Distinct epigenomic patterns exist in important DNA elements of the cardiac genome in human end-stage cardiomyopathy. The epigenome may control the expression of local or distal genes with critical functions in myocardial stress response. If epigenomic patterns track with disease progression, assays for the epigenome may be useful for assessing prognosis in heart failure. Further studies are needed to determine whether and how the epigenome contributes to the development of cardiomyopathy.

Cardiac hypertrophy is a growth response of the heart to increased hemodynamic demand or damage. Accompanying this heart enlargement is a remodeling of Ca 2؉ signaling. Due to its fundamental role in controlling cardiomyocyte contraction during every heartbeat, modifications in Ca 2؉ fluxes significantly impact on cardiac output and facilitate the development of arrhythmias. Using cardiomyocytes from spontaneously hypertensive rats (SHRs), we demonstrate that an increase in Ca 2؉ release through inositol 1,4,5-trisphosphate receptors (InsP 3Rs) contributes to the larger excitation contraction coupling (ECC)-mediated Ca 2؉ transients characteristic of hypertrophic myocytes and underlies the more potent enhancement of ECCmediated Ca 2؉ transients and contraction elicited by InsP3 or endothelin-1 (ET-1). Responsible for this is an increase in InsP 3R expression in the junctional sarcoplasmic reticulum. Due to their close proximity to ryanodine receptors (RyRs) in this region, enhanced Ca 2؉ release through InsP 3Rs served to sensitize RyRs, thereby increasing diastolic Ca 2؉ levels, the incidence of extra-systolic Ca 2؉ transients, and the induction of ECC-mediated Ca 2؉ elevations. Unlike the increase in InsP 3R expression and Ca 2؉ transient amplitude in the cytosol, InsP3R expression and ECC-mediated Ca 2؉ transients in the nucleus were not altered during hypertrophy. Elevated InsP 3R2 expression was also detected in hearts from human patients with heart failure after ischemic dilated cardiomyopathy, as well as in aortic-banded hypertrophic mouse hearts. Our data establish that increased InsP 3R expression is a general mechanism that underlies remodeling of Ca 2؉ signaling during heart disease, and in particular, in triggering ventricular arrhythmia during hypertrophy.calcium ͉ ECC ͉ IP3 ͉ SHR ͉ signalling

Epigenetic mechanisms such as microRNA and histone modification are crucially responsible for dysregulated gene expression in heart failure. In contrast, the role of DNA methylation, another well-characterized epigenetic mark, is unknown. In order to examine whether human cardiomyopathy of different etiologies are connected by a unifying pattern of DNA methylation pattern, we undertook profiling with ischaemic and idiopathic end-stage cardiomyopathic left ventricular (LV) explants from patients who had undergone cardiac transplantation compared to normal control. We performed a preliminary analysis using methylated-DNA immunoprecipitation-chip (MeDIP-chip), validated differential methylation loci by bisulfite-(BS) PCR and high throughput sequencing, and identified 3 angiogenesis-related genetic loci that were differentially methylated. Using quantitative RT-PCR, we found that the expression of these genes differed significantly between CM hearts and normal control (p<0.01). Moreover, for each individual LV tissue, differential methylation showed a predicted correlation to differential expression of the corresponding gene. Thus, differential DNA methylation exists in human cardiomyopathy. In this series of heterogenous cardiomyopathic LV explants, differential DNA methylation was found in at least 3 angiogenesis-related genes. While in other systems, changes in DNA methylation at specific genomic loci usually precede changes in the expression of corresponding genes, our current findings in cardiomyopathy merit further investigation to determine whether DNA methylation changes play a causative role in the progression of heart failure.

