Cardiac Med1 deletion promotes early lethality, cardiac remodeling, and transcriptional reprogramming

The mediator complex, a multisubunit nuclear complex, plays an integral role in regulating gene expression by acting as a bridge between transcription factors and RNA polymerase II. Genetic deletion of mediator subunit 1 (Med1) results in embryonic lethality, due in large part to impaired cardiac development. We first established that Med1 is dynamically expressed in cardiac development and disease, with marked upregulation of Med1 in both human and murine failing hearts. To determine if Med1 deficiency protects against cardiac stress, we generated two cardiac-specific Med1 knockout mouse models in which Med1 is conditionally deleted (Med1cKO mice) or inducibly deleted in adult mice (Med1cKO-MCM mice). In both models, cardiac deletion of Med1 resulted in early lethality accompanied by pronounced changes in cardiac function, including left ventricular dilation, decreased ejection fraction, and pathological structural remodeling. We next defined how Med1 deficiency alters the cardiac transcriptional profile using RNA-sequencing analysis. Med1cKO mice demonstrated significant dysregulation of genes related to cardiac metabolism, in particular genes that are coordinated by the transcription factors Pgc1α, Pparα, and Errα. Consistent with the roles of these transcription factors in regulation of mitochondrial genes, we observed significant alterations in mitochondrial size, mitochondrial gene expression, complex activity, and electron transport chain expression under Med1 deficiency. Taken together, these data identify Med1 as an important regulator of vital cardiac gene expression and maintenance of normal heart function. NEW & NOTEWORTHY Disruption of transcriptional gene expression is a hallmark of dilated cardiomyopathy; however, its etiology is not well understood. Cardiac-specific deletion of the transcriptional coactivator mediator subunit 1 (Med1) results in dilated cardiomyopathy, decreased cardiac function, and lethality. Med1 deletion disrupted cardiac mitochondrial and metabolic gene expression patterns.

Download Full-text

P319Proliferative induced cardiomyocyte precursor cells obtained by direct reprogramming and transcriptional selection

European Heart Journal ◽

10.1093/eurheartj/ehz747.0154 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

D Bachamanda Somesh ◽

K Klose ◽

K Juerchott ◽

U Krueger ◽

D Kunkel ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Cardiac Function ◽

Heart Function ◽

Cardiac Fibroblasts ◽

Gene Expression Pattern ◽

Left Ventricular ◽

Precursor Cells ◽

Left Ventricular Ejection ◽

Ventricular Ejection Fraction

Abstract Objective Enriched populations of proliferative induced cardiomyocyte precursors (iCMPs) generated from cardiac fibroblasts (CF) could represent a potential population for use in therapeutic application. We developed a protocol for generation of iCMPs from cardiac fibroblasts by genetic reprogramming followed by transcriptional selection and induced maturation. Methods Cardiac fibroblasts were reprogrammed into induced cardiomyocyte precursor cells (iCMPs) via transduction with cardiomyogenesis-related transcription factors Gata4, Mef2c, TBX5 and Myocd. Pure population of iCMPs were obtained by molecular beacons (MB) based selection of MYH6/7 expression. The transcriptome was profiled by RNA sequencing. For maturation iCMPs were treated with 5'-azacytidine and cultured in medium containing ascorbic acid and TGFβ1. The cells were monitored regularly for formation of sarcomere structures, cardiac marker expression and contractions. iCMPs were transplanted into MI mice and monitored for 6 weeks. At the end of 6 weeks, the mice were sacrificed and hearts were explanted and cryopreserved to examine the heart structure and scare size. Cardiac function was monitored by echocardiography. Results iCMPs expressed troponin T, α-actinin and myosin heavy chain (MHC) protein observed by immunocytology. These iCMPs could robustly proliferative and maintain a stable phenotype. Global transcriptome analysis revealed that the iCMP gene expression profile is unique from those of the parental CFs and adult CMs. With Gene Ontology (GO) analysis, we found that iCMPs show upregulation of genes associated with cardiac development, differentiation and morphogenesis while they showed downregulation of genes associated to cell-proliferation in comparison to their parental CFs. Evaluation of selected gene sets showed downregulation of non-myocyte genes, upregulation of transcription factors and upregulation of certain functional and structural genes like ion channel genes and contractile genes. Maturation studies showed that iCMPs gradually changed morphology after stimulation with 5-Aza, as seen in immunofluorescence staining. Cells expressing cTnT showed formation of sarcomeric striations. In vivo translational studies showed reduction of heart function in control groups treated with PBS or CF whereas the iCMPs group showed an improvement in function, reflected by left ventricular ejection fraction and fractional shortening as evaluated by echocardiography. Additionally we noticed an improvement in left ventricular wall thickness at diastole in mice transplanted with iCMPs. Conclusions Gene expression pattern suggests that these iCMPs represent an intermediate state of cardiogenic population that can be expanded to yield therapeutic cell doses. When transplanted to mouse hearts following myocardial infarction, they improved cardiac function.

