Assigning Distal Genomic Enhancers to Cardiac Disease-Causing Genes
2020; 142 (9): 910?12
Epigenomes of Human Hearts Reveal New Genetic Variants Relevant for Cardiac Disease and Phenotype
2020; 127 (6): 761?77
A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes
Mitochondrial substrate utilization regulates cardiomyocyte cell-cycle progression
2020; 2 (2): 167-+
Identifying genetic markers for heterogeneous complex diseases such as heart failure is challenging and requires prohibitively large cohort sizes in genome-wide association studies to meet the stringent threshold of genome-wide statistical significance. On the other hand, chromatin quantitative trait loci, elucidated by direct epigenetic profiling of specific human tissues, may contribute toward prioritizing subthreshold variants for disease association.Here, we captured noncoding genetic variants by performing epigenetic profiling for enhancer H3K27ac chromatin immunoprecipitation followed by sequencing in 70 human control and end-stage failing hearts.We have mapped a comprehensive catalog of 47?321 putative human heart enhancers and promoters. Three thousand eight hundred ninety-seven differential acetylation peaks (FDR [false discovery rate], 5%) pointed to pathways altered in heart failure. To identify cardiac histone acetylation quantitative trait loci (haQTLs), we regressed out confounding factors including heart failure disease status and used the G-SCI (Genotype-independent Signal Correlation and Imbalance) test1 to call out 1680 haQTLs (FDR, 10%). RNA sequencing performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression quantitative trait loci), either in cis (180) or through long-range interactions (81), identified by Hi-C (high-throughput chromatin conformation assay) and HiChIP (high-throughput protein centric chromatin) performed on a subset of hearts. Furthermore, a concordant relationship between the gain or disruption of TF (transcription factor)-binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalization of haQTLs with the subthreshold loci of heart-related genome-wide association studies datasets.Disease and phenotype association for 62 unique loci are now implicated. These loci may indeed mediate their effect through modification of enhancer H3K27 acetylation enrichment and their corresponding gene expression differences (bioRxiv: https://doi.org/10.1101/536763). Graphical Abstract: A graphical abstract is available for this article.
View details for DOI 10.1161/CIRCRESAHA.120.317254
View details for Web of Science ID 000567460400010
View details for PubMedID 32529949
Adipose circular RNAs exhibit dynamic regulation in obesity and functional role in adipogenesis
2019; 1 (7): 688?703
The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.
View details for DOI 10.1038/s42255-020-0169-x
View details for Web of Science ID 000522484100006
View details for PubMedID 32617517
View details for PubMedCentralID PMC7331943
Robust CTCF-Based Chromatin Architecture Underpins Epigenetic Changes in the Heart Failure Stress-Gene Response
2019; 139 (16): 1937?56
Non-coding RNAs are emerging as novel regulators in adipocyte differentiation and function. Circular RNAs (circRNAs) are a new class of non-coding transcripts generated across all eukaryotic tissues, but their function in adipose biology remains unknown. Here we perform deep sequencing of visceral and subcutaneous fat to discover thousands of adipose circRNAs, many of which are species conserved, tissue specific and dynamically regulated during adipogenesis and obesity. We identify circTshz2-1 and circArhgap5-2 as indispensable regulators of adipogenesis in vitro. To characterize the function of circRNAs in vivo, we deliver adenoviral shRNA targeting circArhgap5-2 into mouse inguinal tissue and show that the expression of this circRNA is essential in maintaining the global adipocyte transcriptional programme involved in lipid biosynthesis and metabolism. We also demonstrate that the pro-adipogenic function of circArhgap5-2 is conserved in human adipocytes. Our results provide important evidence that circRNAs serve as important regulators in adipocyte differentiation and metabolism.
