can lead to disease. Skeletal-muscle pathology can be caused by abnormal ryanodine receptors (malignant hyperthermia, porcine stress syndrome, central core disease), plasma-membrane Ca2+ channels (hypokalemic periodic paralysis, muscular dysgenesis mice, paraneoplastic Lambert-Eaton myasthenia syndrome) or Ca2+ pumps (Brody disease). Neurologic disorders can be related to altered function of plasma-membrane Ca2+ channels (episodic ataxia type 2, spinocerebellar ataxia type 6, familial hemiplegic migraine, glutamate excitotoxicity, tottering, leaner, lethargic and stargazer mice), IP3 receptors (Lowe's oculocerebrorenal syndrome, manic depression, Alzheimer's disease, opisthotonos mice) and Ca2+ pumps (deafwaddler mouse and wriggle mouse sagami). Two skin diseases are caused by Ca(2+)-pump mutations (Darier disease and Hailey-Hailey disease). Incomplete X-linked congenital stationary night blindness is caused by a mutation in the plasma-membrane Ca2+ channels in rods and cones.

Animals↗
PubMed1999

Calcium handling proteins in isolated spinal motoneurons.

Amyotrophic lateral sclerosis is characterized by motoneuron degeneration, in which glutamate-induced cell death is thought to play a pathogenic role. This excitotoxic process is mediated by cytosolic Ca2+ overload. The glutamatergic ionotropic channel molecules, which constitute a major route of Ca2+ entry, were present on cultured spinal motoneurons. Using ratio RT-PCR, the relative presence in isolated motoneurons of the GluR subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor was evaluated. GluR1 and GluR2 mRNAs were present abundantly, while GluR3 and GluR4 mRNAs were much less abundant. The relative amount of mRNAs encoding the different protein isoforms responsible for Ca2+ uptake into the internal stores and for controlled release of Ca2+ from these stores was also determined. For the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), only the SERCA2b class 4 splice variant was found. The inositol 1,4,5-trisphosphate receptor (IP3R) mRNAs were mainly transcribed from the IP3RI and IP3RII genes. Heterogeneity was also observed for the ryanodine receptors (RyR) as the RyR1, RyR2 and RyR3 mRNAs were present.

Animals↗
PubMed1999

Prolonged passive stretch of rat soleus muscle provokes an increase in the mRNA levels of the muscle regulatory factors distributed along the entire length of the fibers.

The mRNA levels of the adult and the neonatal sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCA1a and SERCA1b, respectively) and those of the muscle regulatory factors (MRFs: myoD, myf-5, myogenin, MRF4) have been assessed by RT PCR in rat soleus muscles immobilized for 3 days in an extended position (passive stretch). The transcript level of the fast type SERCA1a Ca(2+)-transport ATPase decreased to half of its normal value, whereas that of neonatal SERCA1b isoform increased 5-fold above control in stretched muscles. Immunostaining of muscle cross sections showed that the fraction of fibers expressing the SERCA1a protein was decreased evenly along the length of the stretched muscles indicating that a transformation occurred of fast fibers to slow ones. The mRNA levels of MRFs were elevated 3- to 6-fold above the normal level and were distributed evenly along the length of the stretched muscles. However in the controls these transcripts were more abundant at both ends of the muscle. The stretch increased the level of myoD and immunocytochemistry showed the expression of myoD protein in a number of nuclei of the stretched muscles whereas it was practically undetectable by this method in the control muscles. Western blotting did not indicate a significant stretch-induced increase in the level of the myogenin protein, in spite of the fact that immunocytochemistry tended to show more myogenin-positive nuclei in stretched muscles as compared to the controls. These data indicate that after 3 days of passive stretch the central and the terminal parts of the soleus muscle adapt similarly by increasing the levels of the MRFs, by decreasing the overall levels of the fast SERCA1-type of ATPase and by partially re-establishing a neonatal mode of alternative SERCA1 transcript splicing resulting in an increased SERCA1b/1a ratio.

Acetylcholinesterase↗
PubMed1999

Expression patterns of sarco/endoplasmic reticulum Ca(2+)-ATPase and inositol 1,4,5-trisphosphate receptor isoforms in vascular endothelial cells.

