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Early detection of skeletal muscle atrophy is important to prevent further muscle weakness. However, there are few non-invasive biomarkers for skeletal muscle atrophy. Recent studies have reported that the N-terminal fragment (N-titin) of titin, a giant sarcomeric protein, is detected in the urine of patients with muscle damage. In this study, we hypothesized that urinary N-titin would be a potential early biomarker of skeletal muscle atrophy in mice caused by sciatic nerve denervation. Male mice were randomly divided into control and denervation groups, and urinary N-titin levels were assessed daily for 9 days using an enzyme-linked immunosorbent assay system. Despite reduced titin protein levels in atrophic muscles 10 days after denervation, cleaved N-titin fragments were not increased in the urine of mice with denervation-induced muscle atrophy. Furthermore, we found no uptake of Evans blue dye from the extracellular space into the cytoplasm in atrophic muscles, suggesting that the sarcomeric membrane is intact in those muscles. The present results suggest that cleaved N-titin in the urine is not suitable as an early biomarker of skeletal muscle atrophy.

Duchenne muscular dystrophy (DMD) is X-linked recessive myopathy caused by mutations in the dystrophin gene. Although conventional treatments have improved their prognosis, inevitable progressive cardiomyopathy is still the leading cause of death in patients with DMD. To explore novel therapeutic options, a suitable animal model with heart involvement has been warranted.We have generated a rat model with an out-of-frame mutation in the dystrophin gene using CRISPR/Cas9 genome editing (DMD rats). The aim of this study was to evaluate their cardiac functions and pathologies to provide baseline data for future experiments developing treatment options for DMD.In comparison with age-matched wild rats, 6-month-old DMD rats showed no significant differences by echocardiographic evaluations. However, 10-month-old DMD rats showed significant deterioration in left ventricular (LV) fractional shortening (P = 0.024), and in tissue Doppler peak systolic velocity (Sa) at the LV lateral wall (P = 0.041) as well as at the right ventricular (RV) free-wall (P = 0.004). These functional findings were consistent with the fibrotic distributions by histological analysis.Although the cardiac phenotype was milder than anticipated, DMD rats showed similar distributions and progression of heart involvement to those of patients with DMD. This animal may be a useful model with which to develop effective drugs and to understand the underlying mechanisms of progressive heart failure in patients with DMD.

Background Duchenne muscular dystrophy (DMD) is a progressive, degenerative muscular disorder and cognitive dysfunction caused by mutations in the dystrophin gene. It is characterized by excess inflammatory responses in the muscle and repeated degeneration and regeneration cycles. Neutral sphingomyelinase 2/sphingomyelin phosphodiesterase 3 (nSMase2/Smpd3) hydrolyzes sphingomyelin in lipid rafts. This protein thus modulates inflammatory responses, cell survival or apoptosis pathways, and the secretion of extracellular vesicles in a Ca2+-dependent manner. However, its roles in dystrophic pathology have not yet been clarified. Methods To investigate the effects of the loss of nSMase2/Smpd3 on dystrophic muscles and its role in the abnormal behavior observed in DMD patients, we generated mdx mice lacking the nSMase2/Smpd3 gene (mdx:Smpd3 double knockout [DKO] mice). Results Young mdx:Smpd3 DKO mice exhibited reduced muscular degeneration and decreased inflammation responses, but later on they showed exacerbated muscular necrosis. In addition, the abnormal stress response displayed by mdx mice was improved in the mdx:Smpd3 DKO mice, with the recovery of brain-derived neurotrophic factor (Bdnf) expression in the hippocampus. Conclusions nSMase2/Smpd3-modulated lipid raft integrity is a potential therapeutic target for DMD.

