WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

Circulating microRNA-21 is an early predictor of ROS-mediated damage in subjects with high risk of developing diabetes and in drug-naïve T2D.

BACKGROUND: Impaired glucose tolerance (IGT) is a risk factor for the development of diabetes and related complications that ensue. Early identification of at-risk individuals might be beneficial to reduce or delay the progression of diabetes and its related complications. Recently, microRNAs emerged as potential biomarkers of diseases. The aim of the present study was to evaluate microRNA-21 as a potential biomarker for the risk of developing diabetes in adults with IGT and to investigate its downstream effects as the generation of reactive oxygen species (ROS), the induction of manganese-superoxide dismutase-2 (SOD2), and the circulating levels of 4-HNE (4-hydroxynonenal). METHODS: To evaluate the prognostic and predictive values of plasmatic microRNA-21 in identifying metabolic derangements, we tested a selected cohort (n = 115) of subjects enrolled in the DIAPASON Study, whom were selected on ADA criteria for 2hPG. Statistical analysis was performed using ANOVA or the Kruskal-Wallis test as appropriate. ROC curves were drawn for diagnostic accuracy of the tests; positive and negative predictive values were performed, and Youden's index was used to seek the cut-off optimum truncation point. ROS, SOD2 and 4-HNE were also evaluated. RESULTS: We observed significant upregulation of microRNA-21 in IGT and in T2D subjects, and microRNA-21 was positively correlated with glycaemic parameters. Diagnostic performance of microRNA-21 was high and accurate. We detected significant overproduction of ROS by electron paramagnetic resonance (EPR), significant accumulation of the lipid peroxidation marker 4-HNE, and defective SOD2 antioxidant response in IGT and newly diagnosed, drug-naïve T2D subjects. In addition, ROC curves demonstrated the diagnostic accuracy of markers used. CONCLUSIONS: our data demonstrate that microRNA-21 is associated with prediabetic status and exhibits predictive value for early detection of glucose imbalances. These data could provide novel clues for miR-based biomarkers to evaluate diabetes.

Increases in circulating levels of ketone bodies and cardiovascular protection with SGLT2 inhibitors: Is low-grade inflammation the neglected component?

Recent clinical trials have demonstrated a strong cardiovascular (CV) protective effect of sodium/glucose cotransporter (SGLT) 2 inhibitors, a recently introduced class of hypoglycaemic agents. The improvement in glycated haemoglobin and other conventional risk factors explains only a portion of the observed reduction in CV risk. A relevant feature of SGLT2-inhibitor-treated diabetic patients is the increase in circulating levels of ketone bodies, which has been proposed to mediate part of the beneficial effects of this class of drugs, mainly through their bioenergetic properties. However, ketone bodies are emerging as potent anti-inflammatory molecules, and inflammation is a recognized risk factor for the development of CV events. In this framework, we hypothesize that, through their unique mechanism of action and by increasing circulating ketone bodies, SGLT2 inhibitors indirectly target the IL-1ß pathway and thus produce a consistent amelioration of low-grade inflammation, a clinically relevant phenomenon in diabetic patients with high CV risk. This attenuation could slow the progression of CV disease and especially the atherosclerotic process, which is sensitive to environmental changes, even over a short time period. To test this conceptual structure, it would be necessary to measure circulating pro-inflammatory molecules in patients treated with SGLT inhibitors. The addition of inflammatory markers to the list of clinical data measured in FDA-requested, large CV outcome trials could provide supplementary information regarding potential secondary effects of new anti-hyperglycaemic drugs, considering that the inflammatory process is an often neglected cornerstone of CV diseases.

Glucose-sensing microRNA-21 disrupts ROS homeostasis and impairs antioxidant responses in cellular glucose variability.