Download Full-text

Abstract 519: Talin is Required for Normal Adult Heart Function

Circulation Research ◽

10.1161/res.127.suppl_1.519 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Yoshitake Cho ◽

Ruixia Li ◽

Ana M Manso ◽

Robert S Ross

Keyword(s):

Heart Failure ◽

Cardiac Function ◽

Cardiac Dysfunction ◽

Cardiac Development ◽

Heart Function ◽

Left Ventricular ◽

Mass Spectrometry Analysis ◽

Adult Heart ◽

Spectrometry Analysis ◽

Functional Changes

Talin (Tln) is a component of muscle costameres that links integrins to other components of the cellular cytoskeleton and plays an important role in maintaining the cellular integrity of cardiac myocytes (CM). There are two talin genes, Tln1 and Tln2, expressed in the heart. Tln1 is ubiquitously expressed, and Tln2 is dominantly expressed in CM. In our previous study, we show that the global deletion of Tln2 in mice (T2KO) caused no structural or functional changes in the heart, presumably because CM Tln1 became up-regulated. However, we found that mice lacking both CM Tln1 and Tln2 exhibit cardiac dysfunction by 4 weeks (w) of age with 100% mortality by 6 months (m), showing Tln plays an essential role in cardiac development and in maintaining cardiac function. In this study, we produced a tamoxifen (Tamo)-inducible mouse model in which Tln1 could be explicitly reduced in the adult CM (T1icKO), and then generate T1icKO:T2KO (T1/2dKO), so that the function of Tln could be assessed in the postnatal heart. T2KO and Tln1/2dKO mice were injected with Tamo at 8w. Echocardiograms were performed to evaluate cardiac function up to 8w post-Tamo injection. While T2KO mice showed normal cardiac function, T1/2dKO exhibited a gradual decrease in function post-Tamo injection. At 8w post-Tamo injection, T1/2dKO mice showed cardiac hypertrophy, fibrosis, and heart failure. To understand the mechanism by which deletion CM talin leads to cardiac dysfunction, left ventricular tissue protein lysates from T2KO and T1/2dKO mice at 4w post-Tamo when cardiac function (echo) and structure were preserved in dKO. The protein lysates were subjected to quantitative mass spectrometry analysis. We found there are 1,100 proteins differentially expressed in T2KO and T1/2dKO hearts. Pathway analysis was performed, and the results showed that proteins involved in vesicle transport, protein folding, and innate immunity are most up-regulated in the T1/2dKO heart. Taken together, our results show that Tln is required for maintaining proper cardiac function in the adult heart. The deletion of Tln in CM results in the up-regulation of multiple intracellular pathways, and we are currently studying the role of each pathway in the pathogenesis of heart failure induced by CM Tln deletion.

Download Full-text

Long-term levosimendan treatment improves systolic function and myocardial relaxation in mice with cardiomyocyte-specific disruption of the Serca2 gene

Journal of Applied Physiology ◽

10.1152/japplphysiol.01044.2012 ◽

2013 ◽

Vol 115 (10) ◽

pp. 1572-1580 ◽

Cited By ~ 7

Author(s):

Vigdis Hillestad ◽

Frank Kramer ◽

Stefan Golz ◽

Andreas Knorr ◽

Kristin B. Andersson ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Cardiac Function ◽

