a1_We have investigated expression of skeletal calsequestrin (CSQ1) and fiber type composition in normal and regenerated fast and slow skeletal muscles and in the left heart ventricles of euthyroid (EU), hypothyroid (HY) and hyperthyroid (TH) adult inbred Lewis strain rats. The CSQ1 level was determined by SDS-PAGE followed by Western blot analysis. CSQ1 gene expression was assessed using reverse transcription and subsequent real time polymerase chain reaction. Muscle regeneration was achieved by intramuscular grafting of either soleus or extensor digitorum longus (EDL) from 3- to 4-week-old rats to either EDL or soleus muscle of 2-month-old rats. The fiber type composition was assessed by a stereological method applied to stained muscle cross sections. We found that the protein and mRNA levels for CSQ1 were highest in the EDL muscle, the relative CSQ1 protein levels in the soleus muscle were two times lower and the transcript levels more than 5 times lower compared to the EDL. In the left heart ventricle, protein isoform and CSQ1 transcript were also present, although at protein level, CSQ1 was hardly detectable. TH status increased and HY status decreased the expression of CSQ1 in the EDL, but its relative levels in the soleus and in the heart did not change. The regenerated soleus transplanted into EDL, as well as EDL transplanted into soleus exhibited protein and mRNA levels of CSQ1 corresponding to the host muscle and not to the graft source. TH status increased the percentages of the fastest 2X/D and 2B fibers at the expense of slow type 1 and fast 2A fibers in the EDL and that of fast 2A fibers in the soleus at the expense of slow type 1 fibers. HY status led to converse fiber type changes., a2_We suggest that the observed changes in CSQ1 levels in TH and HY compared to EU rats can be related to fiber type changes caused by alteration of the thyroid status rather than to the direct effect of thyroid hormones on CSQ1 gene expression., T. Soukup ... [et al.]., and Obsahuje seznam literatury
We examined the effects of the unilateral heterochronous isotransplantation on the fiber type composition and myosin heavy chain (MyHC) isoform content of unoperated slow soleus and fast extensor digitorum longus muscles of female inbred Lewis strain rats. Comparison was made between “control” unoperated muscles of experimental rats (after intramuscular transplantation surgery) with the corresponding muscles of completely naive (unoperated) rats of three age groups (5-, 8- and 14-month-old). This was done in order to ascertain whether these muscles can be used as reliable controls to the transplanted and host muscles for our ongoing grafting experiments. The fiber type composition was determined by assessing the histochemical reaction for myofibrillar adenosine triphosphatase, the MyHC isoform content was determined immunocytochemically using monoclonal antibodies specific to different MyHC isoforms and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Our experiments show that the heterochronous intramuscular isotransplantation procedure had no significant effect on the fiber type composition and MyHC isoform content of the “control” unoperated muscles of the experimental rats when compared to the corresponding muscles of the naive animals. Furthermore, the duration and type of isotransplantation did not also lead to differences among corresponding “control” muscles of experimental animals. We conclude that the unoperated muscles of the experimental rats can be used as controls in our current transplantation project dealing with long-term grafting experiments., T. Soukup, V. Smerdu, G. Zachařová., and Obsahuje seznam literatury
a1_We analyzed fiber type composition of soleus and extensor digitorum longus (EDL) muscles of 3- to 19-month-old male and female inbred Lewis rats using histochemical demonstration of mATPase activity. The rats were divided into four groups of the mean age of 3, 6, 9 and 14 months. We found that the soleus muscle of 3-month-old rats contained significantly more of fast 2A fibers and less of slow type 1 fibers compared to older rats, while no significant difference was found between female and male rats at any age group. In contrast, we found no significant difference in the EDL fiber type composition among the age groups, but we found that the EDL muscle of female rats contained significantly less 2A fibers and more 2B fibers than that of male animals. Our results thus revealed an age difference in the soleus muscle and a sex difference in the EDL muscle among postnatal Lewis rats. The number of slow type 1 fibers in the soleus muscle varied between 87 and 100 % and that of 2A fibers between 13 and 0 %. In the EDL the percentage of type 1 fibers varied between 2.6 and 8.7 %, that of 2A fibers between 12.6 and 25.8 % and that of 2B fibers between 70.4 and 81.6 %. Both muscles thus exhibited a considerable degree of variability among individual animals even in the same age group. Furthermore, a comparison of the Lewis rats with literature