ABSTRACT
Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.
Subject(s)
Cellular Reprogramming/genetics , Gene Expression Regulation/genetics , Transcription Factors/metabolism , Anti-Inflammatory Agents/pharmacology , Cellular Reprogramming/drug effects , Fibroblasts/physiology , GATA4 Transcription Factor/metabolism , Gene Expression Regulation/drug effects , Genome-Wide Association Study , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , Repressor Proteins , TranscriptomeABSTRACT
Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique.
Subject(s)
Cellular Reprogramming , Fibroblasts/metabolism , Myocytes, Cardiac/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Transcription, Genetic , Animals , Calcium/metabolism , Calcium Signaling , Cell Differentiation , Embryo, Mammalian/cytology , Fibroblasts/cytology , Mice , Myocardial Contraction , Myocytes, Cardiac/cytology , Sequence Analysis, RNA , Time Factors , Transcription Factors/metabolismABSTRACT
Among naturally occurring polymorphisms in the 5' flanking region of the human angiotensinogen (AGT) gene, the -20 and -217 polymorphisms have the strongest effects on AGT regulation in AGT-expressing cells derived from liver, kidney, brain, and fat. These polymorphisms may affect allele-specific transcription factor binding, and the high-expressing alleles are both relatively common. We show herein that the -20C allele has higher transcriptional activity than -20A, and the -20A allele confers no additional transactivation potential beyond that of a mutated vector. Gel-shift assays show that upstream stimulatory factor (USF)1 and USF2 preferentially bind the -20C allele, whereas the -20A allele retains a low affinity USF binding site. Plasmid immunoprecipitation assays confirmed preferential association of USF1 with the -20C allele in transfected HepG2 cells. Chromatin immunoprecipitation confirmed that USF1 binds to the endogenous AGT -20C allele in CCF cells, the only cell line tested that carries the -20C allele, and to the human AGT promoter in liver and adipose tissue from transgenic mice. Transduction of AGT-expressing cells with short hairpin RNAs specifically targeting USF1 or USF2, resulted in cell- and allele-specific attenuation of AGT promoter activity. In vivo, knockdown of USF expression in the liver of transgenic mice expressing the -20C allele of AGT resulted in lower AGT expression, a decrease in circulating human AGT protein but no change in expression of GAPDH or hepatocyte nuclear factor-4alpha. We conclude that USF1 functionally and differentially regulates AGT expression via the -20 polymorphism and that the differential expression exhibited by -20 can be accounted for by differential association with USF1.
Subject(s)
5' Flanking Region , Angiotensinogen/metabolism , Polymorphism, Genetic , Promoter Regions, Genetic , Transcription, Genetic , Transcriptional Activation , Upstream Stimulatory Factors/metabolism , 3T3-L1 Cells , Adipose Tissue/metabolism , Angiotensinogen/genetics , Animals , Cell Line, Tumor , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Humans , Liver/metabolism , Mice , Mice, Transgenic , RNA Interference , RNA, Small Interfering/metabolism , Transfection , Upstream Stimulatory Factors/geneticsABSTRACT
Right-sided heart failure is the most common cause of death in pulmonary hypertension (PH). Echocardiographic measurements of right atrial (RA) size are associated with worse outcome in PH, however the association between RA function and death in PH has not been well-described. 160 PH patients (World Health Organization groups 1-5) underwent cardiac magnetic resonance imaging (cMRI) and right heart catheterization (RHC) within 6 weeks of each other at a tertiary care academic medical center in the United States. We measured cMRI RA maximum and minimum volumes indexed to body surface area and calculated RA emptying fraction (RAEF). We evaluated the relationship between RAEF and clinical variables with death using Cox proportional hazard models. 57 deaths occurred during a median follow-up of 3.5 years (36 % died overall, 10 % per year). RAEF was directly correlated in univariate analyses with right ventricular (RV) ejection fraction, left ventricular (LV) ejection fraction, LV size, cardiac index, absence of tricuspid and pulmonic regurgitation, absence of pericardial effusion, estimated glomerular filtration rate, 6-minute walk distance, and pulmonary arterial oxygen saturation, whereas it was inversely correlated with death, BNP, heart rate, mean RA pressure, mean PA pressure, pulmonary and systemic vascular resistance, RV size, and RA size. Using multivariate analyses, RAEF had a robust inverse association with death after adjusting for measured risk factors (HR per 5 % change in RAEF: 0.83 [95 % CI 0.73-0.94], p = 0.003). In PH patients, decreased RAEF by cMRI is independently associated with worse survival after adjustment for other risk factors.
