microRNAs slow translating ribosomes to prevent protein misfolding in eukaryotes.

Slower translation rates reduce protein misfolding. Such reductions in speed can be mediated by the presence of non-optimal codons, which allow time for proper folding to occur. Although this phenomenon is conserved from bacteria to humans, it is not known whether there are additional eukaryote-specific mechanisms which act in the same way. MicroRNAs (miRNAs), not present in prokaryotes, target both coding sequences (CDS) and 3' untranslated regions (UTR). Given their low suppressive efficiency, it has been unclear why miRNAs are equally likely to bind to a CDS. Here, we show that miRNAs transiently stall translating ribosomes, preventing protein misfolding with little negative effect on protein abundance. We first analyzed ribosome profiles and miRNA binding sites to examine whether miRNAs stall ribosomes. Furthermore, either global or specific miRNA deficiency accelerated ribosomes and induced aggregation of a misfolding-prone polypeptide reporter. These defects were rescued by slowing ribosomes using non-cleaving shRNAs as miRNA mimics. We finally show that proinsulin misfolding, associated with type II diabetes, was resolved by non-cleaving shRNAs. Our findings provide a eukaryote-specific mechanism of co-translational protein folding and a previously unknown mechanism of action to target protein misfolding diseases.

MicroRNA-140 is not involved in sepsis-induced muscle atrophy.

Sepsis is a life-threatening inflammatory response to infection, often accompanied by skeletal muscle atrophy. A previous study demonstrated that the administration of microRNA-140 (miR-140) attenuated lipopolysaccharide (LPS)-induced muscle atrophy, whereas miR-140 knockdown with siRNA promoted atrophy. Therefore, we investigated whether miR-140 is involved in LPS-induced muscle atrophy using a genetic model, miR-140-/- mice. We found that a single injection of LPS induced atrophy both in slow-twitch and fast-twitch muscles. The muscle weights and fiber cross-sectional areas were significantly reduced in both the wild-type (WT) and miR-140-/- mice, with no difference between genotypes. The expression of several proteolysis markers, muscle-specific RING-finger 1 (MuRF1) and MAFbx/atrogin-1, increased in both groups after LPS injection. The ubiquitinated proteins in the miR-140-/- mice were similar to those in the WT mice. Therefore, the deletion of miR-140 did not affect LPS-induced muscle atrophy.NEW & NOTEWORTHY We used miR-140-/- mice to determine the function of miR-140 in LPS-induced skeletal muscle atrophy. To our knowledge, this study is the first to examine slow-twitch muscles in LPS-induced muscle wasting after miR-140 manipulation.

Deficiency of MicroRNA-23-27-24 Clusters Exhibits the Impairment of Myelination in the Central Nervous System.

Several microRNAs (miRNAs), including miR-23 and miR-27a have been reportedly involved in regulating myelination in the central nervous system. Although miR-23 and miR-27a form clusters in vivo and the clustered miRNAs are known to perform complementary functions, the role of these miRNA clusters in myelination has not been studied. To investigate the role of miR-23-27-24 clusters in myelination, we generated miR-23-27-24 cluster knockout mice and evaluated myelination in the brain and spinal cord. Our results showed that 10-week-old knockout mice had reduced motor function in the hanging wire test compared to the wild-type mice. At 4 weeks, 10 weeks, and 12 months of age, knockout mice showed reduced myelination compared to wild-type mice. The expression levels of myelin basic protein and myelin proteolipid protein were also significantly lower in the knockout mice compared to the wild-type mice. Although differentiation of oligodendrocyte progenitor cells to oligodendrocytes was not inhibited in the knockout mice, the percentage of oligodendrocytes expressing myelin basic protein was significantly lower in 4-week-old knockout mice than that in wild-type mice. Proteome analysis and western blotting showed increased expression of leucine-zipper-like transcription regulator 1 (LZTR1) and decreased expression of R-RAS and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the knockout mice. In summary, loss of miR-23-27-24 clusters reduces myelination and compromises motor functions in mice. Further, LZTR1, which regulates R-RAS upstream of the ERK1/2 pathway, a signal that promotes myelination, has been identified as a novel target of the miR-23-27-24 cluster in this study.

Changes in Urinary Titin Fragment in Response to Different Types of Dynamic Eccentric Exercises.