View details for DOI 10.1038/s42255-019-0078-z
View details for Web of Science ID 000500745100008
View details for PubMedID 32694641
Single cardiomyocyte nuclear transcriptomes reveal a lincRNA-regulated de-differentiation and cell cycle stress-response in vivo
2017; 8: 225
The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis-regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress-gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout.We undertook an independent and orthogonal analysis of chromatin organization with mouse pressure-overload model of myocardial stress (transverse aortic constriction) and cardiomyocyte-specific knockout of Ctcf. We also downloaded published data sets of similar cardiac mouse models and subjected them to independent reanalysis.We found that the cardiomyocyte chromatin architecture remains broadly stable in transverse aortic constriction hearts, whereas Ctcf knockout resulted in ?99% abolition of global chromatin loops. Disease gene expression changes correlated instead with differential histone H3K27-acetylation enrichment at their respective proximal and distal interacting genomic enhancers confined within these static chromatin structures. Moreover, coregulated genes were mapped out as interconnected gene sets on the basis of their multigene 3D interactions.This work reveals a more stable genome-wide chromatin framework than previously described. Myocardial stress-gene transcription responds instead through H3K27-acetylation enhancer enrichment dynamics and gene networks of coregulation. Robust and intact CTCF looping is required for the induction of a rapid and accurate stress response.
View details for DOI 10.1161/CIRCULATIONAHA.118.036726
View details for Web of Science ID 000469321500009
View details for PubMedID 30717603
A landscape of circular RNA expression in the human heart
2017; 113 (3): 298?309
Cardiac regeneration may revolutionize treatment for heart failure but endogenous progenitor-derived cardiomyocytes in the adult mammalian heart are few and pre-existing adult cardiomyocytes divide only at very low rates. Although candidate genes that control cardiomyocyte cell cycle re-entry have been implicated, expression heterogeneity in the cardiomyocyte stress-response has never been explored. Here, we show by single nuclear RNA-sequencing of cardiomyocytes from both mouse and human failing, and non-failing adult hearts that sub-populations of cardiomyocytes upregulate cell cycle activators and inhibitors consequent to the stress-response in vivo. We characterize these subgroups by weighted gene co-expression network analysis and discover long intergenic non-coding RNAs (lincRNA) as key nodal regulators. KD of nodal lincRNAs affects expression levels of genes related to dedifferentiation and cell cycle, within the same gene regulatory network. Our study reveals that sub-populations of adult cardiomyocytes may have a unique endogenous potential for cardiac regeneration in vivo.Adult mammalian cardiomyocytes are predominantly binucleated and unable to divide. Using single nuclear RNA-sequencing of cardiomyocytes from mouse and human failing and non-failing adult hearts, See et al. show that some cardiomyocytes respond to stress by dedifferentiation and cell cycle re-entry regulated by lncRNAs.
View details for DOI 10.1038/s41467-017-00319-8
View details for Web of Science ID 000407198800014
View details for PubMedID 28790305
View details for PubMedCentralID PMC5548780
An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility (vol 142, pg 1562, 2020)
2021; 143 (15): E830
Conversion of the death inhibitor ARC to a killer activates pancreatic ? cell death in diabetes.
2021; 56 (6): 747?60.e6
Circular RNA (circRNA) is a newly validated class of single-stranded RNA, ubiquitously expressed in mammalian tissues and possessing key functions including acting as microRNA sponges and as transcriptional regulators by binding to RNA-binding proteins. While independent studies confirm the expression of circRNA in various tissue types, genome-wide circRNA expression in the heart has yet to be described in detail.We performed deep RNA-sequencing on ribosomal-depleted RNA isolated from 12 human hearts, 25 mouse hearts and across a 28-day differentiation time-course of human embryonic stem cell-derived cardiomyocytes. Using purpose-designed bioinformatics tools, we uncovered a total of 15?318 and 3017 cardiac circRNA within human and mouse, respectively. Their abundance generally correlates with the abundance of their cognate linear RNA, but selected circRNAs exist at disproportionately higher abundance. Top highly expressed circRNA corresponded to key cardiac genes including Titin (TTN), RYR2, and DMD. The most abundant cardiac-expressed circRNA is a cytoplasmic localized single-exon circSLC8A1-1. The longest human transcript TTN alone generates up to 415 different exonic circRNA isoforms, the majority (83%) of which originates from the I-band domain. Finally, we confirmed the expression of selected cardiac circRNA by RT-PCR, Sanger sequencing and single molecule RNA-fluorescence in situ hybridization.Our data provide a detailed circRNA expression landscape in hearts. There is a high-abundance of specific cardiac-expressed circRNA. These findings open up a new avenue for future investigation into this emerging class of RNA.