Expression patterns of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) and inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were studied in endothelial cells at the mRNA level by ratio RT-PCR technique and subsequent restriction-enzyme analysis. Three types of cells have been used in the present study: rat adrenal medulla microvascular endothelial cells (RAMEC), rat aortic endothelial cells (RAEC), and human umbilical vein endothelial cells (HUVEC). Our data show the presence of multiple SERCA and IP3R isoforms in each type of endothelial cells. Freshly isolated HUVEC were an exception in this respect since they contained only SERCA3 without SERCA2b messengers. The expression patterns changed upon cell proliferation: SERCA3 and IP3R-1 messengers decreased, while IP3R-3 increased with culturing. Upon cell differentiation, induced by culturing the cells on Matrigel, the expression pattern of the IP3R changed even further in all endothelial cell types: IP3R-1 was reduced in all three cell kinds, while IP3R-3 raised significantly in RAEC and RAMEC. In HUVEC the expression of SERCA returned, upon differentiation, to the levels observed in the freshly isolated cells. Thus, the plasticity of expression of various SERCA and IP3R isoforms shows that possibly different Ca2+ pools may play distinct roles in cell proliferation and differentiation.

Adrenal Medulla↗
PubMed1998

Structure of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene. Promoter analysis and alternative splicing of the SERCA3 pre-mRNA.

Human chromosome 17-specific genomic clones extending over 90 kilobases (kb) of DNA and coding for sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The presence of the D17S1828 genetic marker in the cosmid contig enabled us to map the SERCA3 gene (ATP2A3) 11 centimorgans from the top of the short arm p of chromosome 17, in the vicinity of the cystinosis gene locus. The SERCA3 gene contains 22 exons spread over 50 kb of genomic DNA. The exon/intron boundaries are well conserved between human SERCA3 and SERCA1 genes, except for the junction between exons 8 and 9 which is found in the SERCA1 gene but not in SERCA3 and SERCA2 genes. The transcription start site (+1) is located 152 nucleotides (nt) upstream of the AUG codon. The 5'-flanking region, including exon 1, is embedded in a 1.5-kb CpG island and is characterized by the absence of a TATA box and by the presence of 14 putative Sp1 sites, 11 CACCC boxes, 5 AP-2-binding motifs, 3 GGCTGGGG motifs, 3 CANNTG boxes, a GATA motif, as well as single sites for Ets-1, c-Myc, and TFIIIc. Functional promoter analysis indicated that the GC-rich region (87% G + C) from -135 to -31 is of critical importance in initiating SERCA3 gene transcription in Jurkat cells. Exon 21 (human, 101 base pairs; mouse, 86 base pairs) can be alternatively excluded, partially included, or totally included, thus generating, respectively, SERCA3a (human and mouse, 999 amino acids (aa)), SERCA3b (human, 1043 aa; mouse, 1038 aa), or SERCA3c (human, 1024 aa; mouse, 1021 aa) isoforms with different C termini. Expression of the mouse SERCA3 isoforms in COS-1 cells demonstrated their ability to function as active pumps, although with different apparent affinities for Ca2+.

Alternative Splicing↗
PubMed1998

Regulation of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2 gene transcript in neuronal cells.

The sarco/endoplasmic reticulum Ca(2+)-transport ATPase (SERCA2) pre-mRNA is alternatively processed in a tissue-specific manner. At its 3' end, two 5' splice donor sites compete for the same 3' acceptor splice site (3'A). While the upstream 5' donor splice site (5'D1) is used in muscle cells giving rise to the class 1 mRNA, the downstream one (5'D2) is exclusively used in neuronal cells generating the class 4 mRNA. Using a neuroblastoma cell line and a minigene containing the 3' end of the SERCA2 gene, we have investigated the regulation of the neuronal-type of splicing. We have shown that a strong 3'A is required for splicing because exchanging it for a weaker one abolishes splicing. A second region spanning the entire exon 25 downstream of the 3'A is also necessary for the repression of the muscle-specific splicing in neuronal cells. In addition the tissue-specific (muscle/neuron) selection of the appropriate 5' donor splice site seems to be determined by at least two distinct but adjacent negative cis-active elements located in the last 237 nt of the optional exon 24. The upstream negative element controls the neuronal splicing while the downstream one represses the muscle-specific splicing in neuronal cells. It is suggested that the cis-active elements in the gene transcript are the target of trans-acting factors that are responsible for the repression of neuronal- or muscle-specific splicing in a tissue-specific manner.

Animals↗
PubMed1998

Distribution and isoform diversity of the organellar Ca2+ pumps in the brain.

