ABSTRACT: In order to identify targets for HDAC4, NRVM were infected with adenoviral vectors encoding beta-Galactosidase or Flag- HDAC4, and incubated in serum free or 10% fetal calf serum containing growth medium for 48 hrs. NRVM were infected with adenoviral vectors encoding beta-Galactosidase (control) or Flag- HDAC4 (experiment), and incubated in serum free or 10% fetal calf serum containing growth medium for 48 hrs. 2 biological samples of each condition were analyzed.
Project description:The use of anthracycline antibiotics such as doxorubicin (DOX) has greatly improved the mortality and morbidity of cancer patients. However, the associated risk of cardiomyopathy has limited their clinical application. DOX-associated cardiotoxicity is irreversible and progresses to heart failure (HF). For this reason, a better understanding of the molecular mechanisms underlying these adverse cardiac effects is essential to develop improved regimes that include cardioprotective strategies. MicroRNAs (miRNAs) are short non-coding RNAs that are able to post-trascriptionally regulate gene expression. MiRNAs have been demonstrated to be involved in both cancer and cardiovascular disease. Therefore, we were interested in unveiling the potential role of miRNAs in chemotherapy-induced HF. We used a combination of three different models to recreate this cardiac toxicity (acute in vitro DOX treatment, DOX-induced HF in vivo and a myocardial infarction -MI- leading to failure model) to study the pattern of dysregulated miRNAs. Using RNA from all three conditions, miRNA microarray profiling was performed and a common miRNA signature was identified. Interestingly, these dysregulated miRNAs have been previously identified as involved in the failing heart. Our results suggest that DOX is able to alter the expression of miRNAs implicated in HF, in vitro as well as in vivo. The present study is a microRNA profiling of the damaged cardiac muscle (cardiomyocyte cell population), following either myocardial infarction (MI) induction or doxorubicin (DOX) treatment. Two DOX-treated models were included: ARC exposed to DOX in vitro and a validated DOX-induced heart failure model generated by repeated administration of DOX injections.
Project description:This experiment investigated the effects of the novel bromodomain inhibitor 3i-1248 in neonatal mouse ventricular cardiomyocytes with and without neurohormonal stimulation with ET-1.
Project description:Cardiac hypertrophy consists in the enlargement of cardiomyocytes and alteration of the extracellular matrix organization in response to physiological or pathological stress. In pathological hypertrophy ocuurs myocardial damage, loss of cardiomyocytes, fibrosis, inflammation, sarcomere disorganization and metabolic impairment, leading to cardiac dysfunction.The rodent model treated with isoproterenol induces cardiac hypertrophy due the constant activation of β-adrenergic receptors. We conducted a quantitative label-free proteomic analysis of cardiomyocytes isolated from hearts of mice treated or not with isoproterenol to better understand the molecular bases of cellular response due to isoproterenol-induced injury.
Project description:Central questions like cardiomyocyte subtype emergence during cardiogenesis or availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, characterization and implementation of pure cardiomyocyte subtypes is still challenging due to technical limitations. Our aim was to identify surface markers enabling the selective detection and purification of atrial and ventricular cardiomyocytes from mouse hearts. In a surface marker screen we found differential expression of CD49f in atrial and ventricular embryonic cardiomyocytes (E13.5). By flow cytometry we could correlate a high CD49f expression with MLC-2a on the single cell level; a low CD49f expression corresponded to MLC-2v. Based on the persisting differential CD49f expression we developed purification protocols for cardiomyocytes subtypes from the developing mouse heart. Flow sorting of E15.5 hearts into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cells led to a selective depletion (CD49flow) or enrichment of MLC-2a+ cells (CD49fhigh). We found a corresponding CD49f-dependent distribution of MLC-2a when pre-enriched neonatal cardiomyocytes (P2) were flow-sorted into CD49flow and CD49fhigh. Atrial and ventricular identity was confirmed by expression profiling and patch clamp analysis of sorted embryonic hearts, which unequivocally demonstrated that the sorted cells were viable and functional. For the first time, we introduce a non-genetic, antibody-based approach to specifically isolate atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This newly gained capability of obtaining highly pure, viable cells will facilitate in-depths characterization of the individual cellular subsets and will aid translational research and therapeutic applications. The dataset comprises four different cardiomyocytes subtypes from the developing mouse heart. Embryonic (E15.5) hearts were dissociated and flow-sorted into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cardiomyocytes. Neonatal (P2) hearts were dissociated, contaminating non-myocytes were removed by MACS depletion, and the purified cardiomyocytes were flow-sorted into CD49flow and CD49fhigh cardiomyocytes. Four biological replicates were available for each sample groups. Microarray analysis was conducted on the Agilent Whole Mouse Genome Oligo Microarray 8x60K platform.