Clear cell renal cell carcinoma (ccRCC), a tubular epithelial cell (TEC) malignancy, frequently secretes tumor necrosis factor (TNF). TNF signals via two distinct receptors (TNFRs). TNFR1, expressed in normal kidney primarily on endothelial cells, activates apoptotic signaling kinase 1 and nuclear factor-B (NF-B) and induces cell death, whereas TNFR2, inducibly expressed on endothelial cells and on TECs by injury, activates endothelial/epithelial tyrosine kinase (Etk), which trans-activates vascular endothelial growth factor receptor 2 (VEGFR2) to promote cell proliferation. We investigated TNFR expression in clinical samples and function in short-term organ cultures of ccRCC tissue treated with wild-type TNF or specific muteins selective for TNFR1 (R1-TNF) or TNFR2 (R2-TNF). There is a significant increase in TNFR2 but not TNFR1 expression on malignant TECs that correlates with increasing malignant grade. In ccRCC organ cultures, R1-TNF increases TNFR1, activates apoptotic signaling kinase and NF-B, and promotes apoptosis in malignant TECs. R2-TNF increases TNFR2, activates NF-B, Etk, and VEGFR2 and increases entry into the cell cycle. Wild-type TNF induces both sets of responses. R2-TNF actions are blocked by pretreatment with a VEGFR2 kinase inhibitor. We conclude that TNF, acting through TNFR2, is an autocrine growth factor for ccRCC acting via Etk-VEGFR2 cross-talk, insights that may provide a more effective therapeutic approach to this disease.

BackgroundDNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding.ResultsUsing genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation.ConclusionsOur analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.

Induced pluripotent stem cells (iPSCs) have the potential to generate patient-specific tissues for disease modeling and regenerative medicine applications. However, before iPSC technology can progress to the translational phase, several obstacles must be overcome. These include uncertainty regarding the ideal somatic cell type for reprogramming, the low kinetics and efficiency of reprogramming, and karyotype discrepancies between iPSCs and their somatic precursors. Here we describe the use of late-outgrowth endothelial progenitor cells (L-EPCs), which possess several favorable characteristics, as a cellular substrate for the generation of iPSCs. We have developed a protocol that allows the reliable isolation of L-EPCs from peripheral blood mononuclear cell preparations, including frozen samples. As a proof-of-principle for clinical applications we generated EPC-iPSCs from both healthy individuals and patients with heritable and idiopathic forms of pulmonary arterial hypertension. L-EPCs grew clonally; were highly proliferative, passageable, and bankable; and displayed higher reprogramming kinetics and efficiencies compared with dermal fibroblasts. Unlike fibroblasts, the high efficiency of L-EPC reprogramming allowed for the reliable generation of iPSCs in a 96-well format, which is compatible with high-throughput platforms. Array comparative genome hybridization analysis of L-EPCs versus donor-matched circulating monocytes demonstrated that L-EPCs have normal karyotypes compared with their subject's reference genome. In addition, >80% of EPC-iPSC lines tested did not acquire any copy number variations during reprogramming compared with their parent L-EPC line. This work identifies L-EPCs as a practical and efficient cellular substrate for iPSC generation, with the potential to address many of the factors currently limiting the translation of this technology. STEM CELLS TRANSLATIONAL MEDICINE 2012;1:855-865

Apoptosis is an evolutionarily conserved mode of cell death that is tightly regulated and critical for multicellular organism development and cellular homeostasis. Specific biochemical and morphological changes characterise cells undergoing apoptosis, and reflect the specificity in which activated apoptotic pathways follow. The two best-characterized apoptotic pathways are the extrinsic pathway and the intrinsic pathway, which involve cell surface death receptors and the mitochondria and endoplasmic reticulum respectively. Apoptotic stimuli lead to activation of either or both of these pathways, and involve sequential activation of different cysteine proteases (caspases), and in the case of the intrinsic pathway, activation of a family of Bcl-2 proteins that critically regulate cell death. Conversely, dis-inhibition of endogenous inhibitors is often required for effective apoptotic cell death. Furthermore, an interesting recurring protein-protein interaction within this framework of apoptotic cascades involves interactions between death domain motifs that are present on many of the regulatory proteins in both apoptotic pathways. Cardiomyocyte apoptosis has been demonstrated in human heart failure and in rodents, apoptosis itself directly causes dilated cardiomyopathy. Understanding the intricacies of apoptotic death pathways and determining the relevance of these to cardiomyopathy is therefore essential if cardiomyocyte apoptosis is to be a pharmacological target for heart failure therapy.

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