The urinary level of the titin fragment has been considered a non-invasive and sensitive biomarker for muscle damage in clinical cases. However, there is little evidence regarding changes in the urinary titin fragment in response to exercise-induced muscle damage. In this study, we aimed to investigate whether the urinary titin fragment reflects the magnitude of muscle damage induced by two lower-limb eccentric exercises. In this study, healthy young male subjects performed drop jump (n=9) and eccentric ergometer exercise (n=9). Blood and urine samples were collected at various time points before and after the exercises. Although perceived muscle soreness assessed by sit-to-stand tasks was increased at 24 h and 48 h after both drop jump and the eccentric ergometer exercise groups, the pressure pain threshold was not changed. Changes of the urinary titin fragment, plasma myomesin 3 fragments, creatine kinase (CK), and myoglobin (Mb) after the eccentric exercises were increased but not statistically significant. Meanwhile, we found that the changes in the urinary titin fragment levels in response to both drop jump and the eccentric ergometer exercise were correlated with those of plasma CK and Mb levels. These results provide evidence that the urinary titin fragment level is a non-invasive biomarker reflecting the magnitude of eccentric exercise-induced muscle damage.

An inducible knockout of Dicer in adult mice does not affect endurance exercise-induced muscle adaptation.

The contractile and metabolic properties of adult skeletal muscle change in response to endurance exercise. The mechanisms of transcriptional regulation in exercise-induced skeletal muscle adaptation, including fiber-type switching and mitochondrial biogenesis, have been investigated intensively, whereas the role of microRNA (miRNA)-mediated posttranscriptional gene regulation is less well understood. We used tamoxifen-inducible Dicer1 knockout (iDicer KO) mice to reduce the global expression of miRNAs in adult skeletal muscle and subjected these mice to 2 wk of voluntary wheel running. Dicer mRNA expression was completely depleted in fast-twitch plantaris muscle after tamoxifen injection. However, several muscle-enriched miRNAs, including miR-1 and miR-133a, were reduced by only 30-50% in both the slow and fast muscles. The endurance exercise-induced changes that occurred for many parameters (i.e., fast-to-slow fiber-type switch and increases in succinate dehydrogenase, respiratory chain complex II, and citrate synthase activity) in wild type (WT) also occurred in the iDicer KO mice. Protein expression of myosin heavy chain IIa, peroxisome proliferator-activated receptor-γ coactivator-1α, and cytochrome c complex IV was also increased in the iDicer KO mice by the voluntary running. Furthermore, there was no significant difference in oxygen consumption rate in the isolated mitochondria between the WT and iDicer KO mice. These data indicate that muscle-enriched miRNAs were detectable even after 4 wk of tamoxifen treatment and there was no apparent specific endurance-exercise-induced muscle phenotype in the iDicer KO mice.

Conditional Deletion of Dicer in Adult Mice Impairs Skeletal Muscle Regeneration.

Skeletal muscle has a remarkable regenerative capacity, which is orchestrated by multiple processes, including the proliferation, fusion, and differentiation of the resident stem cells in muscle. MicroRNAs (miRNAs) are small noncoding RNAs that mediate the translational repression or degradation of mRNA to regulate diverse biological functions. Previous studies have suggested that several miRNAs play important roles in myoblast proliferation and differentiation in vitro. However, their potential roles in skeletal muscle regeneration in vivo have not been fully established. In this study, we generated a mouse in which the Dicer gene, which encodes an enzyme essential in miRNA processing, was knocked out in a tamoxifen-inducible way (iDicer KO mouse) and determined its regenerative potential after cardiotoxin-induced acute muscle injury. Dicer mRNA expression was significantly reduced in the tibialis anterior muscle of the iDicer KO mice, whereas the expression of muscle-enriched miRNAs was only slightly reduced in the Dicer-deficient muscles. After cardiotoxin injection, the iDicer KO mice showed impaired muscle regeneration. We also demonstrated that the number of PAX7+ cells, cell proliferation, and the myogenic differentiation capacity of the primary myoblasts did not differ between the wild-type and the iDicer KO mice. Taken together, these data demonstrate that Dicer is a critical factor for muscle regeneration in vivo.

Role of endothelial microRNA-23 clusters in angiogenesis in vivo.

