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.

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.

MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity.

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.

The Impact of Different Amounts of Calcium Intake on Bone Mass and Arterial Calcification in Ovariectomized Rats.

Reduced estrogen secretion and low calcium (Ca) intake are risk factors for bone loss and arterial calcification in female rodents. To evaluate the effects of Ca intake at different amounts on bone mass changes and arterial calcification, 8-wk-old female Wistar rats were randomly placed in ovariectomized (OVX) control and OVX with vitamin D3 plus nicotine (VDN) treatment groups. The OVX with VDN rats were then divided into six groups to receive different amounts of Ca in their diets: 0.01%, 0.1%, 0.3%, 0.6%, 1.2%, or 2.4% Ca. After 8 wk of administration, low Ca intake groups with 0.01% and 0.1% Ca diets had significantly reduced bone mineral density (BMD) and bone mechanical properties as compared with those of the other groups, whereas high Ca intake groups with 1.2% and 2.4% Ca diets showed no differences as compared with the 0.6% Ca intake group. For both the 0.01% and 2.4% Ca intake groups, Ca levels in their thoracic arteries were significantly higher as compared with those of the 0.6% Ca diet group, and that was highly correlated with serum PTH levels. An increase in relative BMP-2 mRNA expression in the arterial tissues of the 0.01% and 2.4% Ca diet groups was also observed. These results suggested that extremely low Ca intake during periods of estrogen deficiency may be a possible risk for the complications of reduced BMD and arterial calcification and that extremely high Ca intake may promote arterial calcification with no changes in BMD.

Mesenchymal-stem-cell-derived exosomes accelerate skeletal muscle regeneration.

Mesenchymal stem cell (MSC) transplantation is used for treatment of many diseases. The paracrine role of MSCs in tissue regeneration is attracting particular attention. We investigate the role of MSC exosomes in skeletal muscle regeneration. MSC exosomes promote myogenesis and angiogenesis in vitro, and muscle regeneration in an in vivo model of muscle injury. Although MSC exosomes had low concentrations of muscle-repair-related cytokines, a number of repair-related miRNAs were identified. This study suggests that the MSC-derived exosomes promote muscle regeneration by enhancing myogenesis and angiogenesis, which is at least in part mediated by miRNAs such as miR-494.

Effects of systemic hypoxia on human muscular adaptations to resistance exercise training.

Hypoxia is an important modulator of endurance exercise-induced oxidative adaptations in skeletal muscle. However, whether hypoxia affects resistance exercise-induced muscle adaptations remains unknown. Here, we determined the effect of resistance exercise training under systemic hypoxia on muscular adaptations known to occur following both resistance and endurance exercise training, including muscle cross-sectional area (CSA), one-repetition maximum (1RM), muscular endurance, and makers of mitochondrial biogenesis and angiogenesis, such as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), citrate synthase (CS) activity, nitric oxide synthase (NOS), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF-1), and capillary-to-fiber ratio. Sixteen healthy male subjects were randomly assigned to either a normoxic resistance training group (NRT, n = 7) or a hypoxic (14.4% oxygen) resistance training group (HRT, n = 9) and performed 8 weeks of resistance training. Blood and muscle biopsy samples were obtained before and after training. After training muscle CSA of the femoral region, 1RM for bench-press and leg-press, muscular endurance, and skeletal muscle VEGF protein levels significantly increased in both groups. The increase in muscular endurance was significantly higher in the HRT group. Plasma VEGF concentration and skeletal muscle capillary-to-fiber ratio were significantly higher in the HRT group than the NRT group following training. Our results suggest that, in addition to increases in muscle size and strength, HRT may also lead to increased muscular endurance and the promotion of angiogenesis in skeletal muscle.

The effect of different amounts of calcium intake on bone metabolism and arterial calcification in ovariectomized rats.

Low calcium (Ca) intake is the one of risk factors for both bone loss and medial elastocalcinosis in an estrogen deficiency state. To examine the effect of different amounts of Ca intake on the relationship between bone mass alteration and medial elastocalcinosis, 6-wk-old female SD rats were randomized into ovariectomized (OVX) control or OVX treated with vitamin D(3) plus nicotine injection (VDN) groups. The OVX treated with VDN group was then divided into 5 groups depending on the different Ca content in their diet, 0.01%, 0.1%, 0.6%, 1.2%, and 2.4% Ca intakes. After 8 wk of experimentation, the low Ca intake groups of 0.01% and 0.1% showed a low bone mineral density (BMD) and bone properties significantly different from those of the other groups, whereas the high Ca intake groups of 1.2% and 2.4% showed no difference compared with the OVX control. Only in the 0.01% Ca intake group, a significantly higher Ca content in the thoracic artery was found compared with that of the OVX control. Arterial tissues of the 0.01% Ca intake group showed an increase of bone-specific alkaline phosphatase (BAP) activity, a marker of bone mineralization, associated with arterial Ca content. However, the high Ca intake did not affect arterial Ca content nor arterial BAP activity. These results suggested that a low Ca intake during periods of rapid bone loss caused by estrogen deficiency might be one possible cause for the complication of both bone loss and medial elastocalcinosis.

