A fast Myosin super enhancer dictates muscle fiber phenotype through competitive interactions with Myosin genes.

The contractile properties of adult myofibers are shaped by their Myosin heavy chain isoform content. Here, we identify by snATAC-seq a 42 kb super-enhancer at the locus regrouping the fast Myosin genes. By 4C-seq we show that active fast Myosin promoters interact with this super-enhancer by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the super-enhancer recapitulates the endogenous spatio-temporal expression of adult fast Myosin genes. In situ deletion of the super-enhancer by CRISPR/Cas9 editing demonstrates its major role in the control of associated fast Myosin genes, and deletion of two fast Myosin genes at the locus reveals an active competition of the promoters for the shared super-enhancer. Last, by disrupting the organization of fast Myosin, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

Polyphenols and Their Effects on Muscle Atrophy and Muscle Health.

Skeletal muscle atrophy is the decrease in muscle mass and strength caused by reduced protein synthesis/accelerated protein degradation. Various conditions, such as denervation, disuse, aging, chronic diseases, heart disease, obstructive lung disease, diabetes, renal failure, AIDS, sepsis, cancer, and steroidal medications, can cause muscle atrophy. Mechanistically, inflammation, oxidative stress, and mitochondrial dysfunction are among the major contributors to muscle atrophy, by modulating signaling pathways that regulate muscle homeostasis. To prevent muscle catabolism and enhance muscle anabolism, several natural and synthetic compounds have been investigated. Recently, polyphenols (i.e., natural phytochemicals) have received extensive attention regarding their effect on muscle atrophy because of their potent antioxidant and anti-inflammatory properties. Numerous in vitro and in vivo studies have reported polyphenols as strongly effective bioactive molecules that attenuate muscle atrophy and enhance muscle health. This review describes polyphenols as promising bioactive molecules that impede muscle atrophy induced by various proatrophic factors. The effects of each class/subclass of polyphenolic compounds regarding protection against the muscle disorders induced by various pathological/physiological factors are summarized in tabular form and discussed. Although considerable variations in antiatrophic potencies and mechanisms were observed among structurally diverse polyphenolic compounds, they are vital factors to be considered in muscle atrophy prevention strategies.

Morin attenuates dexamethasone-mediated oxidative stress and atrophy in mouse C2C12 skeletal myotubes.

Glucocorticoids are the drugs most commonly used to manage inflammatory diseases. However, they are prone to inducing muscle atrophy by increasing muscle proteolysis and decreasing protein synthesis. Various studies have demonstrated that antioxidants can mitigate glucocorticoid-induced skeletal muscle atrophy. Here, we investigated the effect of a potent antioxidative natural flavonoid, morin, on the muscle atrophy and oxidative stress induced by dexamethasone (Dex) using mouse C2C12 skeletal myotubes. Dex (10 µM) enhanced the production of reactive oxygen species (ROS) in C2C12 myotubes via glucocorticoid receptor. Moreover, Dex administration reduced the diameter and expression levels of the myosin heavy chain protein in C2C12 myotubes, together with the upregulation of muscle atrophy-associated ubiquitin ligases, such as muscle atrophy F-box protein 1/atrogin-1, muscle ring finger protein-1, and casitas B-lineage lymphoma proto-oncogene-b. Dex also significantly decreased phosphorylated Foxo3a and increased total Foxo3a expression. Interestingly, Dex-induced ROS accumulation and Foxo3a expression were inhibited by morin (10 µM) pretreatment. Morin also prevented the Dex-induced reduction of myotube thickness, together with muscle protein degradation and suppression of the upregulation of atrophy-associated ubiquitin ligases. In conclusion, our results suggest that morin effectively prevents glucocorticoid-induced muscle atrophy by reducing oxidative stress.

The Prevalence and Diagnostic Ratio of Familial Hypercholesterolemia (FH) and Proportion of Acute Coronary Syndrome in Japanese FH Patients in a Healthcare Record Database Study.