The capillary network is distributed throughout the body, and its reconstruction is induced under various pathophysiological conditions. MicroRNAs are small noncoding RNAs that regulate gene expression via posttranscriptional mechanisms and are involved in many biological functions, including angiogenesis. Previous studies have shown that each microRNA of miR-23 clusters, composed of the miR-23a cluster (miR-23a~27a~24-2) and miR-23b cluster (miR-23b~27b~24-1), regulates angiogenesis in vitro. However, the role of miR-23 clusters, located within a single transcription unit, in angiogenesis in vivo has not been elucidated. In the present study, we generated vascular endothelial cell (EC)-specific miR-23 cluster double-knockout (DKO) mice and demonstrated sprouting angiogenesis under various conditions, including voluntary running exercise, hindlimb ischemia, skin wound healing, and EC sprouting from aorta explants. Here, we demonstrated that EC-specific miR-23 DKO mice are viable and fertile, with no gross abnormalities observed in pups or adults. The capillary number was normally increased in the muscles of these DKO mice in response to 2 wk of voluntary running and hindlimb ischemia. Furthermore, we did not observe any abnormalities in skin wound closure or EC sprouting from aortic ring explants in EC-specific miR-23 cluster DKO mice. Our results suggest that endothelial miR-23 clusters are dispensable for embryonic development and postnatal angiogenesis in vivo. NEW & NOTEWORTHY We generated vascular endothelial cell (EC)-specific miR-23a/b cluster double-knockout mice and determined sprouting angiogenesis under various conditions, including voluntary running exercise, hindlimb ischemia, skin wound healing, and EC sprouting from aorta explants. We demonstrated that the double-knockout mice were viable and fertile, with no gross abnormalities in exercise- and ischemia-induced angiogenesis and skin wound closure or EC sprouting from aortic ring explants.

Heat Stress Modulates Both Anabolic and Catabolic Signaling Pathways Preventing Dexamethasone-Induced Muscle Atrophy In Vitro.

It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid-induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid-induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel-like factor 15 (KLF15). Heat stress recovered the dexamethasone-induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress-induced protection against glucocorticoid-induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. J. Cell. Physiol. 232: 650-664, 2017. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.

MicroRNA expression profiling in skeletal muscle reveals different regulatory patterns in high and low responders to resistance training.

Large variability exists in muscle adaptive response to resistance exercise (RE) training between individuals. Recent studies have revealed a significant role for microRNAs (miRNAs) in skeletal muscle plasticity. In this study, we investigated how RE affects miRNA expression and whether the variability of muscle hypertrophy to RE training may be attributed to differential miRNA regulation in the skeletal muscle. To screen high and low responders to RE, we had 18 young men perform arm curl exercise training. After screening, all the men performed 12 wk of lower body RE training, but only the high or low responders participated in the acute RE test before training. Muscle biopsies were obtained from the vastus lateralis muscle at baseline, 3 h after acute RE, and after the training period. Total RNA was extracted from the skeletal muscle, and miRNA expression (800 miRNAs) was analyzed. RE training increased the cross-sectional area of the biceps brachii (-1.7-26.1%), quadriceps (2.2-16.8%), and hamstrings (1.6-18.4%). Eighty-five and 102 miRNAs were differentially expressed after acute and chronic RE, respectively (P < 0.05). Seventeen miRNAs, especially 23b-3p, 26a-5p, 32-5p, 148b-3p, and 376a-3p, were differentially expressed at baseline, and 23 miRNAs, especially let-7a-5p, 95, 148a-3p, and 376a-3p, and 26 miRNAs, especially 30d-5p and 376a-3p, were differentially regulated after acute and chronic RE, respectively, in the skeletal muscle between high and low responders, indicating that the expression patterns of several miRNAs are altered by acute or chronic RE, and that miRNAs are involved in skeletal muscle adaptation to RE training.

New mouse model of skeletal muscle atrophy using spiral wire immobilization.

INTRODUCTION: Disuse-induced skeletal muscle atrophy is a serious concern; however, there is not an effective mouse model to elucidate the molecular mechanisms. We developed a noninvasive atrophy model in mice. METHODS: After the ankle joints of mice were bandaged into a bilateral plantar flexed position, either bilateral or unilateral hindlimbs were immobilized by wrapping in bonsai steel wire. RESULTS: After 3, 5, or 10 days of immobilization of the hip, knee, and ankle, the weight of the soleus and plantaris muscles decreased significantly in both bilateral and unilateral immobilization. MAFbx/atrogin-1 and MuRF1 mRNA was found to have significantly increased in both muscles, consistent with disuse-induced atrophy. Notably, the procedure did not result in either edema or necrosis in the fixed hindlimbs. CONCLUSIONS: This method allows repeated, direct access to the immobilized muscle, making it a useful procedure for concurrent application and assessment of various therapeutic interventions. Muscle Nerve 54: 788-791, 2016.

