RNA Expression Profiling of Human iPSC-Derived Cardiomyocytes in a Cardiac Hypertrophy Model

<div><p>Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of messenger RNA (mRNA) and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer the opportunity for disease modeling. Here we utilize a previously established <i>in</i><i>vitro</i> model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1) stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the <i>in</i><i>vitro</i> data with human myocardial biopsies detected overlapping expression changes between the <i>in</i><i>vitro</i> samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.</p></div

Cardiac hypertrophy marker expression.

<p>Bar plot showing the expression levels for some of the canonical hypertrophy markers in ET1-CM when compared with control-CM. The y-axis represents the mean + SD log2 fold change values taken from triplicate control-CM and ET1-CM experiments.</p

Known mature miRNA validation with RT-qPCR.

<p>Comparison of RT-qpCR and miRNA-Seq derived log₂ fold change for a subset of known human mature miRNAs between control-CMs and ET1-CMs. The mean values taken from triplicate experiments are plotted with standard deviation error bars.</p

PCA plot comparing <i>in</i><i>vitro</i> hiPSC model with <i>in</i><i>vivo</i> human myocardial biopsies.

<p>Plot of first and second principal components (PC1 and PC2) for microarray expression data comparing ET-1 treated hiPSC-CMs with human myocardial biopsy with and without LVH data.</p

Dose vs. response plot for hypertrophy markers.

<p>RT-qPCR was used to measure the expression levels of (a) NPPB and (b) ACTA1 and NPPA as different ET-1 dose.</p

Characterization of 137 Genomic DNA Reference Materials for 28 Pharmacogenetic Genes: A GeT-RM Collaborative Project

Pharmacogenetic testing is increasingly available from clinical laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, NAT2, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and laboratory developed tests. Through consensus verification, we confirmed the presence of at least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing

Signal transducer of inflammation gp130 modulates atherosclerosis in mice and man

Liver-derived acute phase proteins (APPs) emerged as powerful predictors of cardiovascular disease and cardiovascular events, but their functional role in atherosclerosis remains enigmatic. We report that the gp130 receptor, which is a key component of the inflammatory signaling pathway within hepatocytes, influences the risk of atherosclerosis in a hepatocyte-specific gp130 knockout. Mice on an atherosclerosis-prone genetic background exhibit less aortic atherosclerosis (P <0.05) with decreased plaque macrophages (P <0.01). Translating these findings into humans, we show that genetic variation within the human gp130 homologue, interleukin 6 signal transducer (IL6ST), is significantly associated with coronary artery disease (CAD; P <0.05). We further show a significant association of atherosclerotic disease at the ostium of the coronary arteries (P <0.005) as a clinically important and heritable subphenotype in a large sample of families with myocardial infarction (MI) and a second independent population-based cohort. Our results reveal a central role of a hepatocyte-specific, gp130-dependent acute phase reaction for plaque development in a murine model of atherosclerosis, and further implicate UST as a genetic susceptibility factor for CAD and MI in humans. Thus, the acute phase reaction should be considered an important target for future drug development in the management of CAD