data of other rat strains showed that the number of fast 2A fibers in the soleus muscle of 4-month-old and older animals decreased in this order: SHR > Lister Hooded > Fisher 344 > Sprague- Dawley > Wistar > WBN/Kob > Lewis strain, being almost 20 % in the SHR and less than 2 % in the Lewis rats. In contrast, the “fastest” composition (judged according to the percentage of the fastest 2B fibers) of the EDL muscle was demonstrated by Lewis, Wistar and Fisher 344 rats (about 75 %), while Sprague-Dawley and WBN/Kob rats contained only about 50 % of 2B fibers., a2_The percentage of slow type 1 fibers in the EDL was low in all strains (about 5 %). Our results thus show that the individual, age and sex as well as inter-strain differences in muscle fiber type composition should not be ignored when comparing results of different studies. We also demonstrated that the inbred Lewis strain appears to have more “specialized” muscle composition, as its soleus is the “slowest” and its EDL is the “fastest” among the routinely used rat strains., P. Novák, G. Zachařová, T. Soukup., and Obsahuje bibliografii a bibliografické odkazy
We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDS-PAGE and analyzed it by two subsequent mass spectrometry techniques. After tryptic digestion, the obtained peptides were identified by Matrix-Assisted Laser Desorption/Ionisation reflectron Time of Flight mass spectrometry (MALDI-TOF MS) and sequenced by Liquid chromatography tandem mass spectrometry (ESI LC/MS/MS). The analyzed peptides proportionally covered 30 % of the 2b MyHC isoform sequence. The results suggest that the primary structure is identical with the highest probability to a NCBI database record ref|NP_062198.1|, representing the last updated record of rat 2b isoform. Nonetheless, four peptides carrying amino acid substitution(s) in comparison with the NCBI database record were identified., J. Žurmanová, D. Maláčová, F. Půta, P. Novák, J. Říčný, T. Soukup., and Obsahuje bibliografii a bibliografické odkazy
Previous data suggest that type 1 diabetes mellitus leads to the deterioration of myocardial intercellular communication mediated by connexin-43 (Cx43) channels. We therefore aimed to explore Cx43, PKC signaling and ultrastructure in non -treated and omega-3 fatty acid (omega-3) treated spontaneously diabetic Goto-Kakizaki (GK) rats considered as type 2 diabetes model. Four-week-old GK and non-diabetic Wistar-Clea rats were fed omega -3 (200 mg/kg/day) for 2 months and compared with untreated rats. Realtime PCR and immunoblotting were performed to determine Cx43, PKC- epsilon and PKC-delta expression. In situ Cx43 was examined by immunohistochemistry and subcellular alterations by electr on microscopy. Omega-3 intake reduced blood glucose, triglycerides, and cholesterol in diabetic rats and this was associated with improved integrity of cardiomyocytes and capillaries in the heart. Myocardial Cx43 mRNA and protein levels were higher in diab etic versus non- diabetic rats and were further enhanced by omega-3. The ratio of phosphorylated (functional) to non-phosphorylated Cx43 was lower in diabetic compared to non- diabetic rats but was increased by omega-3, in part due to up -regulation of PKC-epsilon. In addition, proapoptotic PKC-delta expression was decreased. In conclusion, spontaneously diabetic rats at an early stage of disease benefit from omega-3 intake due to its hypoglycemic effect, upregulation of myocardial Cx43, and preservation of cardiovascular ultrastructure. These findings indicates that supplementation of omega-3 may be beneficial also in the management of diabetes in humans., J. Radosinska, L. H. Kurahara, K. Hiraishi, C. Viczenczova, T. Egan Benova, B. Szeiffova Bacova, V: Dosenko, J. Navarova, B. Obsitnik, I. Imanaga, T. Soukup, N. Tribulova., and Obsahuje bibliografii
Recently, we have established that slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles of euthyroid (EU) Lewis rats posses the same proportions between their four myosin heavy chain (MyHC) mRNAs , protein isoforms and fiber types as determined by real time RT-PCR, SDS-PAGE and 2-D stereological fiber type analysis, respectively. In the present paper we investigated if these proportions are maintained in adult Lewis rats with hyperthyroid (HT) and hypothyroid (HY) status. Although HT and HY states change MyHC isoform expression, results from all three methods showed that proportion between MyHC mRNA-1, -2a, -2x/d, -2b , protein isoforms MyHC-1, -2a, -2x/d, -2b and to lesser extent also fiber types 1, 2A, 2X/D, 2B were preserved in both SOL and EDL muscles. Furthermore, in the SOL muscle mRNA expression of slow MyHC-1 remained up to three orders higher compared to fast MyHC transcripts, which explains the predominance of MyHC-1 isoform and fiber type 1 even in HT rats. Although HT status led in the SOL to increased expression of MyHC-2a mRNA, MyHC-2a isoform and 2A fibers, it preserved extremely low expression of MyHC-2x and -2b mRNA and protein isoforms, which explains the absence of pure 2X/D and 2B fibers. HY status, on the other hand, almost completely abolished expression of all three fast MyHC mRNAs , MyHC protein isoforms and fast fiber types in the SOL muscle. Our data present evidence that a correlation between mRNA , protein content and fiber type composition found in EU status is also preserved in HT and HY rats., T. Soukup, M. Diallo., and Obsahuje bibliografii