Project description:Molecular analysis of transcriptional changes in cardiomyocytes induced by Oncostatin M treatment. The rationale of this experiment is described in [Kubin, T., et al. Oncostatin M is a major mediator of cardiomyocyte dedifferentiation and remodeling. Cell stem cell 9, 420-432 (2011)]
Project description:In the current study we examined several proteomic- and RNA-Seq-based datasets of cardiac-enriched, cell-surface and membrane-associated proteins in human fetal and mouse neonatal ventricular cardiomyocytes. By integrating available microarray and tissue expression profiles along with MGI phenotypic analysis, we identified 173 membrane-associated proteins that are cardiac-enriched, conserved amongst eukaryotic species, and have not yet been linked to a ‘cardiac’ Phenotype-Ontology. To highlight the utility of this dataset, we selected several proteins to investigate more carefully, including FAM162A, MCT1, and COX20, to show cardiac enrichment, subcellular distribution and expression patterns in disease. Three-dimensional imaging was used to validate subcellular localization and expression in adult mouse ventricular cardiomyocytes. FAM162A, MCT1, and COX20 were differentially expressed at the transcriptomic and proteomic levels in multiple models of mouse and human heart diseases and may represent potential diagnostic and therapeutic targets for human dilated and ischemic cardiomyopathies. Altogether, we believe this comprehensive cardiomyocyte membrane proteome dataset will prove instrumental to future investigations aimed at characterizing heart disease markers and/or therapeutic targets for heart failure.
Project description:\miR-208a is a cardiac specific microRNA whose expression is dysregulated in several cardiac diseases including myocardial infarction (MI) and dilated cardiomyopathy in which it is associated with adverse outcomes. Given that there is increased apoptosis in these pathologies, we investigated if miR-208a has any effect on apoptosis genes expression. we also investigated its effect on genes in other pathways such as autophagy, ion channels, angiogenesis. Methods and Results The effect of miR-208a on apoptosis during ischemia was studied in cultured neonatal mice myocytes transfected with agomir or antagomir. Differential gene expression induced by miR-208a was assessed using microarrays. Microarray profiling was done on custom made array chips by a service provider ( Ribobio co. Guangzhou China), using total RNA isolated from cultured cardiomyocytes transfected with miR-208a agomir or control for 72hrs. Up and down regulated genes are available as additional files in this submission https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3477/files/
Project description:A novel ppp1r13l sequence variation causes dilated cardiomyopathy and cardiac inflammation. In this experiment we knocked down IASPP (protein product of ppp1r13l) in cardiomyocytes and exposed them to lipopolysaccharide for time interval of 2 and 4 hours. Transcriptome was examined using rna-seq high-throughput sequencing.
Project description:Inflammation is a major factor in heart disease. IκB kinase (IKK) and its downstream target NF-κB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-κB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2 (IKK2-CA). In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b+ cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. To gain insight into proximal events after activation of IKK2/NF-κB, we isolated cardiomyocytes and activated the transgene in vitro, thus avoiding interference of other cell types. Gene expression analysis revealed up-regulation of transcripts of chemotactic cytokines, adhesion molecules, and a striking number of IFN-regulated genes in cardiomyocytes expressing IKK2-CA. In addition, apoptotic and anti-apoptotic genes were up-regulated.
Project description:Identfification of MEF2A target genes using ChIP-exo in skeletla muscle and primary cardiomyocytes. Identfification of MEF2A target genes using ChIP-exo and RNA-seq in skeletal muscle and primary cardiomyocytes. MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells. MEF2A target genes were identified in 48 hr DM C2C12 myoblasts cells and primary cardiomyocytes using ChIP-exo. Binding profiles on MEF2A in each cell type were compared. Cross sectional-analysis between ChIP-exo identified targets and RNA-seq analysis of MEF2A deplted myoblasts was also done.