MicroRNA-23a has minimal effect on endurance exercise-induced adaptation of mouse skeletal muscle.

Skeletal muscles contain several subtypes of myofibers that differ in contractile and metabolic properties. Transcriptional control of fiber-type specification and adaptation has been intensively investigated over the past several decades. Recently, microRNA (miRNA)-mediated posttranscriptional gene regulation has attracted increasing attention. MiR-23a targets key molecules regulating contractile and metabolic properties of skeletal muscle, such as myosin heavy-chains and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α). In the present study, we analyzed the skeletal muscle phenotype of miR-23a transgenic (miR-23a Tg) mice to explore whether forced expression of miR-23a affects markers of mitochondrial content, muscle fiber composition, and muscle adaptations induced by 4 weeks of voluntary wheel running. When compared with wild-type mice, protein markers of mitochondrial content, including PGC-1α, and cytochrome c oxidase complex IV (COX IV), were significantly decreased in the slow soleus muscle, but not the fast plantaris muscle of miR-23a Tg mice. There was a decrease in type IId/x fibers only in the soleus muscle of the Tg mice. Following 4 weeks of voluntary wheel running, there was no difference in the endurance exercise capacity as well as in several muscle adaptive responses including an increase in muscle mass, capillary density, or the protein content of myosin heavy-chain IIa, PGC-1α, COX IV, and cytochrome c. These results show that miR-23a targets PGC-1α and regulates basal metabolic properties of slow but not fast twitch muscles. Elevated levels of miR-23a did not impact on whole body endurance capacity or exercise-induced muscle adaptations in the fast plantaris muscle.

Myelin-Specific microRNA-23a/b Cluster Deletion Inhibits Myelination in the Central Nervous System during Postnatal Growth and Aging.

Microribonucleic acids (miRNAs) comprising miR-23a/b clusters, specifically miR-23a and miR-27a, are recognized for their divergent roles in myelination within the central nervous system. However, cluster-specific miRNA functions remain controversial as miRNAs within the same cluster have been suggested to function complementarily. This study aims to clarify the role of miR-23a/b clusters in myelination using mice with a miR-23a/b cluster deletion (KO mice), specifically in myelin expressing proteolipid protein (PLP). Inducible conditional KO mice were generated by crossing miR-23a/b clusterflox/flox mice with PlpCre-ERT2 mice; the offspring were injected with tamoxifen at 10 days or 10 weeks of age to induce a myelin-specific miR-23a/b cluster deletion. Evaluation was performed at 10 weeks or 12 months of age and compared with control mice that were not treated with tamoxifen. KO mice exhibit impaired motor function and hypoplastic myelin sheaths in the brain and spinal cord at 10 weeks and 12 months of age. Simultaneously, significant decreases in myelin basic protein (MBP) and PLP expression occur in KO mice. The percentages of oligodendrocyte precursors and mature oligodendrocytes are consistent between the KO and control mice. However, the proportion of oligodendrocytes expressing MBP is significantly lower in KO mice. Moreover, changes in protein expression occur in KO mice, with increased leucine zipper-like transcriptional regulator 1 expression, decreased R-RAS expression, and decreased phosphorylation of extracellular signal-regulated kinases. These findings highlight the significant influence of miR-23a/b clusters on myelination during postnatal growth and aging.

Roles of programmed death-1 and muscle innate lymphoid cell-derived interleukin 13 in sepsis-induced intensive care unit-acquired weakness.

