Cardiac Gene Regulation Research Articles

Exploring genetic mapping and co-expression patterns to illuminate significance of Tbx20 in cardiac biology.

The transcription factor Tbx20 is integral to heart development and plays a significant role in various cardiac diseases. Despite its established importance, the regulatory mechanisms and functional significance of Tbx20 remain incompletely understood. To elucidate these mechanisms, we initially conducted eQTL mapping to identify genetic loci associated with Tbx20 expression in heart tissue from BXD mice. Co-expression and enrichment analyses revealed pathways linked to Tbx20, including dilated cardiomyopathy, hypertrophic cardiomyopathy, and FoxO signaling. Additionally, protein-protein interaction studies identified essential cardiac proteins, such as Myl2 and Myl7, along with upstream regulators like Mef2c. To validate our bioinformatic findings, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) to assess the relative mRNA expression levels of TBX20 and Mef2c in the heart tissues of BXD mice compared to their parental strains (B6 and D2). Our results demonstrated significant up-regulation of both TBX20 and Mef2c in the BXD group relative to the parental strains. Conversely, both genes were down-regulated in B6, D2, Control, and Treatment groups when compared to BXD mice. These findings confirm the predicted regulatory roles of TBX20 and Mef2c in cardiac development as suggested by our initial analyses.This study not only reinforces the critical role of Tbx20 in cardiac gene regulation but also highlights its potential as a therapeutic target for cardiovascular disorders. Further investigations into Tbx20 and its interactions will enhance our understanding of heart biology and contribute to the development of targeted therapies for heart diseases.

Circadian Regulation of Cardiac Arrhythmias and Electrophysiology.

Circadian rhythms in physiology and behavior are ≈24-hour biological cycles regulated by internal biological clocks (ie, circadian clocks) that optimize organismal homeostasis in response to predictable environmental changes. These clocks are present in virtually all cells in the body, including cardiomyocytes. Many decades ago, clinicians and researchers became interested in studying daily patterns of triggers for sudden cardiac death, the incidence of sudden cardiac death, and cardiac arrhythmias. This review highlights historical and contemporary studies examining the role of day/night rhythms in the timing of cardiovascular events, delves into changes in the timing of these events over the last few decades, and discusses cardiovascular disease-specific differences in the timing of cardiovascular events. The current understanding of the environmental, behavioral, and circadian mechanisms that regulate cardiac electrophysiology is examined with a focus on the circadian regulation of cardiac ion channels and ion channel regulatory genes. Understanding the contribution of environmental, behavioral, and circadian rhythms on arrhythmia susceptibility and the incidence of sudden cardiac death will be essential in developing future chronotherapies.

NRSF/REST-Mediated Epigenomic Regulation in the Heart: Transcriptional Control of Natriuretic Peptides and Beyond.

Simple SummaryReactivation of the fetal cardiac gene program, such as those encoding atrial and brain natriuretic peptides (ANP and BNP, respectively), is a characteristic feature of failing hearts. We previously revealed that a transcriptional repressor, neuron-restrictive silencer factor (NRSF), also called repressor element-1-silencing transcription factor (REST), plays a crucial role in the transcriptional control of ANP, BNP and other fetal cardiac genes through collaboration with various other transcription factors to maintain physiological cardiac function and electrical stability. Increased production of ANP and BNP prevents the progression of heart failure, but reactivation of Gαo and fetal-type cardiac ion channels (T-type Ca2+ and HCN channels) leads to deteriorated cardiac function and lethal arrhythmias observed in mice with disturbed NRSF function. Epigenetic regulators with which NRSF forms a complex modify histone acetylation and methylation, thereby participating in NRSF-mediated transcriptional regulation. Further comprehensive studies will lead to clarification of the molecular mechanisms underlying the development of cardiac dysfunction and heart failure.Reactivation of fetal cardiac genes, including those encoding atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), is a key feature of pathological cardiac remodeling and heart failure. Intensive studies on the regulation of ANP and BNP have revealed the involvement of numerous transcriptional factors in the regulation of the fetal cardiac gene program. Among these, we identified that a transcriptional repressor, neuron-restrictive silencer factor (NRSF), also named repressor element-1-silencing transcription factor (REST), which was initially detected as a transcriptional repressor of neuron-specific genes in non-neuronal cells, plays a pivotal role in the transcriptional regulation of ANP, BNP and other fetal cardiac genes. Here we review the transcriptional regulation of ANP and BNP gene expression and the role of the NRSF repressor complex in the regulation of cardiac gene expression and the maintenance of cardiac homeostasis.