The gene family of organellar-type Ca2+ transport ATPases consists of three members. SERCA1 is expressed exclusively in fast skeletal muscle; SERCA2 is ubiquitously expressed, whereas SERCA3 is considered to be mainly expressed in cells of the hematopoietic lineage and in some epithelial cells. In the brain, the organellar-type Ca2+ transport ATPases are almost exclusively transcribed from the SERCA2 gene. Four different SERCA2 mRNAs have been described (classes 1-4). However, unlike in nonneuronal cells, which express the class 1, 2, and 3 splice variants, the main SERCA2 mRNA in the brain is the class 4 messenger. Similar to classes 2 and 3, the class 4 codes for the ubiquitously expressed SERCA2b protein. Recently, we have reported the distribution of the SERCA isoforms in the brain (Baba-Aissa et al., 1996a,b). SERCA2b was present in most neurons of all investigated brain regions. The highest levels were found in the Purkinje neurons of the cerebellum and in the pyramidal cells of the hippocampus. Interestingly, SERCA3 and SERCA2a are coexpressed along with SERCA2b in the Purkinje neurons, but are weakly expressed in the other brain regions if present at all. Since these three protein isoforms have a different affinity for Ca2+, their possible roles in relation to Ca2+ stores in neurons are discussed.

Animals↗
PubMed1998

Expression of the sarco/endoplasmic reticulum Ca(2+)-transport ATPase protein isoforms during regeneration from notexin-induced necrosis of rat soleus muscle.

Expression levels of fast-twitch (SERCA1), slow-twitch (SERCA2a) and "housekeeping" (SERCA2b) isoforms of the sarcoplasmic reticulum Ca(2+)-transport ATPase were monitored during regeneration of rat soleus muscles following necrosis induced by the toxin notexin at the tissue level by Western blot analysis and at the cellular level by immunocytochemical analysis. Due to necrosis, levels of muscle-specific SERCA1 and SERCA2a isoforms dropped to low levels on the third day after injection of the toxin. Subsequently, during regeneration both isoforms recovered but with a different time course. Expression of the fast type SERCA1 increased first. This type showed its most pronounced increase between day 3 and 10. Expression of the slow type SERCA2a was biphasic. After an increase to approximately one third of the control value on days 5-10, it showed its main increase up to the control level between day 10 and 21. Expression levels of the house-keeping SERCA2b isoform remained relatively constant throughout the 4 weeks of regeneration. Between day 10 and 28, when new innervation is established, SERCA2a expression spread gradually over almost all fibers whereas the number of SERCA1-expressing fibers decreased and only a limited number of fibers co-expressed SERCA1 and SERCA2a. At 4 weeks of regeneration, expression of the fast isoform was found only in 12% of the fibers, whereas the slow form was found in 98% of the fibers. In the contralateral untreated soleus muscles, 26% SERCA1-positive and 81% SERCA2a-positive fibers were observed. Immunocytochemical analysis showed that SERCA1 and SERCA2a were co-expressed with fast and slow myosin isoforms in fibers of normal muscles but in regenerated muscle only slow myosin and slow SERCA isoforms correlated. The results show that during regeneration levels of fast and slow SERCA proteins change in a similar way as their mRNAs do. However, in regenerated soleus, unlike in normal muscle, expression of slow SERCA is coregulated only with the slow myosin isoform. This finding is in agreement with the fact that the number of slow type fibers is increased in regenerated soleus.

Animals↗
PubMed1998

mRNA levels of myogenic regulatory factors in rat slow and fast muscles regenerating from notexin-induced necrosis.

The transcript levels of the myogenic regulatory factors (myoD, myf5, myogenin and MRF4) were measured by RT PCR in rat soleus (slow) and EDL (fast) muscles which were regenerating from notexin-induced necrosis. Some muscle fibers in the EDL were more resistant to the toxin, therefore the necrosis and the dominance of myoblasts were delayed for two days in EDL compared to soleus. In spite of this shift in time-course of necrosis, both types of muscle presented roughly similar, although variable, changes in the expression pattern of MRF mRNA levels. For both muscles, the myoD mRNA was upregulated on the first day after administration of the toxin, whereas concomitantly myf-5 mRNA disappeared but showed a substantial increase in later stages of regeneration. In contrast, the mRNA levels of the late MRFs myogenin and MRF4 decreased on day one only in the soleus, then increased on day three in both types of muscle. Meanwhile in EDL the level of MRF4 mRNA remained relatively normal. Four weeks after administration of the toxin the mRNA levels for each of the MRFs returned to nearly control levels. This shows that in spite of the different time course of the necrosis and regeneration, also documented by the microscopical morphology and the skeletal actin mRNA levels of the muscles, the level of MRF transcripts changed according to a quite predictable pattern; the upregulation corresponded to myoblast activation and the downregulation to the reinnervation.

Animals↗