Background Previous research indicated that nitric oxide synthase (NOS) is the key molecule for S-nitrosylation of ryanodine receptor 1 (RyR1) in DMD model mice (mdx mice) and that both neuronal NOS (nNOS) and inducible NOS (iNOS) might contribute to the reaction because nNOS is mislocalized in the cytoplasm and iNOS expression is higher in mdx mice. We investigated the effect of iNOS on RyR1 S-nitrosylation in mdx mice and whether transgenic expression of truncated dystrophin reduced iNOS expression in mdx mice or not. Methods Three- to 4-month-old C57BL/6 J, mdx, and transgenic mdx mice expressing exon 45–55-deleted human dystrophin (Tg/mdx mice) were used. We also generated two double mutant mice, mdx iNOS KO and Tg/mdx iNOS KO to reveal the iNOS contribution to RyR1 S-nitrosylation. nNOS and iNOS expression levels in skeletal muscle of these mice were assessed by immunohistochemistry (IHC), qRT-PCR, and Western blotting. Total NOS activity was measured by a citrulline assay. A biotin-switch method was used for detection of RyR1 S-nitrosylation. Statistical differences were assessed by one-way ANOVA with Tukey-Kramer post-hoc analysis. Results mdx and mdx iNOS KO mice showed the same level of RyR1 S-nitrosylation. Total NOS activity was not changed in mdx iNOS KO mice compared with mdx mice. iNOS expression was undetectable in Tg/mdx mice expressing exon 45–55-deleted human dystrophin, but the level of RyR1 S-nitrosylation was the same in mdx and Tg/mdx mice. Conclusion Similar levels of RyR1 S-nitrosylation and total NOS activity in mdx and mdx iNOS KO demonstrated that the proportion of iNOS in total NOS activity was low, even in mdx mice. Exon 45–55-deleted dystrophin reduced the expression level of iNOS, but it did not correct the RyR1 S-nitrosylation. These results indicate that iNOS was not involved in RyR1 S-nitrosylation in mdx and Tg/mdx mice muscles.

Introduction Phospholipids are essential components of cellular membranes and are closely associated with cellular functions, but relationships involving skeletal muscle phospholipid profiles and their physiological phenotypes have remained unclear. Methods We carried out comprehensive phospholipid analyses using liquid chromatography-tandem mass spectrometry to determine the phospholipid profiles of skeletal muscles derived from muscle-wasting mouse models, including denervated and Duchenne muscular dystrophy mouse models (mdx) as well as rescued mdx mice expressing truncated dystrophin. Results Consistent phosphatidylcholine and phosphatidylethanolamine alterations in skeletal muscles isolated from denervated and mdx mice were observed. Notably, the levels of these phospholipids binding polyunsaturated fatty acids were reduced in denervated and mdx muscles. Moreover, rescuing the mdx pathology by expressing truncated dystrophin led to the restoration of phospholipid profiles. Discussion Our findings support the hypothesis that phospholipid profiles of the skeletal muscle may be associated with skeletal muscle function.

Background Dilated cardiomyopathy (DCM) in children is often associated with poor morbidity and mortality and exhibits distinct pathological entities from those of adult DCM. Owing to the limited number of patients and the lack of a good animal model, the molecular mechanisms underlying pediatric DCM remain poorly understood. The purpose of this study is to establish an animal model of neonatal DCM and identify early progression factors. Methods Cardiac phenotypes and comprehensive gene expression profiles in homozygous ΔK210 knock-in (TNNT2ΔK210/ΔK210) mice were analyzed and compared to TNNT2+/ΔK210 and wild-type mice at 0 days and 1 week of age. Results Immediately after birth, the cardiac weight in TNNT2ΔK210/ΔK210 mice was already increased compared to that in TNNT2+/ΔK210 and wild-type mice. Echocardiographic examination of 0-day-old and 1-week-old TNNT2ΔK210/ΔK210 mice revealed similar phenotypes of pediatric DCM. In addition, several genes were significantly upregulated in the ventricular tissues of TNNT2ΔK210/ΔK210 mice, and the KEGG PATHWAY analysis revealed several important pathways such as cancer and focal adhesion that might be associated with the pathogenesis and development of DCM. Conclusions TNNT2ΔK210/ΔK210 mice have already developed DCM at birth, indicating that they should be an excellent animal model to identify early progression factors of DCM. Impact TNNT2ΔK210/ΔK210 mice are excellent animal model for DCM. TNNT2ΔK210/ΔK210 mice are excellent animal model to identify early progression factors of DCM. KEGG PATHWAY analysis revealed that several important pathways such as cancer and focal adhesion might be associated with the pathogenesis and development of neonatal DCM.

Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45–55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45–55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45–55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45–55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.