BACKGROUND: Intensive care unit-acquired weakness (ICU-AW) is a syndrome characterized by a long-term muscle weakness often observed in sepsis-surviving patients during the chronic phase. Although ICU-AW is independently associated with increased mortality, effective therapies have yet to be established. Programmed death-1 (PD-1) inhibitors have attracted attention as potential treatments for reversing immune exhaustion in sepsis; however, its impact on ICU-AW remains to be elucidated. Here, we study how PD-1 deficiency affects sepsis-induced skeletal muscle dysfunction in a preclinical sepsis model. METHODS: Chronic sepsis model was developed by treating wild-type (WT) and PD-1 knockout (KO) mice with caecal slurry, followed by resuscitation with antibiotics and saline. Mice were euthanized on days 15-17. Body weights, muscle weights, and limb muscle strengths were measured. Interleukin 13 (IL-13) and PD-1 expressions were examined by flow cytometry. Messenger RNA (mRNA) expressions of slow-twitch muscles were measured by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). In an in vitro study, C2C12 myotubes were treated with lipopolysaccharide (LPS) and recombinant IL-13 followed by gene expression measurements. RESULTS: WT septic mice exhibited decreased muscle weight (quadriceps, P < 0.01; gastrocnemius, P < 0.05; and tibialis anterior, P < 0.01) and long-term muscle weakness (P < 0.0001), whereas PD-1 KO septic mice did not exhibit any reduction in muscle weights and strengths. Slow-twitch specific mRNAs, including myoglobin (Mb), troponin I type 1 (Tnni1), and myosin heavy chain 7 (Myh7) were decreased in WT skeletal muscle (Mb, P < 0.0001; Tnni1, P < 0.05; and Myh7, P < 0.05) after sepsis induction, but mRNA expressions of Tnni1 and Myh7 were increased in PD-1 KO septic mice (Mb, not significant; Tnni1, P < 0.0001; and Myh7, P < 0.05). Treatment of C2C12 myotube cells with LPS decreased the expression of slow-twitch mRNAs, which was restored by IL-13 (Mb, P < 0.0001; Tnni1, P < 0.001; and Myh7, P < 0.05). IL-13 production was significantly higher in ILC2s compared to T cells in skeletal muscle (P < 0.05). IL-13-producing ILC2s in skeletal muscle were examined and found to increase in PD-1 KO septic mice, compared with WT septic mice (P < 0.05). ILC2-derived IL-13 was increased by PD-1 KO septic mice and thought to protect the muscles from experimental ICU-AW. CONCLUSIONS: Long-term muscle weakness in experimental ICU-AW was ameliorated in PD-1 KO mice. ILC2-derived IL-13 production in skeletal muscles was increased in PD-1 KO mice, thereby suggesting that IL-13 alleviates muscle weakness during sepsis. This study demonstrates the effects of PD-1 blockade in preserving muscle strength during sepsis through an increase in ILC2-derived IL-13 and may be an attractive therapeutic target for sepsis-induced ICU-AW.

Skeletal muscle adaptation in response to mechanical stress in p130cas-/- mice.

Mammalian skeletal muscles undergo adaptation in response to changes in the functional demands upon them, involving mechanical-stress-induced cellular signaling called "mechanotransduction." We hypothesized that p130Cas, which is reported to act as a mechanosensor that transduces mechanical extension into cellular signaling, plays an important role in maintaining and promoting skeletal muscle adaptation in response to mechanical stress via the p38 MAPK signaling pathway. We demonstrate that muscle-specific p130Cas-/- mice express the contractile proteins normally in skeletal muscle. Furthermore, muscle-specific p130Cas-/- mice show normal mechanical-stress-induced muscle adaptation, including exercise-induced IIb-to-IIa muscle fiber type transformation and hypertrophy. Finally, we provide evidence that exercise-induced p38 MAPK signaling is not impaired by the muscle-specific deletion of p130Cas. We conclude that p130Cas plays a limited role in mechanical-stress-induced skeletal muscle adaptation.

Regulation of miRNAs in human skeletal muscle following acute endurance exercise and short-term endurance training.

The identification of microRNAs (miRNAs) has established new mechanisms that control skeletal muscle adaptation to exercise. The present study investigated the mRNA regulation of components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin-5), muscle enriched miRNAs, (miR-1, -133a, -133b and -206), and several miRNAs dysregulated in muscle myopathies (miR-9, -23, -29, -31 and -181). Measurements were made in muscle biopsies from nine healthy untrained males at rest, 3 h following an acute bout of moderate-intensity endurance cycling and following 10 days of endurance training. Bioinformatics analysis was used to predict potential miRNA targets. In the 3 h period following the acute exercise bout, Drosha, Dicer and Exportin-5, as well as miR-1, -133a, -133-b and -181a were all increased. In contrast miR-9, -23a, -23b and -31 were decreased. Short-term training increased miR-1 and -29b, while miR-31 remained decreased. Negative correlations were observed between miR-9 and HDAC4 protein (r=-0.71; P=0.04), miR-31 and HDAC4 protein (r=-0.87; P=0.026) and miR-31 and NRF1 protein (r=-0.77; P=0.01) 3 h following exercise. miR-31 binding to the HDAC4 and NRF1 3 untranslated region (UTR) reduced luciferase reporter activity. Exercise rapidly and transiently regulates several miRNA species in muscle. Several of these miRNAs may be involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Identifying endurance exercise-mediated stress signals regulating skeletal muscle miRNAs, as well as validating their targets and regulatory pathways post exercise, will advance our understanding of their potential role/s in human health.

