RNA-sequencing reveals that STRN, ZNF484 and WNK1 add to the value of mitochondrial MT-COI and COX10 as markers of unstable coronary artery disease

Markers in monocytes, precursors of macrophages, which are related to CAD, are largely unknown. Therefore, we aimed to identify genes in monocytes predictive of a new ischemic event in patients with CAD and/or discriminate between stable CAD and acute coronary syn- drome. We included 66 patients with stable CAD, of which 24 developed a new ischemic event, and 19 patients with ACS. Circulating CD14+ monocytes were isolated with magnetic beads. RNA sequencing analysis in monocytes of patients with (n = 13) versus without (n = 11) ischemic event at follow-up and in patients with ACS (n = 12) was validated with qPCR (n = 85). MT-COI, STRN and COX10 predicted new ischemic events in CAD patients (power for separation at 1% error rate of 0.97, 0.90 and 0.77 respectively). Low MT-COI and high STRN were also related to shorter time between blood sampling and event. COX10 and ZNF484 together with MT-COI, STRN and WNK1 separated ACS completely from sta- ble CAD patients. RNA expressions in monocytes of MT-COI, COX10, STRN, WNK1 and ZNF484 were independent of cholesterol lowering and antiplatelet treatment. They were independent of troponin T, a marker of myocardial injury. But, COX10 and ZNF484 in human plaques correlated to plaque markers of M1 macrophage polarization, reflecting vas- cular injury. Expression of MT-COI, COX10, STRN and WNK1, but not that of ZNF484, PBMCs paired with that in monocytes. The prospective study of relation of MT-COI, COX10, STRN, WNK1 and ZNF484 with unstable CAD is warranted

Gene expression changes of single skeletal muscle fibers in response to modulation of the mitochondrial calcium uniporter (MCU)

The mitochondrial calcium uniporter (MCU) gene codifies for the inner mitochondrial membrane (IMM) channel responsible for mitochondrial Ca2 + uptake. Cytosolic Ca2 + transients are involved in sarcomere contraction through cycles of release and storage in the sarcoplasmic reticulum. In addition cytosolic Ca2 + regulates various signaling cascades that eventually lead to gene expression reprogramming. Mitochondria are strategically placed in close contact with the ER/SR, thus cytosolic Ca2 + transients elicit large increases in the [Ca2 +] of the mitochondrial matrix ([Ca2 +]mt). Mitochondrial Ca2 + uptake regulates energy production and cell survival. In addition, we recently showed that MCU-dependent mitochondrial Ca2 + uptake controls skeletal muscle trophism. In the same report, we dissected the effects of MCU-dependent mitochondrial Ca2 + uptake on gene expression through microarray gene expression analysis upon modulation of MCU expression by in vivo AAV infection. Analyses were performed on single skeletal muscle fibers at two time points (7 and 14 days post-AAV injection). Raw and normalized data are available on the GEO database (http://www.ncbi.nlm.nih.gov/geo/) (GSE60931)

Meta-analysis of expression signatures of muscle atrophy: gene interaction networks in early and late stages