Summary Growing evidences are suggesting that extra-long genes in mammals are vulnerable for full-gene length transcription and dysregulation of long genes is a mechanism underlying human genetic disorders. How long-distance transcription is achieved is a fundamental question to be elucidated. In previous study, we had discovered that RNA-binding protein SFPQ preferentially binds to long pre-mRNAs and specifically regulates the cluster of neuronal genes >100 kbp. Here we investigated the roles of SFPQ for long gene expression, target specificities, and also physiological functions in skeletal muscle. Loss of Sfpq selectively downregulated genes >100 kbp including Dystrophin, which is 2.26 Mbp in length. Sfpq knockout (KO) mice showed progressive muscle mass reduction and metabolic myopathy characterized by glycogen accumulation and decreased abundance of mitochondrial oxidative phosphorylation complexes. Functional clustering analysis identified energy metabolism pathway genes as SFPQ's targets. These findings indicate target gene specificities and tissue-specific physiological functions of SFPQ in skeletal muscle., Graphical Abstract, Highlights • SFPQ is essential for long gene expression, including Dystrophin, in skeletal muscle • Disruption of Sfpq caused severe muscle mass reduction and premature death • SFPQ is required for metabolic pathway gene expression in skeletal muscle • Loss of Sfpq decreased OXPHOS complexes and caused glycogen accumulation, Molecular Biology; Molecular Mechanism of Gene Regulation; Pathophysiology

The effect of 2'-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5'-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2'-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s2TMCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.

Introduction: Sarcopenia characterized by the loss of skeletal muscle mass and strength is prevalent in COPD. Sarcopenia has a huge impact on physical activity and is associated with increased mortality in COPD, but detailed molecular mechanism is still unclear. Increased reactive oxygen species (ROS) production and altered mitochondrial function has been reported in limb skeletal muscle of COPD. Previous study reported that muscle-specific depletion of autophagy was associated with increased ROS production in skeletal muscle. Although we have demonstrated the involvement of impaired Parkin-mediated mitochondria-selective autophagy, mitophagy in COPD pathogenesis, participation of mitophagy in sarcopenia progression associated with cigarette smoke (CS) exposure remains to be elucidated. Methods: Myoblasts were isolated from mouse limbs. CS extracts (CSE) was used to examine the effect of CS on myotube differentiation, reflecting myogenesis ability of myoblasts. Wild type (WT) myoblasts and Parkin knockout (KO) derived myoblasts were treated with 0.5 % CSE. Fusion index (the degree of differentiation from myoblast to myotube) and quantitative reverse transcription (RT)-PCR for myogenin (a marker for myotube differentiation) were performed. Results: Fusion index was significantly reduced by CSE exposure in WT myoblasts. Parkin KO myoblasts also showed reduced fusion index even in the absence of CSE. The expression levels of myogenin mRNA were decreased in CSE-exposed WT myoblasts and in Parkin KO myoblasts without CSE treatment. Conclusion: These results suggest that Parkin-mediated mitophagy may have a regulatory role in myogenesis during CS exposure as a part of sarcopenia pathogenesis in COPD.

Introduction: The effect of acute ventricular volume overload on organelles such as myofibrils and mitochondria are incompletely understood. Purpose: We performed acute overstretch on cardiac papillary muscle to investigate the functional and morphological changes. Methods: We used male SD-rats (10-12 weeks old) and dissected papillary muscles from the right ventricle. A papillary muscle preparation was stretched to Lmax. We overstretched it within 2 seconds up to 110% of Lmax (110%Overstretch; 110%OS) or 120% of Lmax (120%OS), and stimulated it (1Hz, 36°C) with tension measurement, compared with Lmax group. After the measurement of tension, we analyzed the morphological changes in muscle fibers and organelles using a Transmission electron microscopy (TEM). Results: The active tension after overstretch was immediately decreased to 67.8±3.8% in 110%OS, and to 17.3±4.0% in 120%OS. In TEM study, muscle fibers kept their well-organized structures and the sarcomere length was stretched to 2.4 μm in 110%OS, and to 2.6 μm in 120%OS. In contrast, in 110%OS, inner mitochondrial density was lower than that in Lmax, indicating swelled mitochondria. In 120%OS, inner mitochondrial empty space with vacuolation was found in the large area of myocardium, indicating that inner mitochondria cristae were susceptible to mechanical stress-induced deterioration. Conclusions: Acute overstretch of rat papillary muscles causes inner mitochondrial collapsing with preserved the sarcomere structure. It could account for the mechanisms of acute volume-overloaded heart failure.

Spaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are due to mutations in the DMD gene. Previous reports show that in-frame deletion of exons 45–55 produces an internally shorted, but functional, dystrophin protein resulting in a very mild BMD phenotype. In order to elucidate the molecular mechanism leading to this phenotype, we generated exon 45–55 deleted dystrophin transgenic/mdx (Tg/mdx) mice. Muscular function of Tg/mdx mice was restored close to that of wild type (WT) mice but the localization of the neuronal type of nitric oxide synthase was changed from the sarcolemma to the cytosol. This led to hyper-nitrosylation of the ryanodine receptor 1 causing increased Ca2+ release from the sarcoplasmic reticulum. On the other hand, Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) was restored to the level of WT mice, suggesting that the Ca2+ dysregulation had been compensated by SERCA activation. In line with this, expression of sarcolipin (SLN), a SERCA-inhibitory peptide, was upregulated in mdx mice, but strongly reduced in Tg/mdx mice. Furthermore, knockdown of SLN ameliorated the cytosolic Ca2+ homeostasis and the dystrophic phenotype in mdx mice. These findings suggest that SLN may be a novel target for DMD therapy.

Skeletal muscle is the largest tissue in the human body and plays an important role in the regulation of systemic homeostasis and displays remarkable plasticity in their metabolic responses to caloric availability and physical activity. Skeletal muscle maintains muscle mass suitable for the environment according to its own activity state. Skeletal muscles also affect the energy regulation of the whole body by skeletal muscle itself changing muscle fiber composition due to external and internal factors. This review focuses on the transcriptional regulation mechanism of muscle fiber type, which is responsible for overall energy consumption.

Incorporation of 2′-O-methyl-2-thioribothymidine (s2Tm) into antisense oligoribonucleotides significantly enhanced the exon skipping activity in Duchenne muscular dystrophy model mice.

Background Chronic increases in the levels of the inflammatory cytokine interleukin-6 (IL-6) in serum and skeletal muscle are thought to contribute to the progression of muscular dystrophy. Dystrophin/utrophin double-knockout (dKO) mice develop a more severe and progressive muscular dystrophy than the mdx mice, the most common murine model of Duchenne muscular dystrophy (DMD). In particular, dKO mice have smaller body sizes and muscle diameters, and develop progressive kyphosis and fibrosis in skeletal and cardiac muscles. As mdx mice and DMD patients, we found that IL-6 levels in the skeletal muscle were significantly increased in dKO mice. Thus, in this study, we aimed to analyze the effects of IL-6 receptor (IL-6R) blockade on the muscle pathology of dKO mice. Methods Male dKO mice were administered an initial injection (200 mg/kg intraperitoneally (i.p.)) of either the anti-IL-6R antibody MR16-1 or an isotype-matched control rat IgG at the age of 14 days, and were then given weekly injections (25 mg/kg i.p.) until 90 days of age. Results Treatment of dKO mice with the MR16-1 antibody successfully inhibited the IL-6 pathway in the skeletal muscle and resulted in a significant reduction in the expression levels of phosphorylated signal transducer and activator of transcription 3 in the skeletal muscle. Pathologically, a significant increase in the area of embryonic myosin heavy chain-positive myofibers and muscle diameter, and reduced fibrosis in the quadriceps muscle were observed. These results demonstrated the therapeutic effects of IL-6R blockade on promoting muscle regeneration. Consistently, serum creatine kinase levels were decreased. Despite these improvements observed in the limb muscles, degeneration of the diaphragm and cardiac muscles was not ameliorated by the treatment of mice with the MR16-1 antibody. Conclusion As no adverse effects of treatment with the MR16-1 antibody were observed, our results indicate that the anti-IL-6R antibody is a potential therapy for muscular dystrophy particularly for promoting skeletal muscle regeneration. Electronic supplementary material The online version of this article (10.1186/s13395-017-0140-z) contains supplementary material, which is available to authorized users.

