Tomasz Sadkowski

Delineation of signalling pathway leading to antioxidant-dependent inhibition of dexamethasone-mediated muscle cell death.

The molecular mechanism of the cell death-promoting effect of dexamethasone (Dex) was studied during myogenesis (10 days) in L6 muscle cells by making use of several indices such as cell viability (protein synthesis, mitochondrial respiration), mortality (DNA fragmentation, chromatin condensation, structural modifications) and immunocytochemical studies [hydrogen peroxide, m-calpain (calpain 2)]. Dex initially (2 nM) stimulated protein synthesis (P < 0.001), but a further increase (20 nM) did not stimulate, whereas a higher dose (200 nM) inhibited formation of cellular proteins (P < 0.001). The latter, apparently, resulted from impaired cell viability (P < 0.001). From the day 4, structural changes featuring cell death were observed. Antioxidants [sodium ascorbate (ASC), catalase (CAT) or N-acetyl-L-cysteine (NAC)] as well as the inhibition of transcription and translation by actinomycin D abrogated Dex-induced cell death (P < 0.001). Using a fluorescent probe (DCFH-DA) we directly corroborated the working hypothesis of the mediating role of H2O2 in the reduction of cell viability by the excess of glucocorticoids. We also found that tPKC, PLCγ, PLA2 were required to induce Dex-dependent cell death since inactivation of tPKC by H7 completely abolished the cytotoxic effect of Dex, while the blockade of PLCγ and PLA2 by U 73122 partially abolished the effect. Cell death was triggered by Ca2+ influx necessary to activate m-calpain since it was reversed by the calcium chelator EGTA or m-calpain inhibitor ALLN but not EDTA nor ALLM. However, cell viability impaired by Ca2+ ionophore A 23187 (P < 0.001) was neither reversed by EGTA, nor EDTA, nor caspase-3 blocker – Ac DEVD CHO, nor ALLN, nor antioxidants – ASC, NAC, CAT. Specific caspase-3 inhibitor Ac DEVD CHO also did not rescue cells from Dex-induced cell death (P < 0.001), in contrast to m-calpain inhibitor – ALLN. Taken together, these findings suggest that reactive oxygen species inhibit protein synthesis and amplify m-calpain-dependent proteolysis. The events that led to the death of L6 muscle cells most likely resulted from Dex-mediated repression of antioxidative defences on the genomic level.

Comparison of skeletal muscle transcriptional profiles in dairy and beef breeds bulls

A cDNA microarray (18 263 probes) was used for transcriptome analysis of bovine skeletal muscle (m. semitendinosus) in 12-month-old bulls of the beef breed Limousin (LIM) and the typical dairy breed Holstein-Friesian (HF, used as a reference). We aimed to identify the genes whose expression may reflect the muscle phenotype of beef bulls. A comparison of muscle transcriptional profiles revealed significant differences in expression of 393 genes between HF and LIM. We classified biological functions of 117 genes with over 2-fold differences in expression between the examined breeds. Among them, 72 genes were up-regulated and 45 genes were down-regulated in LIM vs. HF. The genes were involved in protein metabolism and modifications (22 genes), signal transduction (15), nucleoside, nucleotide and nucleic acid metabolism (13), cell cycle (9), cell structure and motility (9), developmental processes (9), intracellular protein traffic (7), cell proliferation and differentiation (6), cell adhesion (6), lipid, fatty acid and steroid metabolism (5), transport (5), and other processes. For the purpose of microarray data validation, we randomly selected 4 genes:trip12, mrps30, pycrl, andc-erbb3. Real-time RT-PCR results showed similar trends in gene expression changes as those observed in microarray studies. Basing on results of the present study, we proposed a model of the regulation of skeletal muscle growth and differentiation, with a principal role of the somatotropic pathway. It may explain at least in part the development of muscle phenotype in LIM bulls. We assume that the growth hormone directly or indirectly (through IGF-1) activates the calcium-signaling pathway with calcineurin, which stimulates myogenic regulatory factors (MRFs) and inhibits early growth response gene. The inhibition results in indirect activation of MRFs and impaired activation of TGF-beta1 and myostatin, which finally facilitates terminal muscle differentiation.

Gene expression profiling in skeletal muscle of Holstein-Friesian bulls with single-nucleotide polymorphism in the myostatin gene 5'-flanking region

