Fisetin protects against cardiac cell death through reduction of ROS production and caspases activity

Myocardial infarction (MI) is a leading cause of death worldwide. Reperfusion is considered as an optimal therapy following cardiac ischemia. However, the promotion of a rapid elevation of O2 levels in ischemic cells produces high amounts of reactive oxygen species (ROS) leading to myocardial tissue injury. This phenomenon is called ischemia reperfusion injury (IRI). We aimed at identifying new and effective compounds to treat MI and minimize IRI. We previously studied heart regeneration following myocardial injury in zebrafish and described each step of the regeneration process, from the day of injury until complete recovery, in terms of transcriptional responses. Here, we mined the data and performed a deep in silico analysis to identify drugs highly likely to induce cardiac regeneration. Fisetin was identified as the top candidate. We validated its effects in an in vitro model of MI/IRI in mammalian cardiac cells. Fisetin enhances viability of rat cardiomyocytes following hypoxia/starvation - reoxygenation. It inhibits apoptosis, decreases ROS generation and caspase activation and protects from DNA damage. Interestingly, fisetin also activates genes involved in cell proliferation. Fisetin is thus a highly promising candidate drug with clinical potential to protect from ischemic damage following MI and to overcome IRI.This work was supported by FNR, the Luxembourg National Research Fund, FNR-CORE INFUSED project. At the NorLux Laboratory and the Proteome and Genome Research Unit of LIH, it was also supported by funding from Luxembourg’s Ministry of Higher Education and Research (MESR).S

Transcriptional response to cardiac injury in the zebrafish: systematic identification of genes with highly concordant activity across in vivo models

Background: Zebrafish is a clinically-relevant model of heart regeneration. Unlike mammals, it has a remarkable heart repair capacity after injury, and promises novel translational applications. Amputation and cryoinjury models are key research tools for understanding injury response and regeneration in vivo. An understanding of the transcriptional responses following injury is needed to identify key players of heart tissue repair, as well as potential targets for boosting this property in humans. Results: We investigated amputation and cryoinjury in vivo models of heart damage in the zebrafish through unbiased, integrative analyses of independent molecular datasets. To detect genes with potential biological roles, we derived computational prediction models with microarray data from heart amputation experiments. We focused on a top-ranked set of genes highly activated in the early post-injury stage, whose activity was further verified in independent microarray datasets. Next, we performed independent validations of expression responses with qPCR in a cryoinjury model. Across in vivo models, the top candidates showed highly concordant responses at 1 and 3 days post-injury, which highlights the predictive power of our analysis strategies and the possible biological relevance of these genes. Top candidates are significantly involved in cell fate specification and differentiation, and include heart failure markers such as periostin, as well as potential new targets for heart regeneration. For example, ptgis and ca2 were overexpressed, while usp2a, a regulator of the p53 pathway, was down-regulated in our in vivo models. Interestingly, a high activity of ptgis and ca2 has been previously observed in failing hearts from rats and humans. Conclusions: We identified genes with potential critical roles in the response to cardiac damage in the zebrafish. Their transcriptional activities are reproducible in different in vivo models of cardiac injury. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-852) contains supplementary material, which is available to authorized users

Transforming growth factor β receptor 1 is a new candidate prognostic biomarker after acute myocardial infarction

<p>Abstract</p> <p>Background</p> <p>Prediction of left ventricular (LV) remodeling after acute myocardial infarction (MI) is clinically important and would benefit from the discovery of new biomarkers.</p> <p>Methods</p> <p>Blood samples were obtained upon admission in patients with acute ST-elevation MI who underwent primary percutaneous coronary intervention. Messenger RNA was extracted from whole blood cells. LV function was evaluated by echocardiography at 4-months.</p> <p>Results</p> <p>In a test cohort of 32 MI patients, integrated analysis of microarrays with a network of protein-protein interactions identified subgroups of genes which predicted LV dysfunction (ejection fraction ≤ 40%) with areas under the receiver operating characteristic curve (AUC) above 0.80. Candidate genes included transforming growth factor beta receptor 1 (TGFBR1). In a validation cohort of 115 MI patients, TGBFR1 was up-regulated in patients with LV dysfunction (P < 0.001) and was associated with LV function at 4-months (P = 0.003). TGFBR1 predicted LV function with an AUC of 0.72, while peak levels of troponin T (TnT) provided an AUC of 0.64. Adding TGFBR1 to the prediction of TnT resulted in a net reclassification index of 8.2%. When added to a mixed clinical model including age, gender and time to reperfusion, TGFBR1 reclassified 17.7% of misclassified patients. TGFB1, the ligand of TGFBR1, was also up-regulated in patients with LV dysfunction (P = 0.004), was associated with LV function (P = 0.006), and provided an AUC of 0.66. In the rat MI model induced by permanent coronary ligation, the TGFB1-TGFBR1 axis was activated in the heart and correlated with the extent of remodeling at 2 months.</p> <p>Conclusions</p> <p>We identified TGFBR1 as a new candidate prognostic biomarker after acute MI.</p