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.

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.

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.

DHEA administration activates local bioactive androgen metabolism in cancellous site of tibia of ovariectomized rats.

It is not known whether local androgen metabolism is involved in the mechanisms underlying the dehydroepiandrosterone (DHEA) administration-induced improvement of bone mineral density (BMD) in an estrogen-deficiency state. The aim of the present study was to clarify whether DHEA administration would improve local androgen metabolism and BMD in cancellous site of tibia of ovariectomized (OVX) rats. Twenty-two female rats, 6 weeks old, were randomized into three groups: sham-operated rats, OVX control rats, and OVX rats that received DHEA treatment. DHEA was administered intraperitoneally at 20 mg/kg body weight for 8 weeks. The concentrations of free testosterone and dihydrotestosterone (DHT) in cancellous site of tibia did not change as a result of ovariectomy, while the DHT concentration increased following DHEA administration. We revealed that DHEA administration improved the reduction of 17ß- and 3ß-hydroxysteroid dehydrogenases and clearly reversed the reduction of 5α-reductase types 1 and 2 and androgen receptor in the cancellous site of tibia of OVX rats. DHEA administration suppressed estrogen deficiency relative to the decrease in the cancellous BMD, which was positively associated with local DHT concentration. These findings indicate that DHEA administration enhances local bioactive androgen metabolism in the cancellous tibia of young OVX rats, suggesting that local DHT may play a part in the DHEA administration-induced improvement of cancellous BMD.

Acupuncture ameliorated skeletal muscle atrophy induced by hindlimb suspension in mice.

Preventing skeletal muscle atrophy is critical for maintaining quality of life, but it is often a challenging goal for the elderly and patients with severe conditions. We hypothesized that acupuncture in place of exercise training is an alternative non-pharmacological intervention that can help to prevent muscle atrophy. To elucidate the effects of acupuncture on skeletal muscle atrophy caused by hindlimb suspension (HS), we performed acupuncture on mice according to two different methods: acupuncture with electrical stimulation (EA: electroacupuncture) and without electrical stimulation (MA: manual acupuncture). A needle was retained in the gastrocnemius muscle for 30 min every day for 2 weeks in the EA and MA groups. In the EA group, 30 min of repetitive electrical stimulation (1 Hz, 1 ms pulse width, 6.5 mA intensity) was also applied. HS significantly reduced muscle mass and the cross-sectional area of the soleus muscles. This HS-induced reduction was significantly improved in the EA group, although the level of improvement remained insufficient when compared with the control group. We found that the mRNA expression levels of atrogin-1 and MuRF1, which play a principal role in muscle-specific degradation as E3 ubiquitin ligases, were significantly increased in the HS group compared to the control group. EA and MA reduced the HS-induced upregulation of atrogin-1 (p<0.01 in EA and MA) and MuRF1 (p<0.01 in EA) mRNAs. We also found that the expression levels of PI3K, Akt1, TRPV4, adenosine A1 receptor, myostatin, and SIRT1 mRNAs tended to be increased by HS. EA and MA further increased the HS-induced upregulation of Akt1 (p<0.05 in MA) and TRPV4 (p<0.05 in MA) mRNAs. We concluded that acupuncture partially prevented skeletal muscle atrophy. This effect might be due to an increase in protein synthesis and a decrease in protein degradation.

Modulation of viability of live cells by focused ion-beam exposure.

Introduction of membrane-impermeant substances into living cells is the key method to understand contemporary cellular processes by investigating cellular responses and phenotypes. Here, we performed gold ion beam exposure into live cells by using the focused ion beam implantation method, which was originally developed to precisely control semiconductor device performances. We evaluated the viability of the gold-irradiated cells by measuring the concentration of adenosine triphosphate (ATP), which is an intracellular energy source produced in the mitochondrial membrane. The viability of the irradiated cells was found to be 20% higher than that of the unirradiated control cells. The atoms might promote the energy generating processes within the mitochondrion. Our results suggest that the viability of living cells can be modulated by accurately controlling the dopant atom numbers. Our technique may be considered as a potential tool in life and medical sciences to quantitatively elucidate the dose-dependent effects of dopants.