Inflammatory events occurring in dystrophic muscles contribute to the progression of Duchenne muscular dystrophy (DMD). Low-intensity training (LIT) attenuates the phenotype of mdx mice, an animal model for DMD. Therefore, we postulated that LIT could have anti-inflammatory properties. We assessed levels of inflammatory cytokines and infiltrated immune cells in gastrocnemius muscle of mdx mice after LIT. We detected high levels of complement component C5a, chemokine ligand (CCL) 2, CD68+ monocytes/macrophages, and proinflammatory M1 macrophages in muscles of mdx mice. LIT decreased CCL2 levels, increased CD68+ cell numbers, and shifted the macrophage population to the regenerative M2 type. We investigated whether inhibition of C5a or CCL2 with L-aptamers could mimic the effects of LIT. Although no effect of CCL2 inhibition was detected, treatment with the C5a inhibitor, NOX-D21, rescued the phenotype of nonexercised mdx mice, but not of exercised ones. In both cases, the level of CD68+ cells increased and macrophage populations leaned toward the inflammatory M1 type. In muscles of nonexercised treated mice, the level of IL-1 receptor antagonist increased, damage decreased, and fibers were switched toward the glycolytic fast type; in muscles of exercised mice, fibers were switched to the oxidative slow type. These results reveal the effects of LIT on the inflammatory status of mdx mice and suggest that NOX-D21 could be an anti-inflammatory drug for DMD.

Duchenne muscular dystrophy(DMD)is X-linked genetic disorder caused by a lack of the membrane-associated protein dystrophin. DMD is characterized by progressive muscle wasting secondary to repeated muscle damage and inadequate repair. The mechanisms underlying the functional impairments in dystrophic muscle have not yet been fully determined. However, several recent studies indicate that elevated intracellular Ca

MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level. Several miRNAs are exclusively expressed in skeletal muscle and participate in the regulation of muscle differentiation by interacting with myogenic factors. These miRNAs can be found at high levels in the serum of patients and animal models for Duchenne muscular dystrophy, which is expected to be useful as biomarkers for their clinical conditions. By miRNA microarray analysis, we identified miR-188 as a novel miRNA that is elevated in the serum of the muscular dystrophy dog model, CXMDJ. miR-188 was not muscle-specific miRNA, but its expression was up-regulated in skeletal muscles associated with muscle regeneration induced by cardiotoxin-injection in normal dogs and mice. Manipulation of miR-188 expression using antisense oligo and mimic oligo RNAs alters the mRNA expression of the myogenic regulatory factors, MRF4 and MEF2C. Our results suggest that miR-188 is a new player that participates in the gene regulation process of muscle differentiation and that it may serve as a serum biomarker reflecting skeletal muscle regeneration.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder. Here, we show that the CD63 antigen, which is located on the surface of extracellular vesicles (EVs), is associated with increased levels of muscle-abundant miRNAs, namely myomiRs miR-1, miR-133a, and miR-206, in the sera of DMD patients and mdx mice. Furthermore, the release of EVs from the murine myoblast C2C12 cell line was found to be modulated by intracellular ceramide levels in a Ca2+-dependent manner. Next, to investigate the effects of EVs on cell survival, C2C12 myoblasts and myotubes were cultured with EVs from the sera of mdx mice or C2C12 cells overexpressing myomiRs in presence of cellular stresses. Both the exposure of C2C12 myoblasts and myotubes to EVs from the serum of mdx mice, and the overexpression of miR-133a in C2C12 cells in presence of cellular stress resulted in a significant decrease in cell death. Finally, to assess whether miRNAs regulate skeletal muscle regeneration in vivo, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion) into mdx mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated mdx mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated mdx mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of mdx mice from degeneration.