Abstract P3117: Validating Genetic Risk Markers Causing Doxorubicin-induced Cardiotoxicity In African American And European American Pediatric Patients Using A Hipsc Model

The W.H.O. estimates that globally 400,000 children under 19 are diagnosed with cancer annually. 60% of pediatric cancers are treated with chemotherapy using anthracyclines, mainly doxorubicin. Survivors of childhood cancer are approximately 15 times more likely to be diagnosed with congestive heart failure. African American (AA) survivors had a 2.5-fold increased risk of severe or life-threatening cardiomyopathy vs European Americans (EA). In recent WGS studies, 2 novel loci in chromosome 1 & 6 were identified to be correlated with an increased risk of doxorubicin-induced cardiotoxicity (DIC) in AA and EA population groups respectively and were reduced or absent in the other group. Our study aims to validate these loci using patient specific hiPSC-derived cardiomyocytes (hiPSC-CM), and for that we recruited and generated 20 hiPSC lines from the blood of AA and EA pediatric patients. Using a novel bioreactor system, a large-scale production of hiPSC-CM for this patient specific samples is underway, and the cells will be utilized for phenotypic and genotypic characterization. Our aim is to assess the effect of doxorubicin on cardiomyocyte viability, apoptosis, mitochondrial function, and calcium dynamics. Differential cardiac gene regulation after exposure to doxorubicin will be studied using RNA sequencing. In conclusion, our findings will validate the genetic variants predisposing AA and EA populations to DIC. This study will clinically impact therapeutic approaches on both AA and EA populations and at individual levels while using doxorubicin. The validated hiPSC-CMs could help in identifying personalized cardioprotectants to reduce DIC without modifying chemotherapy. This study could also potentially provide data for genomically-informed drug discovery for novel cardioprotectants.

Developmental changes in cardiac expression of KCNQ1 and SCN5A spliceoforms: Implications for sudden unexpected infant death.

Sudden unexpected infant death (SUID) occurs unpredictably and remains unexplained after scene investigation and autopsy. Approximately 1 in 7 cases of SUID can be related to a cardiac cause, and developmental regulation of cardiac ion channel genes may contribute to SUID. The goal of this study was to investigate the developmental changes in the spliceoforms of SCN5A and KCNQ1, 2 genes implicated in SUID. Using reverse transcription quantitative real-time polymerase chain reaction, we quantified expression of SCN5A (adult and fetal) and KCNQ1 (KCNQ1a and b) spliceoforms in 153 human cardiac tissue samples from decedents that succumbed to SUID ("unexplained") and other known causes of death ("explained noncardiac"). There is a stepwise increase in the adult/fetal SCN5A spliceoform ratio from <2 months (4.55 ± 0.36; n = 51) through infancy and into adulthood (17.41 ± 3.33; n = 5). For KCNQ1, there is a decrease in the ratio of KCNQ1b to KCNQ1a between the <2-month (0.37 ± 0.02; n = 46) and the 2- to 4-month (0.28 ± 0.02; n = 52) age groups. When broken down by sex, race, or cause of death, there were no differences in SCN5A or KCNQ1 spliceoform expression, except for a higher ratio of KCNQ1b to KCNQ1a at 5-12 months of age for SUID females (0.40 ± 0.04; n = 9) than for males (0.25 ± 0.03; n = 6) and at <2 months of age for SUID white (0.42 ± 0.03; n = 19) than for black (0.33 ± 0.05; n = 9) infants. This study documents the developmental changes in SCN5A and KCNQ1 spliceoforms in humans. Our data suggest that spliceoform expression ratios change significantly throughout the first year of life.

Epigenetic Regulation of Cardiac Troponin Genes in Pediatric Patients with Heart Failure Supported by Ventricular Assist Device.