View details for DOI 10.1093/cvr/cvw250
View details for Web of Science ID 000397950900008
View details for PubMedID 28082450
Mechanism of Eccentric Cardiomyocyte Hypertrophy Secondary to Severe Mitral Regurgitation
2020; 141 (22): 1787?99
Loss of insulin-secreting pancreatic ? cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains ? cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce ? cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. ? cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by ? cells. Loss of the serpin ?1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of ? cell anchorage and subsequent cell death. Administration of ?1-antitrypsin to mice with diabetes prevents ? cell death and metabolic abnormalities. These data uncover a pathway for ? cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.
View details for DOI 10.1016/j.devcel.2021.02.011
View details for PubMedID 33667344
Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis
CELL DEATH & DISEASE
2020; 11 (5): 354
Primary valvular heart disease is a prevalent cause of morbidity and mortality in both industrialized and developing countries. Although the primary consequence of valvular heart disease is myocardial dysfunction, treatment of valvular heart diseases centers around valve repair or replacement rather than prevention or reversal of myocardial dysfunction. This is particularly evident in primary mitral regurgitation (MR), which invariably results in eccentric hypertrophy and left ventricular (LV) failure in the absence of timely valve repair or replacement. The mechanism of LV dysfunction in primary severe MR is entirely unknown.Here, we developed the first mouse model of severe MR. Valvular damage was achieved by severing the mitral valve leaflets and chords with iridectomy scissors, and MR was confirmed by echocardiography. Serial echocardiography was performed to follow up LV morphology and systolic function. Analysis of cardiac tissues was subsequently performed to evaluate valve deformation, cardiomyocyte morphology, LV fibrosis, and cell death. Finally, dysregulated pathways were assessed by RNA-sequencing analysis and immunofluorescence.In the ensuing 15 weeks after the induction of MR, gradual LV dilatation and dysfunction occurred, resulting in severe systolic dysfunction. Further analysis revealed that severe MR resulted in a marked increase in cardiac mass and increased cardiomyocyte length but not width, with electron microscopic evidence of sarcomere disarray and the development of sarcomere disruption. From a mechanistic standpoint, severe MR resulted in activation of multiple components of both the mammalian target of rapamycin and calcineurin pathways. Inhibition of mammalian target of rapamycin signaling preserved sarcomeric structure and prevented LV remodeling and systolic dysfunction. Immunohistochemical analysis uncovered a differential pattern of expression of the cell polarity regulator Crb2 (crumbs homolog 2) along the longitudinal axis of cardiomyocytes and close to the intercalated disks in the MR hearts. Electron microscopy images demonstrated a significant increase in polysome localization in close proximity to the intercalated disks and some areas along the longitudinal axis in the MR hearts.These results indicate that LV dysfunction in response to severe MR is a form of maladaptive eccentric cardiomyocyte hypertrophy and outline the link between cell polarity regulation and spatial localization protein synthesis as a pathway for directional cardiomyocyte growth.