<p>Abstract</p> <p>Background</p> <p>Skeletal muscle mass can be markedly reduced through a process called atrophy, as a consequence of many diseases or critical physiological and environmental situations. Atrophy is characterised by loss of contractile proteins and reduction of fiber volume. Although in the last decade the molecular aspects underlying muscle atrophy have received increased attention, the fine mechanisms controlling muscle degeneration are still incomplete. In this study we applied meta-analysis on gene expression signatures pertaining to different types of muscle atrophy for the identification of novel key regulatory signals implicated in these degenerative processes.</p> <p>Results</p> <p>We found a general down-regulation of genes involved in energy production and carbohydrate metabolism and up-regulation of genes for protein degradation and catabolism. Six functional pathways occupy central positions in the molecular network obtained by the integration of atrophy transcriptome and molecular interaction data. They are TGF-β pathway, apoptosis, membrane trafficking/cytoskeleton organization, NFKB pathways, inflammation and reorganization of the extracellular matrix. Protein degradation pathway is evident only in the network specific for muscle short-term response to atrophy. TGF-β pathway plays a central role with proteins SMAD3/4, MYC, MAX and CDKN1A in the general network, and JUN, MYC, GNB2L1/RACK1 in the short-term muscle response network.</p> <p>Conclusion</p> <p>Our study offers a general overview of the molecular pathways and cellular processes regulating the establishment and maintenance of atrophic state in skeletal muscle, showing also how the different pathways are interconnected. This analysis identifies novel key factors that could be further investigated as potential targets for the development of therapeutic treatments. We suggest that the transcription factors SMAD3/4, GNB2L1/RACK1, MYC, MAX and JUN, whose functions have been extensively studied in tumours but only marginally in muscle, appear instead to play important roles in regulating muscle response to atrophy.</p

Statistical Test of Expression Pattern (STEPath): a new strategy to integrate gene expression data with genomic information in individual and meta-analysis studies

<p>Abstract</p> <p>Background</p> <p>In the last decades, microarray technology has spread, leading to a dramatic increase of publicly available datasets. The first statistical tools developed were focused on the identification of significant differentially expressed genes. Later, researchers moved toward the systematic integration of gene expression profiles with additional biological information, such as chromosomal location, ontological annotations or sequence features. The analysis of gene expression linked to physical location of genes on chromosomes allows the identification of transcriptionally imbalanced regions, while, Gene Set Analysis focuses on the detection of coordinated changes in transcriptional levels among sets of biologically related genes.</p> <p>In this field, meta-analysis offers the possibility to compare different studies, addressing the same biological question to fully exploit public gene expression datasets.</p> <p>Results</p> <p>We describe STEPath, a method that starts from gene expression profiles and integrates the analysis of imbalanced region as an <it>a priori </it>step before performing gene set analysis. The application of STEPath in individual studies produced gene set scores weighted by chromosomal activation. As a final step, we propose a way to compare these scores across different studies (meta-analysis) on related biological issues. One complication with meta-analysis is batch effects, which occur because molecular measurements are affected by laboratory conditions, reagent lots and personnel differences. Major problems occur when batch effects are correlated with an outcome of interest and lead to incorrect conclusions. We evaluated the power of combining chromosome mapping and gene set enrichment analysis, performing the analysis on a dataset of leukaemia (example of individual study) and on a dataset of skeletal muscle diseases (meta-analysis approach).</p> <p>In leukaemia, we identified the Hox gene set, a gene set closely related to the pathology that other algorithms of gene set analysis do not identify, while the meta-analysis approach on muscular disease discriminates between related pathologies and correlates similar ones from different studies.</p> <p>Conclusions</p> <p>STEPath is a new method that integrates gene expression profiles, genomic co-expressed regions and the information about the biological function of genes. The usage of the STEPath-computed gene set scores overcomes batch effects in the meta-analysis approaches allowing the direct comparison of different pathologies and different studies on a gene set activation level.</p

New miRNA labeling method for bead-based quantification

<p>Abstract</p> <p>Background</p> <p>microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies.</p> <p>Results</p> <p>Here we have applied with an innovative approach, the Luminex^®xMAP™ technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies.</p> <p>Conclusions</p> <p>We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex^®xMAP™ technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.</p

Single cell analysis reveals the involvement of the long non-coding RNA Pvt1 in the modulation of muscle atrophy and mitochondrial network