The purpose of this study was to elucidate the effects of a main pungent component of wasabi, allyl isothiocyanate (AITC), on the number of lymphocytes, T-lymphocyte, B-lymphocyte and natural killer (NK) cells and plasma corticosterone concentrations in rats. AITC was given as either single dosage [20 mg/kg body weight per day; subcutaneous (s.c.) and oral] or as a daily dosage (10 mg and 20 mg/kg body weight per day; s.c.) for 4 days for the hourly and daily assessment of changes in the numbers of lymphocytes, respectively. A single s.c. injection or oral administration of AITC significantly reduced the number of lymphocytes at 4 hr to approximately 0.68 times, indicating that decreased effects of AITC on the number of lymphocytes are independent on the administration route. Administration of AITC for 4 days reduced dose-dependently the number of lymphocytes, and significantly reduced the number of T-lymphocyte and B-lymphocyte to 0.79 and 0.60 times, respectively. However, the number of NK cells did not change by AITC. Administration of AITC increased plasma corticosterone concentrations at 4 day of post s.c. injection to 5.4-6.0 times. These results suggest that AITC-mediated immunosuppresion is at least in part attributable to changes in the number and distribution of lymphocyte and its subsets.

The subacute effects of dihydrocapsaincin (DHC) and capsaicin (CAP) (dose=3 mg/kg body weight per day for 10 days, subcutaueous) on the number and distribution of white blood cells (WBCs) were studied in male adult rats. The administration of DHC and CAP for 10 days decreased significantly the number of total WBCs, lymphocytes and monocyte, and increased significantly the number of the neutrophil and eosinophil without changing the number of basophil. The administration of DHC significantly decreased thymus weight and increased adrenal weight, showing that DHC induced thymus atrophy and adrenal hypertrophy. These results suggest that capsaicinoids induced the decrease of acquired immunity responses and these phenomena may have in part participated in capsaicinoids-induced stress-responses in rats.

The purpose of this study was to elucidate the effects of dexamethasone, a synthetic glucocorticoid, on the immune system by analyzing the number of white blood cells (WBCs) over the course of hours and days of dexamethasone administration. Dexamethasone was given as either a single dosage [1.0 mg/kg body weight (BW); subcutaneous injection (s.c.)] or as a daily dosage (1.0 mg/kg BW per day; s.c.) for 10 days for the hourly and daily assessment of changes in the number of white blood cells, respectively. A single administration of dexamethasone markedly decreased the number of total WBCs, as well as the number of lymphocyte, monocyte, neutrophil and eosinophil subsets with a nadir at 8 hr post-injection. The number of these cells recovered to the control levels at 24 hr. The numbers of total WBCs, lymphocytes, monocyte, eosinophil and basophil were reduced by the daily administration of dexamethasone. However, the number of neutrophil was significantly higher at days 2 and 8 after the injection. These results suggest that glucocorticoid-mediated immunosuppressions are at least partly attributable to quantitative changes in the number of circulating WBCs.

Administration of the beta(2)-agonist clenbuterol has been shown to reduce the expression of beta(2)-adrenoceptor (AR) mRNA in fast-twitch fiber-rich (extensor digitorum longus, EDL) muscle without changing that in slow-twitch fiber-rich (soleus, SOL) muscle in rats. However, the regulatory mechanism for muscle fiber type-dependent down-regulation of the expression of beta(2)-AR mRNA induced by clenbuterol is still unclear. Therefore, mRNA expression of transcriptional and post-transcriptional regulatory factors for beta(2)-AR mRNA levels in fast-twitch fiber-rich (EDL and plantaris, PLA) and slow-twitch fiber-rich (SOL) muscles in clenbuterol-administered (1.0 mg/kg body weight/day for 10 days, subcutaneous) rats was studied by real-time reverse transcription-polymerase chain reaction. Administration of clenbuterol significantly reduced expression of beta(2)-AR mRNA in EDL and PLA muscles without changing that in SOL muscle. Administration of clenbuterol also significantly reduced the mRNA expression of transcriptional regulatory factor (glucocorticoid receptor) and mRNA stabilizing factor (Hu antigen R) in EDL and PLA muscles without changing those in SOL muscle. These results suggest that muscle fiber type-dependent effects of clenbuterol on expression of beta(2)-AR mRNA are closely related to the down-regulation of mRNA expression of transcriptional and post-transcriptional regulatory factors for beta(2)-AR mRNA levels.