BACKGROUND: Antioxidant enzymes play a fundamental role in counteracting oxidative stress induced by high glucose. Although mitochondrial superoxide dismutase (SOD2) is the principal defence against the toxicity of superoxide anions, the mechanism of its inactivation in diabetic subjects is still poorly understood. Recently, microRNA-21 has been associated with diabetes, although its function remains unclear. We sought to explore the mechanism underlying defective SOD2 antioxidant response in HUVECs during exposures to constant high glucose and oscillating glucose (as glucose variability model, GV) and the role of miR-21 in increasing the susceptibility to oxidative stress by disrupting reactive oxygen species (ROS) homeostasis. METHODS: HUVECs exposed for 1 week to constant high glucose and GV were subjected to quantitative electron paramagnetic resonance for ROS measurements. Superoxide anions, SOD2 protein levels and mitochondrial membrane potential (ΔΨm) were also evaluated. Endogenous miR-21 and its putative ROS-homeostatic target genes (KRIT1, FoxO1, NFE2L2 and SOD2) were tested using mimic-miR-21 and quantified by qPCR. Luciferase assays were performed to test miR-21/3'-UTR-SOD2 binding. RESULTS: We observed upregulation of microRNA-21, overproduction of superoxide anions and total ROS generation, depolarisation of the mitochondrial membrane potential (ΔΨm) and defective SOD2 antioxidant response in HUVECs subjected to constant high glucose and GV exposures. We also found that exogenous mimic-microRNA-21 targeted putative microRNA-21 ROS-homeostatic target genes, e.g., KRIT1, NRF2 and SOD2, which were significantly downregulated. All these effects were reverted by a microRNA-21 inhibitor, which improved SOD2 and KRIT1 expression, reduced the levels of ROS production and ameliorated ΔΨm. CONCLUSIONS: Our data demonstrate the association of microRNA-21 with oscillating and high glucose and early mitochondrial dysfunction. We found that microRNA-21 may promote the suppression of homeostatic signalling that normally limits ROS damage. These data provide novel clues about the inhibition of microRNA-21 as a new therapeutic approach to protect against cellular oxidative injury in glucose variability and diabetes.

Novel insights into the regulation of miRNA transcriptional control: implications for T2D and related complications.

In recent years, epigenetics has emerged as an important form of biological regulation involving chromatin control of gene expression. The mechanisms of this fine-tuned regulation are susceptible to changes forced by environmental stimuli and nutritional factors and may be potentially reversible. Dysregulation of epigenetic processes has important consequences for the pathogenesis of complex and multifactorial diseases such as type 2 diabetes (T2D) and vascular complications. Along with DNA methylation (DNA-me), histone modifications and RNA-based mechanisms as the major epigenetic controllers, small non-coding RNAs known as microRNAs (miRNAs) have their own important implications for the pathogenesis of diabetes. There is increasing evidence supporting the role of miRNAs in modulating gene expression, cumulatively contributing to epigenetic gene silencing by acting either on the methylation status of the cells or in alternative roles. Although significant progress has been made in the characterization of miRNA functions, most miRNA promoters have not yet been characterized, and the transcriptional regulation of miRNAs remains elusive. The present work is centred on the new biological insights pertaining to the epigenetics-miRNA regulatory axis, focusing on the development of T2D and cardiovascular complications, and the ability of these mechanisms to interact in a network of DNA-me regulation. The genomic organization of inter- and intragenic miRNA genes is discussed, and the mutual connections between pre-mRNA splicing and miRNA biogenesis are summarized, along with the discovery of novel miRNA transcriptional regulation sites.

Polycomb repressive complex 1 provides a molecular explanation for repeat copy number dependency in FSHD muscular dystrophy.