Deletion of the protein kinase A/protein kinase G target SMTNL1 promotes an exercise-adapted phenotype in vascular smooth muscle.

In vivo protein kinases A and G (PKA and PKG) coordinately phosphorylate a broad range of substrates to mediate their various physiological effects. The functions of many of these substrates have yet to be defined genetically. Herein we show a role for smoothelin-like protein 1 (SMTNL1), a novel in vivo target of PKG/PKA, in mediating vascular adaptations to exercise. Aortas from smtnl1(-/-) mice exhibited strikingly enhanced vasorelaxation before exercise, similar in extent to that achieved after endurance training of wild-type littermates. Additionally, contractile responses to alpha-adrenergic agonists were greatly attenuated. Immunological studies showed SMTNL1 is expressed in smooth muscle and type 2a striated muscle fibers. Consistent with a role in adaptations to exercise, smtnl1(-/-) mice also exhibited increased type 2a fibers before training and better performance after forced endurance training compared smtnl1(+/+) mice. Furthermore, exercise was found to reduce expression of SMTNL1, particularly in female mice. In both muscle types, SMTNL1 is phosphorylated at Ser-301 in response to adrenergic signals. In vitro SMTNL1 suppresses myosin phosphatase activity through a substrate-directed effect, which is relieved by Ser-301 phosphorylation. Our findings suggest roles for SMTNL1 in cGMP/cAMP-mediated adaptations to exercise through mechanisms involving direct modulation of contractile activity.

Eccentric muscle contractions induce greater oxidative stress than concentric contractions in skeletal muscle.

The purpose of this study was to examine oxidative stress in skeletal muscle after eccentric and concentric muscle contractions. Eight-week-old Institute of Cancer Research (ICR) mice (n = 90) were divided into 3 groups: eccentric muscle contraction group (ECC, n = 42), concentric muscle contraction group (CON, n = 42), and control group (pre, n = 6). The tibialis anterior muscle was stimulated via the peroneal nerve to contract either eccentrically or concentrically. The tibialis anterior muscle was isolated before and 0, 6, 12, 18, 24, 72, and 168 h after muscle contraction. Immediately after muscle contractions, thiobarbituric acid reactive substances (TBARS) in skeletal muscle significantly increased (p < 0.05) in both ECC and CON conditions. However, in the ECC group alone, the TBARS level peaked at 12 and 72 h after the contractions. There was greater migration of mononuclear cells in ECC than in CON muscle. In addition, there was a correlation between TBARS in skeletal muscle and migration of mononuclear cells in ECC muscle (r = 0.773, p < 0.01), but this correlation was not apparent in CON muscle (r = 0.324, p = 0.12). The increased mononuclear cells may reflect inflammatory cells. These data suggest that eccentric muscle contraction induces greater oxidative stress in skeletal muscle, which may in turn be due to enhanced generation of reactive oxygen species (ROS) by migrating inflammatory cells.

Peroxisome proliferator-activated receptor-gamma co-activator 1alpha-mediated metabolic remodeling of skeletal myocytes mimics exercise training and reverses lipid-induced mitochondrial inefficiency.

Peroxisome proliferator-activated receptor-gamma co-activator 1alpha (PGC1alpha) is a promiscuous co-activator that plays a key role in regulating mitochondrial biogenesis and fuel homeostasis. Emergent evidence links decreased skeletal muscle PGC1alpha activity and coincident impairments in mitochondrial performance to the development of insulin resistance in humans. Here we used rodent models to demonstrate that muscle mitochondrial efficiency is compromised by diet-induced obesity and is subsequently rescued by exercise training. Chronic high fat feeding caused accelerated rates of incomplete fatty acid oxidation and accumulation of beta-oxidative intermediates. The capacity of muscle mitochondria to fully oxidize a heavy influx of fatty acid depended on factors such as fiber type and exercise training and was positively correlated with expression levels of PGC1alpha. Likewise, an efficient lipid-induced substrate switch in cultured myocytes depended on adenovirus-mediated increases in PGC1alpha expression. Our results supported a novel paradigm in which a high lipid supply, occurring under conditions of low PGC1alpha, provokes a disconnect between mitochondrial beta-oxidation and tricarboxylic acid cycle activity. Conversely, the metabolic remodeling that occurred in response to PGC1alpha overexpression favored a shift from incomplete to complete beta-oxidation. We proposed that PGC1alpha enables muscle mitochondria to better cope with a high lipid load, possibly reflecting a fundamental metabolic benefit of exercise training.