Diastolic Dysfunction ◽

Systolic Function ◽

Cytosolic Calcium ◽

Left Ventricular ◽

Pressure Measurements ◽

Human Heart Failure

In human heart failure (HF), reduced cardiac function has, at least partly, been ascribed to altered calcium homeostasis in cardiomyocytes. The effects of the calcium sensitizer levosimendan on diastolic dysfunction caused by reduced removal of calcium from cytosol in early diastole are not well known. In this study, we investigated the effect of long-term levosimendan treatment in a murine model of HF where the sarco(endo)plasmatic reticulum ATPase ( Serca) gene is specifically disrupted in the cardiomyocytes, leading to reduced removal of cytosolic calcium. After induction of Serca2 gene disruption, these mice develop marked diastolic dysfunction as well as impaired contractility. SERCA2 knockout (SERCA2KO) mice were treated with levosimendan or vehicle from the time of KO induction. At the 7-wk end point, cardiac function was assessed by echocardiography and pressure measurements. Vehicle-treated SERCA2KO mice showed significantly diminished left-ventricular (LV) contractility, as shown by decreased ejection fraction, stroke volume, and cardiac output. LV pressure measurements revealed a marked increase in the time constant (τ) of isovolumetric pressure decay, showing impaired relaxation. Levosimendan treatment significantly improved all three systolic parameters. Moreover, a significant reduction in τ toward normalization indicated improved relaxation. Gene-expression analysis, however, revealed an increase in genes related to production of the ECM in animals treated with levosimendan. In conclusion, long-term levosimendan treatment improves both contractility and relaxation in a heart-failure model with marked diastolic dysfunction due to reduced calcium transients. However, altered gene expression related to fibrosis was observed.

Download Full-text

Abstract W P73: Feasibility and Preliminary Results of Whole Blood RNA-Sequencing Analysis in Patients With Intracranial Aneurysms

Stroke ◽

10.1161/str.45.suppl_1.wp73 ◽

2014 ◽

Vol 45 (suppl_1) ◽

Author(s):

Blake Haas ◽

Nestor R Gonzalez ◽

Elina Nikkola ◽

Mark Connolly ◽

William Hsu ◽

...

Keyword(s):

Gene Expression ◽

Network Analysis ◽

Rna Sequencing ◽

Whole Blood ◽

Intracranial Aneurysms ◽

Small Sample Size ◽

Expression Patterns ◽

Small Sample ◽

Cellular Respiration ◽

Sequencing Analysis

Introduction: Intracranial aneurysms (IA) growth and rupture have been associated with chronic remodeling of the arterial wall. However, the pathobiology of this process remains poorly understood. The objective of the present study was to evaluate the feasibility of analyzing gene expression patterns in peripheral blood of patients with ruptured and unruptured saccular IAs. Materials and Methods: We analyzed human whole blood transcriptomes by performing paired-end, 100 bp RNA-sequencing (RNAseq) using the Illumina platform. We used STAR to align reads to the genome, HTSeq to count reads, and DESeq to normalize counts across samples. Self-reported patient information was used to correct expression values for ancestry, age, and sex. We utilized weighted gene co-expression network analysis (WGCNA) to identify gene expression network modules associated with IA size and rupture. The DAVID tool was employed to search for Gene Ontology enrichment in relevant modules. Results: Samples from 12 patients (9 females, age 57.6 +/-12) with IAs were analyzed. Four had ruptured aneurysms. RNA isolation and application of the methodology described above was successful in all samples. Although the small sample size prevents us from drawing definite conclusions, we observed promising novel co-expression networks for IAs: WCGNA analysis showed down-regulation of two transcript modules associated with ruptured IA status (r=-0.78, p=0.008 and r=-0.77, p=0.009), and up-regulation of two modules associated with aneurysm size (r=0.86, p=0.002 and r=0.9, p=4e-04), respectively. DAVID analyses showed that genes upregulated in an IA size-associated module were enriched with genes involved in cellular respiration and translation, while genes involved in transcription were down-regulated in a module associated with ruptured IAs. Conclusions: Whole blood RNAseq analysis is a feasible tool to capture transcriptome dynamics and achieve a better understanding of the pathophysiology of IAs. Further longitudinal studies of patients with IAs using network analysis are justified.

Download Full-text

Abstract 314: BRaf Plays a Major Role in Gene Expression in Cardiomyocytes in Mouse Hearts in vivo

Circulation Research ◽

10.1161/res.121.suppl_1.314 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Michelle A Hardyman ◽

Stephen J Fuller ◽

Daniel N Meijles ◽

Kerry A Rostron ◽

Sam J Leonard ◽

...

