Isolated cardiomyocyte and cardiac fibroblast expression analysis
ABSTRACT: Some cell type-specific gene expression is maintained in the maturation of cardiomyocytes, where DNA hypomethylation of gene body regions of a set of specific genes. We used microarrays to detail the global gene expression program underlying the maintenance of cardiomyocyte function and maturation and compared it with DNA methylation status. Cardiomyocytes and cardiac fibroblasts were carefully isolated from neonatal and adult hearts and used fresh for the analysis.
Project description:Cardiomyocyte-like cells can be reprogrammed from somatic fibroblasts by combinations of genes, providing a new avenue for cardiac regenerative therapy. Here we show that functional cardiomyocytes can be rapidly and efficiently generated from human fibroblasts by specific combination small molecules. Microarray analysis has been used to compare the expression profile of cardiomyocyte-like cells derived from human foreskin and lung fibroblasts, and human ES cell-derived cardiomyocytes. Cardiomyocytes generated from different origins were metabolically purified under glucose-depleted and lactate-abundant conditions for RNA extraction and hybridization on Affymetrix microarrays.
Project description:During development the fetal heart undergoes a rapid and dramatic transition to adult function through transcriptional and post-transcriptional mechanisms, including alternative splicing (AS). We performed deep RNA-sequencing for high-resolution analysis of transcriptome changes during postnatal mouse heart development using RNA from ventricles and freshly isolated cardiomyocytes (CM) and cardiac fibroblasts (CF). Extensive changes in gene expression and AS occur primarily between postnatal days 1 and 28. CM and CF showed reciprocal regulation of gene expression during postnatal development reflecting differences in proliferative capacity, cell adhesion functions, and mitochondrial metabolism. We found that AS plays a novel role in vesicular trafficking and membrane organization during postnatal CM development. Interestingly, these AS transitions are enriched among targets of two RNA-binding proteins, Celf1 and Mbnl1, which undergo developmentally regulated change in expression. Vesicular traffic genes affected by AS during normal development where Celf1 is down-regulated, showed a reversion to neonatal AS patterns when Celf1 was over-expressed in adults. RNA-seq was performed in RNA samples of ventricles, cardiomyocytes or cardiac fibroblast at different developmental stages; embryonic day 17, postnatal day (PN) 1, 10, 28 and 90 for ventricles, PN1-3, PN28 and PN60 for cardiac fibroblasts, and PN1-2, PN30, and PN67 for cardiomyocytes
Project description:Recent studies have highlighted the role of adrenal corticosteroid signaling in cardiac physiology and pathophysiology. It is known that glucocorticoids and aldosterone are able to bind glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), and these ligand-receptor interactions are redundant. Therefore, it has been impossible to delineate how these nuclear receptors couple with corticosteroid ligands and differentially regulate gene expression for operation of their distinct functions in the heart. Here, to particularly define the role of GR, we applied ligand-based approach involving GR-specific agonist cortivazol (CVZ) and GR antagonist RU486, and performed microarray analysis using rat neonatal cardiomyocytes. We indicated that glucocorticoids appear to be a major determinant of GR-mediated gene expression when compared with aldosterone. Moreover, expression profiles of these genes highlighted numerous roles of glucocorticoids in various aspects of cardiac physiology. Experiment Overall Design: We analyzed gene expression changes after exposure of cells to corticosterone (COR), aldosterone (ALD), and cortivazol (CVZ) in the absence or presence of GR antagonist RU486. Since our preliminary experiments using several cell lines showed that expression of many GR target genes was induced by glucocorticoids in 3 h and to avoid secondary effects of the products of GR-regulated genes, we in the present study set the time periods of exposure to these ligands as 3 h. DNA microarray experiments were performed twice with the same protocol except for RU486 treatment.
Project description:NimbleGen Mouse DNA Methylation 3x720K CpG Islands Pus RefSeq Promoter Array was used to perform a DNA methylome analysis to unravel the molecular mechanism underlying the dedifferentiation and cell cycle reentry of mouse adult cardiomyocytes (ACMs). A total of six DNA samples (10 ng each) derived from the population cells from either control adult cardiomyocytes or mCPCs were subject to whole genome amplification and then 2.5 µg of amplified products were used for DNA methylome analysis. There were three biological replicates in each group (both ACM controls and mCPCs). The whole genome methylome data using NimbleGen Mouse 2.1M array and genomic DNA derived from population cells for both control adult cardiomyocytes and mCPC can be found in E-MTAB-3984. Single-cell whole-transcriptome microarray data using Affymetrix Mouse Genome 430 2.0 Array can be found in E-MTAB-3981.
Project description:NimbleGen Mouse DNA Methylation 2.1M Array (CHARM design) was used to perform a DNA methylome analysis to unravel the molecular mechanism underlying the dedifferentiation and cell cycle reentry of mouse adult cardiomyocytes (ACMs). A total of six DNA samples (10 ng each) derived from population cells were subjected to whole genome amplification using Sigma WGA kit, and then 2.5 µg amplified DNA product was used for DNA methylome analysis. Three biological replicates of population ACM cells were used as controls and three biological replicates of population myocyte-derived progenitor cells (mCPCs) were used in the experimental group. Methylome microarray data using NimbleGen Mouse DNA Methylation 3x720K CpG Islands Pus RefSeq Promoter Array and DNA derived from population cells for both control cardiomyocytes and mCPCs can be found in E-MTAB-3982. Single-cell whole-transcriptome data using Affymetrix Mouse Genome 430 2.0 Array can be found in E-MTAB-3981.