View details for DOI 10.1161/CIRCULATIONAHA.119.043939
View details for Web of Science ID 000538162700014
View details for PubMedID 32272846
Persistent changes in liver methylation and microbiome composition following reversal of diet-induced non-alcoholic-fatty liver disease
CELLULAR AND MOLECULAR LIFE SCIENCES
2019; 76 (21): 4341?54
Human pluripotent stem cells (hPSCs)-derived cardiovascular progenitor cells (CVPCs) are a promising source for myocardial repair, while the mechanisms remain largely unknown. Extracellular vesicles (EVs) are known to mediate cell-cell communication, however, the efficacy and mechanisms of hPSC-CVPC-secreted EVs (hCVPC-EVs) in the infarct healing when given at the acute phase of myocardial infarction (MI) are unknown. Here, we report the cardioprotective effects of the EVs secreted from hESC-CVPCs under normoxic (EV-N) and hypoxic (EV-H) conditions in the infarcted heart and the long noncoding RNA (lncRNA)-related mechanisms. The hCVPC-EVs were confirmed by electron microscopy, nanoparticle tracking, and immunoblotting analysis. Injection of hCVPC-EVs into acutely infracted murine myocardium significantly improved cardiac function and reduced fibrosis at day 28 post MI, accompanied with the improved vascularization and cardiomyocyte survival at border zones. Consistently, hCVPC-EVs enhanced the tube formation and migration of human umbilical vein endothelial cells (HUVECs), improved the cell viability, and attenuated the lactate dehydrogenase release of neonatal rat cardiomyocytes (NRCMs) with oxygen glucose deprivation (OGD) injury. Moreover, the improvement of the EV-H in cardiomyocyte survival and tube formation of HUVECs was significantly better than these in the EV-N. RNA-seq analysis revealed a high abundance of the lncRNA MALAT1 in the EV-H. Its abundance was upregulated in the infarcted myocardium and cardiomyocytes treated with hCVPC-EVs. Overexpression of human MALAT1 improved the cell viability of NRCM with OGD injury, while knockdown of MALAT1 inhibited the hCVPC-EV-promoted tube formation of HUVECs. Furthermore, luciferase activity assay, RNA pull-down, and manipulation of miR-497 levels showed that MALAT1 improved NRCMs survival and HUVEC tube formation through targeting miR-497. These results reveal that hCVPC-EVs promote the infarct healing through improvement of cardiomyocyte survival and angiogenesis. The cardioprotective effects of hCVPC-EVs can be enhanced by hypoxia-conditioning of hCVPCs and are partially contributed by MALAT1 via targeting the miRNA.
View details for DOI 10.1038/s41419-020-2508-y
View details for Web of Science ID 000534345100015
View details for PubMedID 32393784
View details for PubMedCentralID PMC7214429
Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf
2019; 125 (9): 834?46
Non-alcoholic fatty liver disease (NAFLD) is a metabolic liver disease that is thought to be reversible by changing the diet. To examine the impact of dietary changes on progression and cure of NAFLD, we fed mice a high-fat diet (HFD) or high-fructose diet (HFrD) for 9 weeks, followed by an additional 9 weeks, where mice were given normal chow diet. As predicted, the diet-induced NAFLD elicited changes in glucose tolerance, serum cholesterol, and triglyceride levels in both diet groups. Moreover, the diet-induced NAFLD phenotype was reversed, as measured by the recovery of glucose intolerance and high cholesterol levels when mice were given normal chow diet. However, surprisingly, the elevated serum triglyceride levels persisted. Metagenomic analysis revealed dietary-induced changes of microbiome composition, some of which remained altered even after reversing the diet to normal chow, as illustrated by species of the Odoribacter genus. Genome-wide DNA methylation analysis revealed a "priming effect" through changes in DNA methylation in key liver genes. For example, the lipid-regulating gene Apoa4 remained hypomethylated in both groups even after introduction to normal chow diet. Our results support that dietary change, in part, reverses the NAFLD phenotype. However, some diet-induced effects remain, such as changes in microbiome composition, elevated serum triglyceride levels, and hypomethylation of key liver genes. While the results are correlative in nature, it is tempting to speculate that the dietary-induced changes in microbiome composition may in part contribute to the persistent epigenetic modifications in the liver.
View details for DOI 10.1007/s00018-019-03114-4
View details for Web of Science ID 000494964400010
View details for PubMedID 31119300
Following hearts, one cell at a time: recent applications of single-cell RNA sequencing to the understanding of heart disease
2018; 9: 4434
A Transcriptomic and Epigenomic Comparison of Fetal and Adult Human Cardiac Fibroblasts Reveals Novel Key Transcription Factors in Adult Cardiac Fibroblasts.
JACC. Basic to translational science
2016; 1 (7): 590?602
Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM.To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM.We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-? (transforming growth factor-?)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGF?/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts.Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.
View details for DOI 10.1161/CIRCRESAHA.119.314794
View details for Web of Science ID 000489760200008
View details for PubMedID 31495264
View details for PubMedCentralID PMC7336364
Incidentalome from Genomic Sequencing: A Barrier to Personalized Medicine?