Long non-coding RNAs (lncRNAs) are emerging as important players in the regulation of several aspects of cellular biology. For a better comprehension of their function, it is fundamental to determine their tissue or cell specificity and to identify their subcellular localization. In fact, the activity of lncRNAs may vary according to cell and tissue specificity and subcellular compartmentalization. Myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism. How lncRNAs are expressed in different myofibers, participate in metabolism regulation and muscle atrophy or how they are compartmentalized within a single myofiber is still unknown. We compiled a comprehensive catalog of lncRNAs expressed in skeletal muscle, associating the fiber-type specificity and subcellular location to each of them, and demonstrating that many lncRNAs can be involved in the biological processes de-regulated during muscle atrophy. We demonstrated that the lncRNA Pvt1, activated early during muscle atrophy, impacts mitochondrial respiration and morphology and affects mito/autophagy, apoptosis and myofiber size in vivo. This work corroborates the importance of lncRNAs in the regulation of metabolism and neuromuscular pathologies and offers a valuable resource to study the metabolism in single cells characterized by pronounced plasticity

Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic arteries

<p>Abstract</p> <p>Background</p> <p>Atherosclerosis affects aorta, coronary, carotid, and iliac arteries most frequently than any other body vessel. There may be common molecular pathways sustaining this process. Plaque presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading to plaque rupture and thrombosis.</p> <p>Results</p> <p>We used DNA microarrays and meta-analysis to study how the presence of calcified plaque modifies human coronary and carotid gene expression. We identified a series of potential human atherogenic genes that are integrated in functional networks involved in atherosclerosis. Caveolae and JAK/STAT pathways, and S100A9/S100A8 interacting proteins are certainly involved in the development of vascular disease. We found that the system of caveolae is directly connected with genes that respond to hormone receptors, and indirectly with the apoptosis pathway.</p> <p>Cytokines, chemokines and growth factors released in the blood flux were investigated in parallel. High levels of RANTES, IL-1ra, MIP-1alpha, MIP-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-17, PDGF-BB, VEGF and IFN-gamma were found in plasma of atherosclerotic patients and might also be integrated in the molecular networks underlying atherosclerotic modifications of these vessels.</p> <p>Conclusion</p> <p>The pattern of cytokine and S100A9/S100A8 up-regulation characterizes atherosclerosis as a proinflammatory disorder. Activation of the JAK/STAT pathway is confirmed by the up-regulation of IL-6, STAT1, ISGF3G and IL10RA genes in coronary and carotid plaques. The functional network constructed in our research is an evidence of the central role of STAT protein and the caveolae system to contribute to preserve the plaque. Moreover, Cav-1 is involved in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the atherosclerotic phenotype.</p

RNA-sequencing reveals that STRN, ZNF484 and WNK1 add to the value of mitochondrial MT-COI and COX10 as markers of unstable coronary artery disease

Markers in monocytes, precursors of macrophages, which are related to CAD, are largely unknown. Therefore, we aimed to identify genes in monocytes predictive of a new ischemic event in patients with CAD and/or discriminate between stable CAD and acute coronary syndrome. We included 66 patients with stable CAD, of which 24 developed a new ischemic event, and 19 patients with ACS. Circulating CD14+ monocytes were isolated with magnetic beads. RNA sequencing analysis in monocytes of patients with (n = 13) versus without (n = 11) ischemic event at follow-up and in patients with ACS (n = 12) was validated with qPCR (n = 85). MT-COI, STRN and COX10 predicted new ischemic events in CAD patients (power for separation at 1% error rate of 0.97, 0.90 and 0.77 respectively). Low MT-COI and high STRN were also related to shorter time between blood sampling and event. COX10 and ZNF484 together with MT-COI, STRN and WNK1 separated ACS completely from stable CAD patients. RNA expressions in monocytes of MT-COI, COX10, STRN, WNK1 and ZNF484 were independent of cholesterol lowering and antiplatelet treatment. They were independent of troponin T, a marker of myocardial injury. But, COX10 and ZNF484 in human plaques correlated to plaque markers of M1 macrophage polarization, reflecting vascular injury. Expression of MT-COI, COX10, STRN and WNK1, but not that of ZNF484, PBMCs paired with that in monocytes. The prospective study of relation of MT-COI, COX10, STRN, WNK1 and ZNF484 with unstable CAD is warranted