Etude de la fonction de la sous-unité d'intégrine a6 dans les cellules de peau

Les intégrines sont les principaux récepteurs de la matrice extracellulaire. Les souris a6-/- meurent à la naissance et présentent un décollement généralisé de l'épiderme, dû à une absence d'hémidesmosomes dans les tissus mutants. Afin d'analyser plus précisément la fonction d'a6 dans la peau, nous avons généré des lignées de kératinocytes immortalisés de manière conditionnelle, à partir d'embryons de souris a6+/- et a6-/-. Les cellules mutantes adhèrent moins bien sur laminine 5, le composant majeur de la lame basale dans la peau, et sur les laminines 10/11 et 2. Des expériences de puces à ADN ont révélé que l'expression de différentes familles de gènes est modifiée dans les cellules mutantes: protéines de la matrice extracellulaire (laminine 5), molécules d'adhérence (intégrine b4 ou collagène XVII), ou des voies de signalisation (les protéines de la famille TGFb . Nous avons également observé une augmentation de l'expression des marqueurs de la différentiation épidermique dans les tissus mutants. Notre hypothèse est que ces altérations pourraient être liées à une surexpression de la voie TGFb/Jun/Fos dans les kératinocytes a6-/-. Nous avons enfin réalisé une étude structure/fonction d'a6. Pour cela nous avons transfecté de manière stable les lignées de kératinocytes mutants avec les isoformes a6A et a6B. L'analyse de nos lignées cellulaires a révélé que la sous-unité d'intégrine b4 s'accumule dans le réticulum endoplasmique où elle colocalise avec la calnexine lorsque son partenaire a6 est absent, alors qu'elle est transportée à la membrane plasmique d' a6A mais aussi ' a6B. Les propriétés d'adhérence des cellules transfectées par a6B sont cependant altérées sur les substrats de laminine testés précédemment.Integrins are major receptors for the extracellular matrix. a6-/- mice die at birth with a severe skin blistering, due to a total absence of hemidesmosomes in mutant cells. To further study the function of a6 in the skin, we generated conditionally immortalized cell lines from the epidermis of a6+/- and a6-/- mouse embryos. Mutant cells present a decreased adhesion on laminin 5, the major component of the basement membrane in the skin, and on laminin 10/11 and 2. A DNA array analysis revealed alteration in the expression of different families of genes, such as extracellular matrix components (laminin 5), membrane receptors (b4 integrin or collagen XVII) or signalling molecules (TGFb family of proteins). An increase of several epidermal differentiation markers is observed in mutant tissues. Our hypothesis is that this may be linked to an up-regulation of the TGFb/Jun/Fos pathway in a6-/- keratinocytes. We finally studied the function of the cytoplasmic tail of a6 by analysing the behaviour of our a6-/- keratinocytes transfected with either the a6A or the a6B isoform. Results revealed that b4 integrin accumulates in the endoplasmic reticulum where it colocalizes with calnexin when a6 is missing, while it is translocated at the plasma membrane in presence of either a6A or a6B. Adherence properties of mutant cells transfected with a6B are however still altered.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Matrix Metalloproteinase 9 Polymorphism and Outcome after Myocardial Infarction

Matrix metalloproteinase 9 (MMP9) is functionally implicated in the process of infarct healing. Several genetic variation of the MMP9 gene have been described, among which the MMP9 Arg668Gln polymorphism. In the present study, we assessed whether this polymorphism influences outcome after acute myocardial infarction (MI). One thousand forty-nine patients undergoing coronary angiography were genotyped for the MMP9 Arg668Gln polymorphism by TaqMan allelic discrimination assay. This population included 154 controls, 161 patients with non ST-elevation MI (NSTEMI), 504 patients with ST-elevation MI (STEMI), and 230 patients with angina. Frequency of the MMP9 Arg668Gln polymorphism in the global population was 25.1%, and was comparable between all groups. STEMI patients had higher creatine phosphokinase (CPK), troponin T (TnT) and MMP9 plasma levels and had lower ejection fraction (EF) than NSTEMI patients. However, the polymorphism was not associated with infarct severity as determined by peak CPK and TnT levels, nor with LV remodeling and outcome as assessed by 1-month EF and NYHA class, as well as 2- year mortality. In silico molecular modeling simulations predicted that the MMP9 polymorphism may decrease MMP9 activity, but this could not be verified by plasma determinations. This study investigated for the first time the association between the MMP9 Arg668Gln polymorphism and clinical outcome after acute MI. Our results indicate that the polymorphism does not seem to be associated with clinical outcome and in particular with the development of left ventricular dysfunction and heart failure