Repression of repetitive elements is crucial to preserve genome integrity and has been traditionally ascribed to constitutive heterochromatin pathways. FacioScapuloHumeral Muscular Dystrophy (FSHD), one of the most common myopathies, is characterized by a complex interplay of genetic and epigenetic events. The main FSHD form is linked to a reduced copy number of the D4Z4 macrosatellite repeat on 4q35, causing loss of silencing and aberrant expression of the D4Z4-embedded DUX4 gene leading to disease. By an unknown mechanism, D4Z4 copy-number correlates with FSHD phenotype. Here we show that the DUX4 proximal promoter (DUX4p) is sufficient to nucleate the enrichment of both constitutive and facultative heterochromatin components and to mediate a copy-number dependent gene silencing. We found that both the CpG/GC dense DNA content and the repetitive nature of DUX4p arrays are important for their repressive ability. We showed that DUX4p mediates a copy number-dependent Polycomb Repressive Complex 1 (PRC1) recruitment, which is responsible for the copy-number dependent gene repression. Overall, we directly link genetic and epigenetic defects in FSHD by proposing a novel molecular explanation for the copy number-dependency in FSHD pathogenesis, and offer insight into the molecular functions of repeats in chromatin regulation.

DNA methylation analysis of the macrosatellite repeat associated with FSHD muscular dystrophy at single nucleotide level.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common inherited diseases of the skeletal muscle. It is characterized by asymmetric muscle weakness and variable penetrance. FSHD is linked to a reduction in copy number of the D4Z4 3.3 kb macrosatellite repeat, located in 4q35. This causes the epigenetic de-repression of FSHD candidate genes leading to disease. Nevertheless, the molecular mechanism responsible for silencing of FSHD candidate genes in healthy subjects is not fully understood. While a role for DNA methylation has been suggested, so far there is limited information regarding the methylation status of the 325 CpGs contained in each D4Z4 unit. Using a human/rodent monochromosomal hybrid cell line containing a single human chromosome 4, we performed an in depth analysis of DNA methylation for the majority of the CpGs inside D4Z4 at single nucleotide level. We found that D4Z4 is not uniformly methylated and that the level of DNA methylation does not correlate with the density of CpG dinucleotides. Moreover, in several D4Z4 regions characterized by near complete methylation, we found specific unmethylated CpGs. These elements are enriched in transcription factor binding sites that could be involved in muscle-specific D4Z4 activity. Our approach also detected differential methylation among different D4Z4 units, suggesting that the D4Z4 array is a mosaic of euchromatic and heterochromatic domains. Finally, we found that DNA methylation and histone de-acetylation are required to maintain FSHD candidate genes repressed. Taken together, our data underscore new players involved in the epigenetic regulation of the FSHD locus that could be targeted for therapeutic purposes.

Identification of the molecular site of ivabradine binding to HCN4 channels.

Ivabradine is a specific heart rate-reducing agent approved as a treatment of chronic stable angina. Its mode of action involves a selective and specific block of HCN channels, the molecular components of sinoatrial "funny" (f)-channels. Different studies suggest that the binding site of ivabradine is located in the inner vestibule of HCN channels, but the molecular details of ivabradine binding are unknown. We thus sought to investigate by mutagenesis and in silico analysis which residues of the HCN4 channel, the HCN isoform expressed in the sinoatrial node, are involved in the binding of ivabradine. Using homology modeling, we verified the presence of an inner cavity below the channel pore and identified residues lining the cavity; these residues were replaced with alanine (or valine) either alone or in combination, and WT and mutant channels were expressed in HEK293 cells. Comparison of the block efficiency of mutant vs WT channels, measured by patch-clamp, revealed that residues Y506, F509 and I510 are involved in ivabradine binding. For each mutant channel, docking simulations correctly explain the reduced block efficiency in terms of proportionally reduced affinity for ivabradine binding. In summary our study shows that ivabradine occupies a cavity below the channel pore, and identifies specific residues facing this cavity that interact and stabilize the ivabradine molecule. This study provides an interpretation of known properties of f/HCN4 channel block by ivabradine such as the "open channel block", the current-dependence of block and the property of "trapping" of drug molecules in the closed configuration.