In order to re-evaluate the presence and relative quantity of 2b and 2x/d myosin heavy chain (MyHC) transcripts in rat slow soleus muscle by using real time RT-PCR we have compared the available relevant cDNA sequences and designed a new set of primers having similar melting temperatures, matching separate MyHC exons in the regions of maximal differences in MyHC coding sequences, and containing G or C at the 3́-end. These also yielded PCR products of corresponding length, which is an important requirement for real time RT-PCR quantification. The experiments were performed on 8- month-old inbred female Lewis strain rats used in our current study of regenerating transplanted muscles. The real time RT-PCR measurement confirmed the expression of all four MyHC mRNAs (type 1, 2a, 2x/d and 2b) in both fast extensor digitorum longus and slow soleus muscles, although in the soleus muscle of adult rats, only type 1 and 2a protein isoforms can be usually detected, J. Žurmanová, F. Půta, R. Stopková, T. Soukup., and Obsahuje bibliografii a bibliografické odkazy
Red palm oil (RPO) is a rich natural source of antioxidant vitamins, namely carotenes, tocopherols and tocotrienols. However, it contains approximately 50 % saturated fatty acids the regular consumption of which could negatively modify lipid profile. The aim of our study was to test whether 7 weeks of RPO supplementation (1 g/kg body weight/day) would affect blood glucose and lipid metabolism in adult male Wistar rats with altered thyroid status. We induced hypothyroidism and hyperthyroidism in rats by oral administration of either methimazole or mixture of thyroid hormones. Different thyroid status (EU - euthyroid, HY - hypothyroid and HT - hyperthyroid) was characterized by different serum thyroid hormones levels (total and free thyroxine and triiodothyronine), changes in the activity of a marker enzyme of thyroid status - liver mitochondrial glycerol-3-phosphate dehydrogenase, and altered absolute and relative heart weights. Fasting blood glucose levels were higher in HT rats in comparison with EU and HY rats, but the changes caused by RPO supplementation were not significant. The achievement of the HY status significantly increased serum levels of total cholesterol, as well as with high-density lipoproteincholesterol and low-density lipoprotein-cholesterol: 2.43±0.15, 1.48±0.09, 0.89±0.08 mmol/l, compared to EU: 1.14±0.06, 0.77±0.06, 0.34±0.05 mmol/l and HT: 1.01±0.06, 0.69±0.04, 0.20±0.03 mmol/l, respectively. RPO supplementation did not increase significantly levels of blood lipids but tended to increase glutathione levels in the liver. In conclusion, RPO supplementation did not induce the presumed deterioration of glucose and lipid metabolism in rats with three well-characterized alterations in thyroid status., H. Rauchová, M. Vokurková, S. Pavelka, I. Vaněčková, N. Tribulová, T. Soukup., and Seznam literatury
Expression of the muscle phenotype is the result of interaction between intrinsic and extrinsic factors, the latter including innervation, mechanical influences and hormonal signals. This minireview summarizes some of the current knowledge regarding the regulation of myosin heavy chain (MHC) isoform transitions during muscle development and regeneration. It describes the role of genetic factors, neural and mechanical influences and it focuses on the contribution of thyroid hormones to the differentiation of muscle fiber phenotypes as shown by the regulation of the expression of MHC isoforms and development of myofibrillar ATPase activity. Finally, it shortly summarizes results regarding the differentiation of MHC isoforms in regenerated muscle fibers of the graft after heterochronous isotransplantation in rats with different thyroid status., T. Soukup, I. Jirmanová., and Obsahuje bibliografii
Agonist-induced subcellular redistribution of G-protein coupled receptors (GPCR) and of trimeric guanine-nucleotide binding regulatory proteins (G-proteins) represent mechanisms of desensitization of hormone response, which have been studied in our laboratory since 1989. This review brings a short summary of these results and also presents information about related literature data covering at least small part of research carried out in this area. We have also mentioned sodium plus potassium dependent adenosine triphosp hatase (Na, K-ATPase) and 3H-ouabain binding as useful reference standard of plasma membrane purity in the brain., Z. Drastichová, L. Bouřová, V. Lisý, L. Hejnová, V. Rudajev, J. Stöhr, D. Durchánková, P. Ostašov, J. Teisinger, T. Soukup, J. Novotný, P. Svoboda., and Obsahuje bibliografii a bibliografické odkazy