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of low-density lipoprotein cholesterol (LDL-C). Because of underdiagnosis, acute coronary syndrome (ACS) is often the first clinical manifestation of FH. In Japan, there are few reports on the prevalence and diagnostic ratios of FH and the proportion of ACS among FH patients in clinical settings. METHODS: This retrospective, observational study used anonymized data from electronic healthcare databases between April 2001 and March 2015 of patients who had ≥2 LDL-C measurements recorded after April 2009. The index date was defined as the date of the first LDL-C measurement after April 2009. The primary endpoint was the prevalence of definite or suspected FH; secondary endpoints included the proportion of FH patients hospitalized for ACS, the proportion of patients using lipid-lowering drugs (LLDs), and LDL-C levels. RESULTS: Of the 187,781 patients screened, 1547 had definite or suspected FH (0.8%) based on data from the entire period; 832 patients with definite (n = 299, 0.16%) or suspected FH (n = 533, 0.28%) before the index date were identified in the main analysis cohort. LLDs were used in 214 definite FH patients (71.6%) and 137 suspected FH patients (25.7%). Among definite or suspected FH patients with ACS (n = 84) and without ACS (n = 748), 32.1% and 30.1% with definite FH and 3.2% and 2.4% with suspected FH had LDL-C levels < 2.6 mmol/L (<100 mg/dL), respectively. Sixty patients (7.2%) were hospitalized due to ACS at the index date. CONCLUSIONS: The prevalence of FH in this Japanese cohort of patients with ≥2 LDL-C measurements at hospitals was 0.8%, which is higher than that currently reported in epidemiological studies (0.2-0.5%). Patients with suspected FH, with or without ACS, had poorly controlled LDL-C levels and were undertreated. The proportion of FH patients who were hospitalized due to ACS was 7.2%.

GPRC5A facilitates cell proliferation through cell cycle regulation and correlates with bone metastasis in prostate cancer.

The prognosis of patients with progressive prostate cancers that are hormone refractory and/or have bone metastasis is poor. Multiple therapeutic targets to improve prostate cancer patient survival have been investigated, including orphan GPCRs. In our study, we identified G Protein-Coupled Receptor Class C Group 5 Member A (GPRC5A) as a candidate therapeutic molecule using integrative gene expression analyses of registered data sets for prostate cancer cell lines. Kaplan-Meier analysis of TCGA data sets revealed that patients who have high GPRC5A expression had significantly shorter overall survival. PC3 prostate cancer cells with CRISPR/Cas9-mediated GPRC5A knockout exhibited significantly reduced cell proliferation both in vitro and in vivo. RNA-seq revealed that GPRC5A KO PC3 cells had dysregulated expression of cell cycle-related genes, leading to cell cycle arrest at the G2/M phase. Furthermore, the registered gene expression profile data set showed that the expression level of GPRC5A in original lesions of prostate cancer patients with bone metastasis was higher than that without bone metastasis. In fact, GPRC5A KO PC3 cells failed to establish bone metastasis in xenograft mice models. In addition, our clinical study revealed that GPRC5A expression levels in prostate cancer patient samples were significantly correlated with bone metastasis as well as the patient's Gleason score (GS). Combined assessment with the immunoreactivity of GPRC5A and GS displayed higher specificity for predicting the occurrence of bone metastasis. Together, our findings indicate that GPRC5A can be a possible therapeutic target and prognostic marker molecule for progressive prostate cancer.

Microdetermination of Sialic Acids in Blood Samples by Hydrophilic Interaction Chromatography Coupled to Post-column Derivatization and Fluorometric Detection.

An analytical method for the determination of sialic acids in biological samples has been developed and applied to fetal bovine serum (FBS), newborn calf serum and adult bovine serum. The hydrolysis of sera was carried out and the liberated sialic acids were quantified using a rapid and sensitive HPLC. The HPLC includes the separation and detection of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) using hydrophilic interaction liquid chromatography and a fluorometric post-column reaction with 2-cyanoacetamide. The calibration graphs for Neu5Ac and Neu5Gc were linear over the range of 10 pmol - 5 nmol. The concentrations of sialic acids in FBS, newborn calf serum and adult bovine serum were 5.06, 3.79 and 1.64 mM, respectively. The ratios of Neu5Gc and Neu5Ac changed dramatically according to the development stages. The present method has a satisfactory sensitivity in the quantification of Neu5Ac and Neu5Gc in serum samples. It seems that this analytical system can therefore be applied for routine use in clinical investigations of serum sialylation changes in cancer patients.

QRFP-Deficient Mice Are Hypophagic, Lean, Hypoactive and Exhibit Increased Anxiety-Like Behavior.

