HDAC4 is necessary for satellite cell differentiation and muscle regeneration

In response to injury, skeletal muscle exhibits high capacity to regenerate and epigenetics controls multiple steps of this process (Giordani et al., 2013). It has been demonstrated in vitro that completion of muscle differentiation requires shuttling of histone deacetylase 4 (HDAC4), a member of class IIa HDACs, from the nucleus to the cytoplasm and consequent activation of MEF2-dependent differentiation genes (McKinsey et al., 2000). In vivo, HDAC4 expression is up-regulated in skeletal muscle upon injury, suggesting a role for this protein in muscle regeneratio

Histone deacetylase 4 is crucial for proper skeletal muscle development and disease

Epigenetics plays a pivotal role in modulating gene response to physiological or pathological stimuli. Histone Deacetylase inhibitors (HDACi) have been used in the treatment of various cancers1, are ef-fective in several animal models of neurodegenerative diseases, including amyotrophic lateral scle-rosis (ALS), and are currently in clinical trial to promote muscle repair in muscular dystrophies2. However, long-term use of pan-HDAC inhibitors is not tolerated3. The assignment of distinct biologi-cal functions to individual HDACs in skeletal muscle is a prerequisite to improve the efficacy of pharmacological treatments based on HDACi. HDAC4 is a member of class II HDACs that mediates many cellular responses. Clinical reports suggest that inhibition of HDAC4 can be beneficial to cancer cachexia, dystrophic or ALS patients. All the above conditions are characterized by progressive mus-cle wasting and up-regulation of HDAC4 expression in skeletal muscle, suggesting a potential role for this protein in regulating these diseases. To study the role of HDAC4 with a genetic approach, we generated several models of muscle disease in mice lacking HDAC4 in skeletal muscle: cancer ca-chexia, by implanting Lewis lung carcinoma (LLC), muscular dystrophy, by using mdx mice, or ALS, by using SODG93A mice. Lack of HDAC4 worsens skeletal muscle atrophy induced by both LLC and ALS, demonstrated by a reduction in muscle mass and myofibers size. Conversely, dystrophic mice lacking HDAC4 in skeletal muscle show an increased number of necrotic myofibers and run less efficiently than mdx mice. The aggravation of the dystrophic phenotype may be partially due to the impairment in skeletal muscle regeneration observed in mice lacking HDAC4 in skeletal muscle. Our results indi-cate that HDAC4 is necessary for maintaining skeletal muscle homeostasis and function. Current studies aim to investigate the molecular mechanisms underlying the role of HDAC4 in skeletal mus-cle maintenance in response to cancer cachexia, ALS or muscular dystrophy

HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes

Skeletal muscle exhibits a high regenerative capacity, mainly due to the ability of satellite cells to replicate and differentiate in response to appropriate stimuli. Epigenetic control is effective at different stages of this process. It has been shown that the chromatin-remodeling factor HDAC4 is able to regulate satellite cell proliferation and commitment. However, its molecular targets are still uncovered. To explain the signaling pathways regulated by HDAC4 in satellite cells, we generated tamoxifen-inducible mice with conditional inactivation of HDAC4 in Pax7+ cells (HDAC4 KO mice). We found that the proliferation and differentiation of HDAC4 KO satellite cells were compromised, although similar amounts of satellite cells were found in mice. Moreover, we found that the inhibition of HDAC4 in satellite cells was sufficient to block the differentiation process. By RNA-sequencing analysis we identified P21 and Sharp1 as HDAC4 target genes. Reducing the expression of these target genes in HDAC4 KO satellite cells, we also defined the molecular pathways regulated by HDAC4 in the epigenetic control of satellite cell expansion and fusion

Further experimental data supporting the safety of potassium polyaspartate used as a food additive in wine stabilization

Potassium polyaspartate (KPPA) is a food additive used for wine stabilization. KPAA was included in the list of additives allowed in EU, with the Commission Regulation (EU) 2017/1399, having received a positive opinion by EFSA (European Food Safety Authority) in 2016. KPAA is rich in negative charges, which are essential for its enological function consisting in binding positive ions in order to reduce wine instability. Concerns were raised on the fact that the same binding properties could be responsible for a reduction of microelement bioavailability in humans. For this reason and for the protection of consumers' health, the binding properties of potassium polyaspartate versus three minerals (calcium, iron, magnesium) was assayed using the Size-Exclusion Chromatography (SEC). Aliquots obtained by SEC were analysed for their content in polyaspartate (by microbiuret method) and bound minerals (by ICP-OES method). The results obtained by this research shows that, when potassium polyaspartate is added to wine, the negative charges of the additive are saturated, as expected by the specific role of KPAA in tartaric stabilization. In conclusions, the effect on mineral bioavailability must be considered negligible

Toxicologic evaluation of potassium polyaspartate (A-5D K/SD) : genotoxicity and subchronic toxicity