Allosteric modulators of α4ß2 nicotinic acetylcholine receptors: a new direction for antidepressant drug discovery.

Allosteric modulation of ligand-gated ion channels has been intensively studied in the past three decades and is now an established strategy to control receptor function in numerous disease states. Allosteric sites on the GABA(A) receptor are targets for widely prescribed drugs that are used for a variety of pathophysiological states including insomnia and epilepsy. Modulators might be especially valuable to control receptors for which the design of selective orthosteric drugs has proven difficult due to safety issues (e.g., α4ß2 nicotinic acetylcholine receptors and might have several advantages over orthosteric ligands. Modulators influence the action of the endogenous agonist but generally have no effect of their own on the unoccupied receptor. Moreover, the higher subtype selectivity exerted by modulators and that the effects of modulators depend on the simultaneous presence of agonist help to overcome safety problems by preventing over-dosage compared with the administration of orthosteric drugs.

Deep bradycardia and heart block caused by inducible cardiac-specific knockout of the pacemaker channel gene Hcn4.

Cardiac pacemaking generation and modulation rely on the coordinated activity of several processes. Although a wealth of evidence indicates a relevant role of the I(f) ("funny," or pacemaker) current, whose molecular constituents are the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and particularly HCN4, work with mice where Hcn genes were knocked out, or functionally modified, has challenged this view. However, no previous studies used a cardiac-specific promoter to induce HCN4 ablation in adult mice. We report here that, in an inducible and cardiac-specific HCN4 knockout (ciHCN4-KO) mouse model, ablation of HCN4 consistently leads to progressive development of severe bradycardia (∼50% reduction of original rate) and AV block, eventually leading to heart arrest and death in about 5 d. In vitro analysis of sinoatrial node (SAN) myocytes isolated from ciHCN4-KO mice at the mean time of death revealed a strong reduction of both the I(f) current (by ∼70%) and of the spontaneous rate (by ∼60%). In agreement with functional results, immunofluorescence and Western blot analysis showed reduced expression of HCN4 protein in SAN tissue and cells. In ciHCN4-KO animals, the residual I(f) was normally sensitive to ß-adrenergic receptor (ß-AR) modulation, and the permanence of rate response to ß-AR stimulation was observed both in vivo and in vitro. Our data show that cardiac HCN4 channels are essential for normal heart impulse generation and conduction in adult mice and support the notion that dysfunctional HCN4 channels can be a direct cause of rhythm disorders. This work contributes to identifying the molecular mechanism responsible for cardiac pacemaking.

Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node.

Several studies of the pacemaker mechanisms in mammalian cells, most of which were carried out in cells isolated from the rabbit sinoatrial node (SAN), have highlighted the role of the I(f) current. While the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels, is known at the mRNA level, the identification of f-channel proteins in this tissue is still undetermined. Here we investigate HCN protein expression in the rabbit pacemaker region. We found that HCN4 is the main isoform, and set therefore to analyze its distribution within the SAN and surrounding areas with the aim of correlating protein expression and pacemaking function. The analysis was carried out in tissue slices and single cells of the intercaval area, which includes the crista terminalis (CT), the SAN, and the septum interatrialis (SI). Immunolabeling, in situ hybridization, qRT-PCR analysis, and electrophysiological recordings identified the SAN as a region characterized by high HCN4 signal and current levels, while the expression in the CT and in the SI was either negligible or absent. Detailed analysis of the central SAN area showed that cells are predominantly distributed in islets interconnected by cell prolongations, and single-cell HCN4 labeling suggested sites of channel clustering. Our data indicate that in the rabbit SAN, HCN4 proteins are major constituents of native f-channels, and their distribution matches closely the SAN as defined morphologically and electrophysiologically. Until recently, the SAN was identified as the region where Cx43 and atrial natriuretic peptide are not expressed; we propose here that expression of HCN4 is an appropriate tool to map and identify the cardiac SAN pacemaker region.