How the hypothalamus transmits hunger information to other brain regions to govern whole brain function to orchestrate feeding behavior has remained largely unknown. Our present study suggests the importance of a recently found lateral hypothalamic neuropeptide, QRFP, in this signaling. Qrfp-/- mice were hypophagic and lean, and exhibited increased anxiety-like behavior, and were hypoactive in novel circumstances as compared with wild type littermates. They also showed decreased wakefulness time in the early hours of the dark period. Histological studies suggested that QRFP neurons receive rich innervations from neurons in the arcuate nucleus which is a primary region for sensing the body's metabolic state by detecting levels of leptin, ghrelin and glucose. These observations suggest that QRFP is an important mediator that acts as a downstream mediator of the arcuate nucleus and regulates feeding behavior, mood, wakefulness and activity.

AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity.

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that plays a central role in skeletal muscle metabolism. We used skeletal muscle-specific AMPKα1α2 double-knockout (mdKO) mice to provide direct genetic evidence of the physiological importance of AMPK in regulating muscle exercise capacity, mitochondrial function, and contraction-stimulated glucose uptake. Exercise performance was significantly reduced in the mdKO mice, with a reduction in maximal force production and fatigue resistance. An increase in the proportion of myofibers with centralized nuclei was noted, as well as an elevated expression of interleukin 6 (IL-6) mRNA, possibly consistent with mild skeletal muscle injury. Notably, we found that AMPKα1 and AMPKα2 isoforms are dispensable for contraction-induced skeletal muscle glucose transport, except for male soleus muscle. However, the lack of skeletal muscle AMPK diminished maximal ADP-stimulated mitochondrial respiration, showing an impairment at complex I. This effect was not accompanied by changes in mitochondrial number, indicating that AMPK regulates muscle metabolic adaptation through the regulation of muscle mitochondrial oxidative capacity and mitochondrial substrate utilization but not baseline mitochondrial muscle content. Together, these results demonstrate that skeletal muscle AMPK has an unexpected role in the regulation of mitochondrial oxidative phosphorylation that contributes to the energy demands of the exercising muscle.-Lantier, L., Fentz, J., Mounier, R., Leclerc, J., Treebak, J. T., Pehmøller, C., Sanz, N., Sakakibara, I., Saint-Amand, E., Rimbaud, S., Maire, P., Marette, A., Ventura-Clapier, R., Ferry, A., Wojtaszewski, J. F. P., Foretz, M., Viollet, B. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity.

Analysis of DNA repair and protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after exposure to UVC radiation.

Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVC-induced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m(2) of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy.

Fasting-induced hypothermia and reduced energy production in mice lacking acetyl-CoA synthetase 2.

Acetate is activated to acetyl-CoA by acetyl-CoA synthetase 2 (AceCS2), a mitochondrial enzyme. Here, we report that the activation of acetate by AceCS2 has a specific and unique role in thermogenesis during fasting. In the skeletal muscle of fasted AceCS2(-/-) mice, ATP levels were reduced by 50% compared to AceCS2(+/+) mice. Fasted AceCS2(-/-) mice were significantly hypothermic and had reduced exercise capacity. Furthermore, when fed a low-carbohydrate diet, 4-week-old weaned AceCS2(-/-) mice also exhibited hypothermia accompanied by sustained hypoglycemia that led to a 50% mortality. Therefore, AceCS2 plays a significant role in acetate oxidation needed to generate ATP and heat. Furthermore, AceCS2(-/-) mice exhibited increased oxygen consumption and reduced weight gain on a low-carbohydrate diet. Our findings demonstrate that activation of acetate by AceCS2 plays a pivotal role in thermogenesis, especially under low-glucose or ketogenic conditions, and is crucially required for survival.

Cooperative interaction between hepatocyte nuclear factor 4 alpha and GATA transcription factors regulates ATP-binding cassette sterol transporters ABCG5 and ABCG8.

Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4alpha (HNF4alpha) in the ABCG5/ABCG8 intergenic promoter, through which HNF4alpha strongly activated the expression of a reporter gene in both directions. The HNF4alpha-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4alpha. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4alpha leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4alpha and GATA were essential for maximal synergism. We also show that HNF4alpha, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4alpha and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4alpha acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.