Keyword(s):

Gene Expression ◽

Cardiac Hypertrophy ◽

Cardiac Function ◽

Left Ventricular ◽

Braf Mutations ◽

Gene Markers ◽

Lv Mass ◽

Cardiac Volumes

Introduction: Raf kinases lie upstream of ERK1/2 with BRaf being the most highly expressed and having the highest basal activity. V600E BRaf mutations constitutively activate ERK1/2 and are common in cancer. The role of BRaf in the adult heart is yet to be established. ERK1/2 regulate cardiomyocyte gene expression, promoting cardiac hypertrophy and cardioprotection, but effects of ERK1/2 may depend on signal strength. Hypothesis: Our hypotheses are that BRaf is critical in regulating ERK1/2 signaling in cardiomyocytes and, whilst moderate ERK1/2 activity is beneficial, excessive ERK1/2 activity is detrimental to the heart. Methods: We generated heterozygote mice for tamoxifen- (Tam-) inducible cardiomyocyte-specific knockin of V600E in the endogenous BRaf gene. Mice (12 wks) received 2 injections of Tam or vehicle on consecutive days (n=4-10 per group). Kinase activities and mRNA expression were assessed by immunoblotting and qPCR. Echocardiography was performed (Vevo2100). M-mode images (short axis view) were analyzed; data for each mouse were normalized to the mean of 2 baseline controls. Results: V600E knockin did not affect overall BRaf or cRaf levels in mouse hearts, but significantly increased ERK1/2 activities within 48 h (1.51±0.05 fold). Concurrently, mRNAs for hypertrophic gene markers including BNP and immediate early genes (IEGs) increased signficantly. At 72 h, expression of BNP, Fosl1, Myc, Ereg and CTGF increased further, other IEGs (Jun, Fos, Egr1, Atf3) declined, and ANF was upregulated. In contrast, expression of α and β myosin heavy chain mRNAs was substantially downregulated (0.46/0.41±0.05 relative to controls). Within 72 h, left ventricular (LV) mass and diastolic LV wall thickness had increased (1.23±0.05 relative to controls), but cardiac function was severely compromised with significant decreases in ejection fraction and cardiac output (0.53/0.68±0.09 relative to controls) associated with increased LV internal diameters and cardiac volumes. Conclusions: Endogenous cardiomyocyte BRaf is sufficient to activate ERK1/2 in mouse hearts and induce cardiac hypertrophy associated with dynamic temporal changes in gene expression. However, excessive activation of ERK1/2 in isolation is detrimental to cardiac function.

Download Full-text

Mapping-based all-RNA-information sequencing analysis (ARIseq) pipeline simultaneously revealed taxonomic composition, gene expression, and their correlation in an acidic stream ecosystem

10.1101/159293 ◽

2017 ◽

Author(s):

Arisa Tsuboi ◽

Misao Itoga ◽

Yuichi Hongoh ◽

Shigeharu Moriya

Keyword(s):

Gene Expression ◽

Expression Patterns ◽

Taxonomic Composition ◽

Rrna Genes ◽

Reference Database ◽

Similar Distribution ◽

Sequencing Analysis ◽

Protein Coding ◽

Microbial Ecosystems ◽

Sulfur Oxidizing

AbstractWe developed a new pipeline for simultaneous analyses of both rRNA profile as a taxonomic marker and mRNA profile as a functional marker, to understand microbial ecosystems in natural environments. Our pipeline, named All-RNA-Information sequencing (ARIseq), comprises a high-throughput sequencing of reverse transcribed total RNA and several widely used computational tools, and generates quantitatively reliable information on both community structures and gene expression patterns, which were verified by quantitative PCR analyses in this study. Particularly, correlation network analysis in the pipeline can reveal microbial taxa and expressed genes that share patterns of dynamics among different time and/or geographical points. The pipeline is primarily mapping-based, using a public database for small subunit rRNA genes and obtained contigs as the reference database for protein-coding genes. We applied this pipeline to biofilm samples, as examples, collected from an acidic spring water stream in the Chyatsubomi-goke Park in Gunma prefecture, Japan. Our analyses revealed the predominance of iron and sulfur-oxidizing bacteria and Pinnularia diatoms, and also indicated that the distributions of the iron-sulfur-oxidizing bacterial consortium and the Pinnularia diatoms largely overlapped but showed distinct patterns. In addition, our analyses showed that the iron-oxidizing bacterial genus Acidithiobacillus and co-occurring Acidiphilium shared similar distribution pattern whereas another iron-oxidizing genus Leptospirillum exhibited a distinct pattern. Our pipeline enables researchers to more easily capture the outline of microbial ecosystems based on the taxonomic composition, protein-coding gene expression, and their correlations.