2016; 5: 211?16
Cardiovascular disease remains the number one global cause of death and presents as multiple phenotypes in which the interplay between cardiomyocytes and cardiac fibroblasts (CFs) has become increasingly highlighted. Fetal and adult CFs influence neighboring cardiomyocytes in different ways. Thus far, a detailed comparison between the two is lacking. Using a genome-wide approach, we identified and validated 2 crucial players for maintaining the adult primary human CF phenotype. Knockdown of these factors induced significant phenotypical changes, including senescence and reduced collagen gene expression. These may now represent novel therapeutic targets against deleterious functions of CFs in adult cardiovascular disease.
View details for DOI 10.1016/j.jacbts.2016.07.007
View details for PubMedID 30167544
View details for PubMedCentralID PMC6113540
Tricho-hepato-enteric syndrome (THE-S): two cases and review of the literature
EUROPEAN JOURNAL OF PEDIATRICS
2015; 174 (10): 1405?11
In Western cohorts, the prevalence of incidental findings (IFs) or incidentalome, referring to variants in genes that are unrelated to the patient's primary condition, is between 0.86% and 8.8%. However, data on prevalence and type of IFs in Asian population is lacking.In 2 cohorts of individuals with genomic sequencing performed in Singapore (total n = 377), we extracted and annotated variants in the 56 ACMG-recommended genes and filtered these variants based on the level of pathogenicity. We then analyzed the precise distribution of IFs, class of genes, related medical conditions, and potential clinical impact.We found a total of 41,607 variants in the 56 genes in our cohort of 377 individuals. After filtering for rare and coding variants, we identified 14 potential variants. After reviewing primary literature, only 4 out of the 14 variants were classified to be pathogenic, while an additional two variants were classified as likely pathogenic. Overall, the cumulative prevalence of IFs (pathogenic and likely pathogenic variants) in our cohort was 1.6%.The cumulative prevalence of IFs through genomic sequencing is low and the incidentalome may not be a significant barrier to implementation of genomics for personalized medicine.
View details for DOI 10.1016/j.ebiom.2016.01.030
View details for PubMedID 27077130
View details for PubMedCentralID PMC4816806
Tricho-hepato-enteric syndrome (THE-S) is characterized by severe infantile diarrhea, failure to thrive, dysmorphism, woolly hair, and immune or hepatic dysfunction. We report two cases of East Asian descent with THE-S who had remained undiagnosed despite extensive investigations but were diagnosed on whole exome sequencing (WES). Both cases presented with chronic diarrhea, failure to thrive, and recurrent infections. Case 1 had posteriorly rotated low set ears, mild retrognathia, and fine curly hypopigmented hair. She was managed with prolonged total parenteral nutrition and intravenous immunoglobulin infusions. Case 2 had sparse coarse brown hair as well as multiple lentigines and café-au-lait macules. She was managed with amino acid-based formula. For both cases, routine investigations were inconclusive. WES in both cases showed biallelic truncating mutations in TTC37 (c.3507T>G;p.Y1169X and c.3601C>T;p.R1201X in case 1 and c.3507T>G;p.Y1169X and c.154G>T;p.E52X in case 2), suggesting a diagnosis of THE-S.We present novel mutations in the TTC37 gene in two individuals of East Asian descent with the rare THE-S, detected by WES. Future identification of patients with THE-S and establishing genotype-phenotype correlations will aid in counseling the patients and their families.? Tricho-Hepato-Enteric syndrome (THE-S) is characterized by severe infantile diarrhea, failure to thrive, dysmorphism, woolly hair, and immune or hepatic dysfunction. ? Complex patients with diagnostic dilemmas undergo extensive investigations. What is New: ? This is a report of novel mutations in TTC37 in individuals of East Asian descent. ? Whole exome sequencing (WES) can be useful in certain complex cases with diagnostic dilemmas.
View details for DOI 10.1007/s00431-015-2563-z
View details for Web of Science ID 000361649400018
View details for PubMedID 25976726