Subject(s)
Atrial Function, Right , Heart Failure/mortality , Hypertension, Pulmonary/mortality , Adult , Aged , Cardiac Catheterization , Cause of Death , Female , Heart Failure/diagnosis , Heart Failure/physiopathology , Humans , Hypertension, Pulmonary/diagnosis , Hypertension, Pulmonary/physiopathology , Kaplan-Meier Estimate , Magnetic Resonance Imaging, Cine , Male , Middle Aged , Multivariate Analysis , Predictive Value of Tests , Prognosis , Proportional Hazards Models , Retrospective Studies , Risk Factors , Stroke Volume , Tertiary Care Centers , Texas , Time Factors , Ventricular Function, Left , Ventricular Function, RightABSTRACT
Genetic and functional data support a role for angiotensinogen in blood pressure control, and many population studies have suggested that polymorphisms in the angiotensinogen gene contribute to hypertension. Two common haplotypes of the human angiotensinogen gene are -6A/235T and -6G/235M. To study their contributions to blood pressure regulation in a controlled model system, we developed triple-transgenic mice expressing either -6A/235T or -6G/235M human angiotensinogen, expressing either an overexpressed and poorly regulated (REN9) or a tightly regulated (PAC160) human renin, and all carrying a null mutation in the endogenous murine angiotensinogen gene. These humanized mice were then examined for blood pressure differences at baseline and after a high-salt diet, changes in cardiovascular organ weight, and differences in angiotensinogen and renin gene expression. Mice expressing the -6G/235M haplotype on the PAC160 background exhibited increased blood pressure and cardiac hypertrophy at baseline. In contrast, all of the mice with the REN9 background had equivalent baseline blood pressures. On the REN9 background, there was a greater increase in blood pressure in -6A/235T in response to a high-salt diet, providing evidence it may be a susceptibility allele. There were no differences in angiotensinogen expression between haplotypes on either background strain. The data suggest that the impact of angiotensinogen haplotypes on cardiovascular end points may be dependent on renin status and environmental influences, such as dietary sodium. These insights may help explain the discrepancies among observational studies that have examined roles for the -6A/235T and -6G/235M angiotensinogen haplotypes in varied human populations.
Subject(s)
Angiotensinogen/genetics , Blood Pressure/genetics , Alleles , Angiotensinogen/metabolism , Animals , Genetic Variation , Genotype , Haplotypes , Hypertension/genetics , Hypertension/metabolism , Mice , Mice, Transgenic , Renin/genetics , Renin/metabolism , Sodium, Dietary , TelemetryABSTRACT
A number of naturally occurring polymorphisms exist in the human angiotensinogen locus, some of which have been associated with essential hypertension, preeclampsia, and other medical disorders. However, to date there has been no comprehensive determination of the significance of specific haplotypes in relation to the regulation of angiotensinogen expression. We cloned the promoters extending from -1219 to +125 bp from 11 ethnically diverse individuals to acquire a representative cross-section of known haplotype diversity. Eight nonredundant haplotypes were identified, fused to luciferase, and studied for their effect on transcriptional regulation in human astrocyte, proximal tubule, and hepatocyte cell lines endogenously expressing angiotensinogen and in a mouse adipocyte cell line. The studies were carried out under baseline conditions, in the presence of the angiotensinogen enhancer, and in response to hormonal stimulation by dexamethasone, beta-estradiol, or testosterone. A statistical model was then constructed to assess the significance of individual polymorphisms. The polymorphisms with the greatest effect on transcription in these cell lines were located at -20 and -217. There were modest haplotype-specific effects of the angiotensinogen enhancer and no haplotype-specific effects of beta-estradiol, dexamethasone, or testosterone treatment. We conclude the following: (1) the -20 and -217 polymorphisms have the largest influence on angiotensinogen transcription, (2) other polymorphisms have a much smaller impact on angiotensinogen transcription, and (3) the transcriptional influence of the promoter polymorphisms may act cell specifically. Therefore, our data support a hypothesis that polymorphisms in the angiotensinogen promoter may act cell specifically to differentially regulate the level of angiotensinogen transcription in angiotensin-producing tissues.