Myostatin (GDF-8) is a key protein responsible for skeletal muscle growth and development, thus mutations in themstn gene can have major economic and breeding consequences. The aim of the present study was to investigate myostatin gene expression and transcriptional profile in skeletal muscle of Holstein-Friesian (Black-and-White) bulls carrying a polymorphism in the 5’-flanking region of themstn gene (G/C transversion at position -7828). Real-time qRT-PCR and cDNA microarray revealed significantly lowermstn expression in muscle of bulls with the CC genotype, as compared to GG and GC genotypes. The direct comparison of skeletal muscle transcriptional profiles between the CC genotype and GG and GC genotypes resulted in identification of genes, of which at least some can be putative targets for myostatin. Using cDNA microarray, we identified 43 common genes (includingmstn) with significantly different expression in skeletal muscle of bulls with the CC genotype, as compared to GG and GC genotypes, 15 of which were upregulated and 28 were downregulated in the CC genotype. Classification of molecular function of differentially expressed genes revealed the highest number of genes involved in the expression of cytoskeleton proteins (9), extracellular matrix proteins (4), nucleic acid-binding proteins (4), calcium-binding proteins (4), and transcription factors (4). The biological functions of the largest number of genes involved: protein metabolism and modification (10), signal transduction (10), cell structure (8), and developmental processes (8). The main identified signaling pathways were: Wnt (4), chemokines and cytokines (4), integrin (4), nicotine receptor for acetylocholine (3), TGF-beta (2), and cytoskeleton regulation by Rho GTPase (2). We identified previously unrecognized putatively myostatin-dependent genes, encoding transcription factors (EGR1, Nf1b, ILF1), components of the proteasomal complex (PSMB7, PSMD13) and proteins with some other molecular function in skeletal muscle (ITGB1BP3, Pla2g1b, ISYNA1, TNFAIP6, MST1, TNNT1, CALB3, CACYBP, and CTNNA1).

A Novel Antioxidant‐Inhibited Dexamethasone‐Mediated and Caspase‐3‐Independent Muscle Cell Death

Abstract: Dexamethasone (Dex)‐mediated cell death is associated with repression of survival factors (AP‐1, c‐myc, NF‐κB). Dex suppressed the activity of genes encoding antioxidant enzymes leading to impaired viability and apoptotic cell death. These findings suggest that reactive oxygen species inhibit protein synthesis and amplify m‐calpain‐dependent proteolysis. The events that led to death of L6 muscle cells were most likely triggered by Dex‐mediated repression of antioxidative defenses on the genomic level.

Characterisation of equine satellite cell transcriptomic profile response to β-hydroxy-β-methylbutyrate (HMB).

β-Hydroxy-β-methylbutyrate (HMB) is a popular ergogenic aid used by human athletes and as a supplement to sport horses, because of its ability to aid muscle recovery, improve performance and body composition. Recent findings suggest that HMB may stimulate satellite cells and affect expressions of genes regulating skeletal muscle cell growth. Despite the scientific data showing benefits of HMB supplementation in horses, no previous study has explained the mechanism of action of HMB in this species. The aim of this study was to reveal the molecular background of HMB action on equine skeletal muscle by investigating the transcriptomic profile changes induced by HMB in equine satellite cells in vitro. Upon isolation from the semitendinosus muscle, equine satellite cells were cultured until the 2nd day of differentiation. Differentiating cells were incubated with HMB for 24 h. Total cellular RNA was isolated, amplified, labelled and hybridised to microarray slides. Microarray data validation was performed with real-time quantitative PCR. HMB induced differential expressions of 361 genes. Functional analysis revealed that the main biological processes influenced by HMB in equine satellite cells were related to muscle organ development, protein metabolism, energy homoeostasis and lipid metabolism. In conclusion, this study demonstrated for the first time that HMB has the potential to influence equine satellite cells by controlling global gene expression. Genes and biological processes targeted by HMB in equine satellite cells may support HMB utility in improving growth and regeneration of equine skeletal muscle; however, the overall role of HMB in horses remains equivocal and requires further proteomic, biochemical and pharmacokinetic studies.

Breed-dependent microRNA expression in the primary culture of skeletal muscle cells subjected to myogenic differentiation

BackgroundSkeletal muscle in livestock develops into meat, an important source of protein and other nutrients for human consumption. The muscle is largely composed of a fixed number of multinucleated myofibers determined during late gestation and remains constant postnatally. A population of postnatal muscle stem cells, called satellite cells, gives rise to myoblast cells that can fuse with the existing myofibers, thus increasing their size. This requires a delicate balance of transcription and growth factors and specific microRNA (miRNA) expressed by satellite cells and their supporting cells from the muscle stem cell niche. The role of transcription and growth factors in bovine myogenesis is well-characterized; however, very little is known about the miRNA activity during this process. We have hypothesized that the expression of miRNA can vary between primary cultures of skeletal muscle cells isolated from the semitendinosus muscles of different cattle breeds and subjected to myogenic differentiation.ResultsAfter a 6-day myogenic differentiation of cells isolated from the muscles of the examined cattle breeds, we found statistically significant differences in the number of myotubes between Hereford (HER)/Limousine (LIM) beef breeds and the Holstein-Friesian (HF) dairy breed (p ≤ 0.001). The microarray analysis revealed differences in the expression of 23 miRNA among the aforementioned primary cultures. On the basis of a functional analysis, we assigned 9 miRNA as molecules responsible for differentiation progression (miR-1, -128a, -133a, -133b, -139, -206, -222, -486, and -503). The target gene prediction and functional analysis revealed 59 miRNA-related genes belonging to the muscle organ development process.ConclusionThe number of myotubes and the miRNA expression in the primary cultures of skeletal muscle cells derived from the semitendinosus muscles of the HER/LIM beef cattle breeds and the HF dairy breed vary when cells are subjected to myogenic differentiation. The net effect of the identified miRNA and their target gene action should be considered the result of the breed-dependent activity of satellite cells and muscle stem cell niche cells and their mutual interactions, which putatively can be engaged in the formation of a larger number of myotubes in beef cattle-related cells (HER/LIM) during in vitro myogenesis.