Project description:Affymetrix Mouse Genome 430 2.0 Array was used to perform single-cell transcriptome analysis to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse adult cardiomyocytes (ACMs). A total of six samples were used for single-cell transcriptome analysis using Affymetrix MG 430 2.0 Array coupled with a microfluidic chip for single cell isolation and NuGen Ovation Single-Cell Amplification technologies. Three biological replicates of adult cardiomyocyte single cells were used as controls and three biological replicates of myocyte-derived progenitor single cells (mCPCs) were used in the experimental group. The whole genome methylome microarray data using NimbleGen Mouse DNA Methylation 3x720K CpG Island Plus RefSeq Promoter Array and genomic DNA derived from population cells for both control adult cardiomyocytes and mCPCs can be found in E-MTAB-3982. The whole genome methylome data using NimbleGen Mouse 2.1M array and genomic DNA derived from population cells for both control adult cardiomyocytes and mCPC can be found in E-MTAB-3984.
Project description:Evaluate the change in transcription factors that have a role in human mesenchymal stem cell (hMSC) commitment to a cardiomyocyte lineage when co-cultured for 4 days with rat neonatal cardiomyocytes and before acquiring a recognizable cardiac phenotype. A myocardial microenvironment was generated by dissociating neonatal rat hearts and establishing cardiomyocyte primary cultures. HumanMSCs constitutively labeled with dsRed localized to the cell's mitochondria were either grown separately (control) or added to the cardiomyocyte primary cultures and grown for 4 days. dsRed fluorescent hMSCs were harvested from co-cultures at 4 days using a FACscan flow cytometer. The RNA for the microarray analysis was prepared from three biologically separate samples of hMSCs co-cultured for 4 days and from hMSCs grown separately for 4 days (control).
Project description:Human pluripotent stem cell-derived cardiomyocytes (CMs) are a promising tool for cardiac cell therapy. To optimize graft cells for cardiac reconstruction, we compared the engraftment efficiency of intramyocardially-injected undifferentiated-induced pluripotent stem cells (iPSCs), day4 mesodermal cells, and day8, day20, and day30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day20 CMs was significantly higher compared to other cells. Transplantation of day20 CMs into the infarcted hearts of immunodeficient mice showed significant functional improvement. Moreover, the imaging signal and ratio of Ki67-positive CMs at 3 months post injection indicated engrafted CMs proliferated in the host heart. Although this graft growth reached a plateau at 3 months, histological analysis confirmed progressive maturation from 3 to 6 months. These results suggested that day20 CMs had very high engraftment, proliferation, and therapeutic potential in host mouse hearts. Differentiated cells, N=10 Undifferentiated pluripotent stem cells, N=1 Heart samples, N=6
Project description:The differentiation to cardiomyocytes is a prerequisite and an important part of heart development. A good understanding of the complicated cardiomyocyte differentiation process benefits cardiogenesis study. Embryonic stem cells (ESCs), cell lines with infinite ability to proliferate and to be differentiated into all cell types of the adult body, are important research tools for investigation of differentiation and meanwhile good models for developmental research. In the current study, genome-wide gene expression of ESCs is profiled through high throughput platform during cardiomyocyte-specific differentiation and maturation. Gene expression patterns of undifferentiated ESCs and ESC-derived cardiomyocytes provide a global overview of genes involved in cardiomyocyte-specific differentiation, whereas marker gene expression profiles of both ESC-related genes and cardiac-specific genes presented the expression pattern shift during differentiation in a pure ESC-derived cardiomyocyte cell culture system. Transgenic mouse ESC clone with α-MHC–Pac–IRES–EGFP vector containing the EGFP gene and the PuromycinR (Pac) cassette under control of the cardiac α-myosin heavy chain (α-MHC) promoter was cultured and induced into differentiation. Puromycin was applied after differentiation start to enable cardiomyocyte-specific differentiation. Cells were harvested at 4 time points after differentiation start (day0, day12, day19 and day26). Two biological replicates were taken for every time point.
Project description:We examined molecular responses using transcriptome profiling in isolated left ventricular murine cardiomyocytes to 90 cGy, 1 GeV proton (1H) and 15 cGy, 1 GeV/nucleon (n) proton (56Fe) particles 1, 3, 7, 14 and 28 days after exposure. Unsupervised clustering analysis of gene expression segregated samples according to the radiation (IR) response, and time after exposure with 56Fe-IR showing the greatest level of gene modulation. 1H-IR exposures showed little differential transcript modulation. Network analysis categorized the major differentially expressed genes into cell cycle, oxidative responses and transcriptional regulation functional groups. Transcriptional networks identified key nodes regulating expression. Individual transcription factors were inferred to be active at 1, 3, 7, 14 and 28 days after exposure. Validation of the signal transduction network by protein analysis showed that particle IR clearly regulates a long lived signaling mechanism for p38 MAPK signaling and NFATc4 activation. Electrophoresis mobility shift assays supported the role of additional key transcription factors GATA-4, STAT-3 and NF-κB as regulators of the response at specific time points. These data suggest that the molecular response to 56Fe-IR is unique and shows long-lasting gene expression in cardiomyocytes, up to 28 days after exposure. Additionally, proteins involved in signal transduction and transcriptional activation via DNA binding play a role in the response to high charge (Z) and energy (E) particles (HZE). Our study may have implications for NASA’s efforts to develop heart disease risk estimates for astronauts safety via identification of specific HZE-IR molecular markers and for patients receiving conventional and particle radiotherapy. Transcriptome profiling in isolated left ventricular murine cardiomyocytes to 90 cGy, 1 GeV proton (1H) and 15 cGy, 1 GeV/nucleon (n) proton (56Fe) particles 1, 3, 7, 14 and 28 days after exposure.