Glucocorticoids are known to increase the density and mRNA levels of beta-adrenoceptors (beta-AR) via the glucocorticoid receptor (GR) in many tissues. However, the effects of these changes in the skeletal and cardiac muscles remain relatively unknown. We have investigated the effects of dexamethasone on the expression of the beta(1)-, beta(2)-, and beta(3)-AR mRNAs and GR mRNA in fast-twitch fiber-rich extensor digitorum longus (EDL), slow-twitch fiber-rich soleus (SOL), and left ventricle (LV) muscles by real-time quantitative RT-PCR. Male rats were divided into a dexamethasone group and control group. The weight, RNA concentration, and total RNA content of EDL muscle were 0.76-, 0.85-, and 0.65-fold lower, respectively, in the dexamethasone group than in the control group. The weight, RNA concentration, and total RNA content of SOL muscle were 0.92-, 0.87-, and 0.81-fold lower, respectively, in the dexamethasone group than in the control group; these differences were significant. However, the weight/body weight and total RNA content/body weight of LV muscle were 1.38- and 1.39-fold higher, respectively, in the dexamethasone group than in the control group, respectively; these differences were also significant. Dexamethasone significantly decreased GR mRNA expression in EDL muscle without changing the expression of the beta(1)-, beta(2)-, and beta(3)-AR mRNAs. However, dexamethasone significantly decreased the expressions of beta(2)-AR and GR mRNAs in SOL muscle and significantly increased beta(1)-AR mRNA expression in LV muscle-without changing GR mRNA expression. These results suggest that the effects of dexamethasone on the expression of beta(1)- and beta(2)-AR mRNAs and muscle mass depend on the muscle contractile and/or constructive types.

The acute effects of dihydrocapsaicin (DHC) and capsaicin (CAP) on the number of white blood cells (WBCs), neutrophils, eosinophils, basophils, monocytes, lymphocytes, T lymphocytes, B lymphocytes and NK cells, and serum corticosterone levels were studied in rats. Male 7-wk-old SD rats were divided into DHC (3.0 mg/kg BW), CAP (3.0 mg/kg BW) and control (CON) groups. The number of total WBCs was 1.30-1.42 times significantly higher in the DHC group than in the CON group at 6-12 h. The number of neutrophils was 1.62 times significantly higher in the DHC group than in the CON group at 12 h. The number of total WBCs and neutrophils, however, showed no significant changes between the CAP and CON groups. The number of lymphocytes was 0.61 and 0.70 times significantly lower in the DHC and CAP groups than in the CON group at 3 h. The number of T lymphocytes and B lymphocytes was 0.74 and 0.54 times lower in the DHC group than in the CON group, respectively. CAP, however, did not significantly change the number of T lymphocytes or B lymphocytes. No significant changes in the number of NK cells were observed among the three groups. CAP and DHC did not change the number of monocytes, eosinophils or basophils. No significant changes of the serum corticosterone levels were observed among the three groups. In conclusion, capsaicinoids decreased the number of acquired immunity cells, and increased the number of total WBCs and neutrophils without changing the number of monocytes, eosinophils or basophils. The magnitude of these effects was relatively higher in DHC than in CAP.

Clenbuterol [CLE: 4-amino-α(t-butyl-amino)methyl-3,5-dichlorobenzyl alcohol] is well known as a potent β2-adrenergic agonist and non-steroidal anabolic drug, and thus it is generally used for sports doping and asthma therapy. Although the functions of immune cells such as white blood cells (WBCs) have shown to be modulated through β2-adrenoceptors, the effects of CLE on immune-responsive systems have not been elucidated systematically. Therefore, the effects of CLE on the number of WBCs were studied in rats. Male adult rats were divided into CLE-administered group and the control group to compare the number of total WBCs, neutrophils, monocytes, lymphocytes, eosinophils, and basophils. The administration (dose = 1.0 mg ⋅ kg−1 body weight ⋅ day−1, s.c.) of CLE was maintained for 30 days. CLE did not change the number of total WBCs during the experimental period. However, CLE increased significantly the number of neutrophils and monocytes, while CLE decreased drastically the number of lymphocytes and eosinophils. There was no significant change in the number of basophils between both groups. These results suggest that the administration of CLE induces drastic redistribution of WBCs in circulation without changing the number of total WBCs, and these responses of WBCs during the administration of CLE are sustained for at least 30 days. Keywords:: β2-agonist, clenbuterol, immune-responsive system, white blood cell, lymphocyte