Disruption of skeletal muscle mitochondrial network genes and miRNAs in amyotrophic lateral sclerosis.

Skeletal muscle mitochondrial dysfunction is believed to play a role in the progression and severity of amyotrophic lateral sclerosis (ALS). The regulation of transcriptional co-activators involved in mitochondrial biogenesis and function in ALS is not well known. When compared with healthy control subjects, patients with ALS, but not neurogenic disease (ND), had lower levels of skeletal muscle peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA and protein and estrogen-related receptor-α (ERRα) and mitofusin-2 (Mfn2) mRNA. PGC-1ß, nuclear respiratory factor-1 (NRF-1) and Mfn1 mRNA as well as cytochrome C oxidase subunit IV (COXIV) mRNA and protein were lower in patients with ALS and ND. Both patient groups had reductions in citrate synthase and cytochrome c oxidase activity. Similar observations were made in skeletal muscle from transgenic ALS G93A transgenic mice. In vitro, PGC-1α and PGC-1ß regulated Mfn1 and Mfn2 in an ERRα-dependent manner. Compared to healthy controls, miRNA 23a, 29b, 206 and 455 were increased in skeletal muscle of ALS patients. miR-23a repressed PGC-1α translation in a 3' UTR dependent manner. Transgenic mice over expressing miR-23a had a reduction in PGC-1α, cytochome-b and COXIV protein levels. These results show that skeletal muscle mitochondrial dysfunction in ALS patients is associated with a reduction in PGC-1α signalling networks involved in mitochondrial biogenesis and function, as well as increases in several miRNAs potentially implicated in skeletal muscle and neuromuscular junction regeneration. As miR-23a negatively regulates PGC-1α signalling, therapeutic inhibition of miR-23a may be a strategy to rescue PGC-1α activity and ameliorate skeletal muscle mitochondrial function in ALS.

Influence of food restriction combined with voluntary running on bone morphology and strength in male rats.

Athletes, in particular endurance athletes and dancers, are chronically exposed to a state of low energy availability due to insufficient dietary energy intake and massive exercise energy expenditure. Low energy availability sometimes causes bone fragility, thereby increasing the risk of bone disorders. Although the decrease in energy availability shows no sexual dimorphism, epidemiological studies have reported that bone disorders are less frequent in male athletes than in female athletes. We hypothesized that bone tissue was not affected by low energy availability in males. The purpose of this study was to examine the influence of food restriction combined with voluntary running training on bone morphology and strength in adult male rats. Fourteen-week-old male Sprague-Dawley rats were divided randomly into four groups: control (C) group, food restriction (R) group, exercise (Ex) group, and food restriction plus exercise (REx) group. For the R and REx groups, 30 % food restriction was carried out in comparison with the C group. Bone strength, bone mineral density (BMD), bone architecture, and bone turnover rate were measured after a 13-week experimental period. Bone strength was not significantly lower in the REx group compared with the C group. BMD and trabecular bone volume showed no difference among groups. These findings indicate that bone morphology and strength were little affected by food restriction combined with exercise training in adult male rats.

Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells.

Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling.

The Mohawk homeobox gene is a critical regulator of tendon differentiation.

Mohawk (Mkx) is a member of the Three Amino acid Loop Extension superclass of atypical homeobox genes that is expressed in developing tendons. To investigate the in vivo functions of Mkx, we generated Mkx(-/-) mice. These mice had hypoplastic tendons throughout the body. Despite the reduction in tendon mass, the cell number in tail tendon fiber bundles was similar between wild-type and Mkx(-/-) mice. We also observed small collagen fibril diameters and a down-regulation of type I collagen in Mkx(-/-) tendons. These data indicate that Mkx plays a critical role in tendon differentiation by regulating type I collagen production in tendon cells.

Functional Analysis of MicroRNAs in Skeletal Muscle.

MicroRNAs (miRNAs) are small non-coding RNAs that are highly conserved in vertebrates and play important roles in diverse biological processes. miRNAs function to fine-tune gene expression by accelerating the degradation of mRNA and/or by inhibiting protein translation. Identification of muscle-specific miRNAs has extended our knowledge of the molecular network in skeletal muscle. Here we describe methods that are commonly used to analyze the function of miRNAs in skeletal muscle.