Potassium polyaspartate (A-5D K/SD) is proposed for use as a stabiliser in wine, with a maximum use level of 300 mg/L and typical levels in the range of 100-200 mg/L. Potassium polyaspartate (A-5D K/SD) tested negative in a bacterial reverse mutation assay performed in accordance with OECD TG 471 and in an in vitro mammalian cell micronucleus test performed in accordance with OECD TG 487. From a 90-day oral toxicity study in male and female Wistar rats performed in accordance with OECD TG 408, a no observed adverse effect level (NOAEL) was set at 1000 mg/kg bw per day, the highest dose tested. In its opinion adopted on 9 March 2016, the EFSA-ANS Panel (European Food Safety Authority - Panel on Food Additives and Nutrient Sources added to Food), considering these data, concluded that "there was no safety concern from the proposed use and use levels of potassium polyaspartate (A-5D K/SD)"

Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy

Background: Histone deacetylase 4 (HDAC4) is a stress-responsive factor that mediates multiple cellular responses. As a member of class IIa HDACs, HDAC4 shuttles between the nucleus and the cytoplasm; however, HDAC4 cytoplasmic functions have never been fully investigated. Duchenne muscular dystrophy (DMD) is a genetic, progressive, incurable disorder, characterized by muscle wasting, which can be treated with the unspecific inhibition of HDACs, despite this approach being only partially effective. More efficient strategies may be proposed for DMD only after the different HDAC members will be characterized. Methods: To fully understand HDAC4 functions, we generated dystrophic mice carrying a skeletal muscle-specific deletion of HDAC4 (mdx;KO mice). The progression of muscular dystrophy was characterized in mdx and age-matched mdx;KO mice by means of histological, molecular, and functional analyses. Satellite cells (SCs) from these mice were differentiated in vitro, to identify HDAC4 intrinsic functions influencing the myogenic potential of dystrophic SCs. Gain-of-function experiments revealed the cytoplasmic functions of HDAC4 in mdx;KO muscles. Results: Histone deacetylase 4 increased in the skeletal muscles of mdx mice (~3-fold; P < 0.05) and of DMD patients (n = 3, males, mean age 13.3 ± 1.5 years), suggesting that HDAC4 has a role in DMD. Its deletion in skeletal muscles importantly worsens the pathological features of DMD, leading to greater muscle fragility and degeneration over time. Additionally, it impairs SC survival, myogenic potential, and muscle regeneration, ultimately compromising muscle function (P < 0.05–0.001). The impaired membrane repair mechanism in muscles and SCs accounts for the mdx;KO phenotype. Indeed, the ectopic expression of Trim72, a major player in the membrane repair mechanism, prevents SC death (~20%; P < 0.01) and increases myogenic fusion (~40%; P < 0.01) in vitro; in vivo it significantly reduces myofibre damage (~10%; P < 0.005) and improves mdx;KO muscle function (P < 0.05). The mdx;KO phenotype is also fully rescued by restoring cytoplasmic levels of HDAC4, both in vitro and in vivo. The protective role of HDAC4 in the cytoplasm of mdx;KO muscles is, in part, independent of its deacetylase activity. HDAC4 expression correlates with Trim72 mRNA levels; furthermore, Trim72 mRNA decays more rapidly (P < 0.01) in mdx;KO muscle cells, compared with mdx ones. Conclusions: Histone deacetylase 4 performs crucial functions in the cytoplasm of dystrophic muscles, by mediating the muscle repair response to damage, an important role in ensuring muscle homeostasis, probably by stabilizing Trim72 mRNA. Consequently, the cytoplasmic functions of HDAC4 should be stimulated rather than inhibited in muscular dystrophy treatments, a fact to be considered in future therapeutic approaches

Antitumor effect of miR-197 targeting in p53 wild-type lung cancer

Lung cancer is the leading cause of tumor-related death. The lack of effective treatments urges the development of new therapeutic approaches able to selectively kill cancer cells. The connection between aberrant microRNA (miRNA-miR) expression and tumor progression suggests a new strategy to fight cancer by interfering with miRNA function. In this regard, LNAs (locked nucleic acids) have proven to be very promising candidates for miRNA neutralization. Here, we employed an LNA-based anti-miR library in a functional screening to identify putative oncogenic miRNAs in non-small-cell lung cancer (NSCLC). By screening NIH-H460 and A549 cells, miR-197 was identified as a new functional oncomiR, whose downregulation induces p53-dependent lung cancer cell apoptosis and impairs the capacity to establish tumor xenografts in immunodeficient mice. We further identified the two BH3-only proteins NOXA and BMF as new miR-197 targets responsible for induction of apoptosis in p53 wild-type cells, delineating miR-197 as a key survival factor in NSCLC. Thus, we propose the inhibition of miR-197 as a novel therapeutic approach against lung cancer. © 2014 Macmillan Publishers Limited All rights reserved