Single-cell transcriptomics of human iPSC differentiation dynamics reveal a core molecular network of Parkinson’s disease

Parkinson’s disease (PD) is the second-most prevalent neurodegenerative disorder, characterized by the loss of dopaminergic neurons (mDA) in the midbrain. The underlying mechanisms are only partly understood and there is no treatment to reverse PD progression. Here, we investigated the disease mechanism using mDA neurons differentiated from human induced pluripotent stem cells (hiPSCs) carrying the ILE368ASN mutation within the PINK1 gene, which is strongly associated with PD. Single-cell RNA sequencing (RNAseq) and gene expression analysis of a PINK1-ILE368ASN and a control cell line identified genes differentially expressed during mDA neuron differentiation. Network analysis revealed that these genes form a core network, members of which interact with all known 19 protein-coding Parkinson’s disease-associated genes. This core network encompasses key PD-associated pathways, including ubiquitination, mitochondrial function, protein processing, RNA metabolism, and vesicular transport. Proteomics analysis showed a consistent alteration in proteins of dopamine metabolism, indicating a defect of dopaminergic metabolism in PINK1-ILE368ASN neurons. Our findings suggest the existence of a network onto which pathways associated with PD pathology converge, and offers an inclusive interpretation of the phenotypic heterogeneity of PD

A comprehensive integrative analysis of the transcriptional network underlying the zebrafish heart regeneration

Despite a notable reduction in incidence of acute myocardial infarction (MI), patients who experienced it remain at risk for premature death and cardiac malfunction. The human cardiomyocytes are not able to achieve extensive regeneration upon MI. Remarkably, the adult zebrafish is able to achieve complete heart regeneration following amputation, cryoinjury or genetic ablation. This raises new potential opportunities on how to boost heart healing capacity in humans. The objective of our research is to characterize the transcriptional network of the zebrafish heart regeneration and underlying regulatory mechanisms. To conduct our investigation, we used microarray data from zebrafish at 6 post-cryoinjury time points (4 hours, and 1, 3, 7, 14 and 90 days) and control samples. We thereon looked for the gene co-expression patterns in the data and, based on that, constructed a weighted gene co-expression network. To detect candidate functional sub-networks (modules), we used two different network clustering approaches: a density-based (ClusterONE) and a topological overlap-based (Hybrid Dynamic Branch Cut) algorithms. The visualization of the expression changes of the candidate modules reflected the dynamics of the recovery process. Also we aimed to identify candidate “hub” genes that might regulate the behavior of the biological modules and drive the regeneration process. We identified eighteen distinct modules associated with heart recovery upon cryoinjury. Functional enrichment analysis displayed that the modules are involved in different cellular processes crucial for heart regeneration, including: cell fate specification (p-value < 0.006) and migration (p-value < 0.047), ribosome biogenesis (p-value < 0.004), cardiac cell differentiation (p-value < 3E-04), and various signaling events (p-value < 0.037). The visualization of the modules’ expression profiles confirmed the relevance of these functional enrichments. For instance, the genes of the module involved in regulation of endodermal cell fate specification were up-regulated upon injury until 3 days. Among the candidate hub genes detected in the network, there are genes relevant to atherosclerosis treatment and inflammation during cardiac arrest. These and other findings are currently undergoing deeper computational analyses. The top promising targets will be independently validated using our zebrafish (in vivo) model. In conclusion, our findings provide insights into the complex regulatory mechanisms involved during heart regeneration in the zebrafish. These data will be useful for modelling specific network-based responses to heart injury, and for finding sensitive network points that may trigger or boost heart regeneration

The Talin Rod IBS2 α-Helix Interacts with the β3 Integrin Cytoplasmic Tail Membrane-proximal Helix by Establishing Charge Complementary Salt Bridges*S⃞

Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a second, IBS2, that maps to helix 50 of the talin rod domain and is essential for linking integrin β subunits to the cytoskeleton (Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J. Biol. Chem. 282, 17280-1728817430904). Through the combined approach of mutational analysis of the β3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies, as well as site-specific antibody inhibition experiments, we provide evidence that the integrin β3-talin rod interaction relies on a helix-helix association between α-helix 50 of the talin rod domain and the membrane-proximal α-helix of the β3 integrin cytoplasmic tail. Moreover, charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin β3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the β3 subunit cytoplasmic helix as the integrin αIIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the β3 subunit following integrin activation