Download Full-text

Abstract 17402: Single Nuclei RNA-sequencing of Human Hypertrophic Cardiomyopathy Myectomy Samples Reveals Common Novel Mechanisms of Pathogenesis and Potential Therapeutic Targets Regardless of Genotype

Circulation ◽

10.1161/circ.142.suppl_3.17402 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Amy Larson ◽

Hassan Rastegar ◽

Gordon S Huggins ◽

Ethan J Rowin ◽

Martin S Maron ◽

...

Keyword(s):

Gene Expression ◽

Hypertrophic Cardiomyopathy ◽

Rna Sequencing ◽

Alternative Treatment ◽

Expression Patterns ◽

Therapeutic Targets ◽

Treatment Strategies ◽

Ventricular Outflow Tract ◽

Left Ventricular ◽

Surgical Myectomy

Introduction: Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease, often resulting in left ventricular outflow tract obstruction, relieved by surgical myectomy. Current treatments are largely palliative and do not target the root causes. Understanding the molecular drivers of the disease could lead to alternative treatment strategies through identification of novel therapeutic targets. Methods: We performed single nuclei RNA-sequencing (snRNA-seq) on thousands of nuclei from 9 patient myectomy samples and septal tissue from 4 unused donor hearts selected randomly without regard to genotype to identify the cell populations and determine the gene expression patterns in individual cells. Each sample was processed individually using Seurat v3 for quality control and normalization. Next, all 13 samples were integrated into a combined dataset for clustering and differential gene expression analysis to identify markers specific to each cluster and to assign cell identities. Results: Our results revealed several clusters of cardiomyocytes with differences in sarcomeric and metabolic gene expression. Several fibroblast populations were also observed. Numerous genes were differentially expressed between the HCM and normal samples. For example, RARRES1 expression was observed in many of the fibroblast populations in the normal samples, but was absent in the HCM samples. RARRES1 is involved in regulating fatty acid metabolism and autophagy, both of which are altered in HCM. Additionally, expression of PLA2G2A was absent in the HCM samples but was present in almost every cell type in the normal controls. PLA2G2A is involved in suppression of RTK mediated hypertrophic signaling, impacts lipid signaling, and has tumor suppressor properties. Thus, both RARRES1 and PLA2G2A may represent novel targets in HCM. Conclusions: This approach reveals novel potential therapeutic targets within common final HCM pathological pathways independent of genotype that have the potential to guide development of alternative treatment strategies. Further analysis of larger datasets using this approach will likely identify even more common pathway targets and identify additional common mechanisms in the pathogenesis of obstructive HCM.

Download Full-text

Cardiac function in spontaneously hypertensive diabetic rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1986.251.3.h571 ◽

1986 ◽

Vol 251 (3) ◽

pp. H571-H580 ◽

Cited By ~ 8

Author(s):

B. Rodrigues ◽

J. H. McNeill

Keyword(s):

Cardiac Function ◽

Heart Function ◽

Myocardial Dysfunction ◽

Diabetic Rats ◽

Left Ventricular ◽

Spontaneously Hypertensive ◽

Wistar Kyoto ◽

Developed Pressure ◽

Working Heart ◽

Diabetes Induction

The isolated perfused working heart was used to study hypertensive diabetes-induced alterations in cardiac function at 6 and 12 wk after diabetes was induced. At 6 wk after diabetes induction, cardiac performance was depressed in the diabetic animals. However, there was no difference in cardiac function between normotensive Wistar and spontaneously hypertensive (SHR) diabetic rats. Wistar-Kyoto (WKY) rats were also included as normotensive controls in our 12-wk study. Hearts from 12-wk SHR and Wistar diabetic animals exhibited a depressed left ventricular developed pressure and positive and negative dP/dt when compared with control animals. However, this depression was not seen in the WKY diabetic animals. In addition, quantitation of various parameters of heart function revealed highly significant differences between SHR diabetic animals and all other groups associated with an increased mortality. Serum lipids were elevated in SHR and Wistar and were unaffected in WKY diabetic rats. Furthermore, thyroid hormone levels were not depressed in WKY diabetic rats as seen in the other two diabetic groups. This normal lipid metabolism and thyroid status could, in part, explain the lack of cardiac dysfunction in these animals. The data provide further evidence that the combination of hypertension and diabetes mellitus produces greater myocardial dysfunction than with either disease alone and is associated with a significant mortality.

Download Full-text