Ventricular Assist Device (VAD) therapy is considered as a part of standard care for end-stage Heart Failure (HF) children unresponsive to medical management, but the potential role of miRNAs in response to VAD therapy on molecular pathways underlying LV remodeling and cardiac function in HF is unknown. The aims of this study were to evaluate the effects of VAD on miRNA expression profile in cardiac tissue obtained from HF children, to determine the putative miRNA targets by an in-silico analysis as well as to verify the changes of predicated miRNA target in the same cardiac samples. The regulatory role of selected miRNAs on predicted targets was evaluated by a dedicated in vitro study. miRNA profile was determined in cardiac samples obtained from 13 HF children [median: 29 months; 19 LVEF%; 9 Kg] by NGS before VAD implant (pre-VAD) and at the moment of heart transplant (Post-VAD). Only hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-1246 were differentially expressed at post-VAD when compared to pre-VAD, and validated by real-time PCR. Putative targets of the selected miRNAs were involved in regulation of sarcomere genes, such as cardiac troponin (cTns) complex. The expression levels of fetal ad adult isoforms of cTns resulted significantly higher after VAD in cardiac tissue of HF pediatric patients when compared with HF adults. An in vitro study confirmed a down-regulatory effect of hsa-miR-19a-3p on cTnC expression. The effect of VAD on sarcomere organization through cTn isoform expression may be epigenetically regulated, suggesting for miRNAs a potential role as therapeutic targets to improve heart function in HF pediatric patients.

Identification of a Novel Copy Number Variation of EYA4 Causing Autosomal Dominant Non-syndromic Hearing Loss.

Eyes absent 4 (EYA4) is the causative gene of autosomal dominant non-syndromic hereditary hearing loss, DFNA10. We aimed to identify a copy number variation of EYA4 in a non-syndromic sensory neural hearing loss pedigree. A Japanese family showing late-onset and progressive hearing loss was evaluated. A pattern of autosomal dominant inheritance of hearing loss was recognized in the pedigree. No cardiac disease was observed in any of the individuals. Targeted exon sequencing was performed using massively parallel DNA sequencing (MPS) analysis. Scanning of the array comparative genomic hybridization (aCGH) was completed and the copy number variation (CNV) data from the aCGH analysis was confirmed by matching all CNV calls with MPS analysis. Breakpoint detection was performed by whole-genome sequencing and direct sequencing. Sequencing results were examined, and co-segregation analysis of hearing loss was completed. We identified a novel hemizygous indel that showed CNV in the EYA4 gene from the position 133,457,057 to 133,469,892 on chromosome 6 (build GRCh38/hg38) predicted as p.(Val124_Pro323del), and that was segregated with post-lingual and progressive autosomal dominant sensorineural hearing loss by aCGH analysis. Based on the theory of genotype-phenotype correlation with EYA4 mutations in terms of hearing loss and comorbid dilated cardiomyopathy, the region of amino acids 124 to 343 is hypothesized not to be the pathogenic region causing dilated cardiomyopathy. Additionally, the theory of genotype-phenotype correlation about the prevalence of dilated cardiomyopathy is thought to be rejected because of no correlation of deleted amino acid region with the prevalence of dilated cardiomyopathy. These results will help expand the research on both the coordination of cochlear transcriptional regulation and normal cardiac gene regulation via EYA4 transcripts and provide information on the genotype-phenotype correlations of DFNA10 hearing loss.

Abstract 15916: Role of Small-cajal Body Associated RNAs (scaRNAs) in Regulation of Cardiomyogenesis

Introduction: The requirement of non-coding RNAs particularly scaRNAs are playing a critical role in alternative splicing and maturation of mRNAs. Dysregulated splicing of mRNAs has been shown to cause heart defects. In this study, we are comparing the role of scaRNAs during differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (iCMCs) from normal individual and Noonan syndrome (NS) patient. We have selected NS patient cells because it is an autosomal dominant genetic disorder which leads to cardiomyopathy and congenital heart defects in humans. Hypothesis: We hypothesize that scaRNA1 and scaRNA20 have a significant role in the development of cardiomyocytes, and these scaRNAs are dysfunctional in iCMCs derived from NS. Methods and Results: We have compared the normal skin fibroblast-derived iPSCs (N-iPSCs) and N-iCMCs with NS patient-derived NS-iPSCs and NS-iCMCs using quantitative RT-PCR, Western blot and immunofluorescence analyses. We also used the knockdown and overexpression of scaRNA1 and scaRNA20 approaches to delineate the importance of these scaRNAs during cardiomyogenesis. Our qRT-PCR data showed a significantly lower expression of scaRNA1 and scaRNA20 (Fig. A) as well as the cardiac-specific genes CTT and GATA4 (Fig. B) in NS-iCMCs when compared to the normal iCMCs. Furthermore, the qRT-PCR data from the scaRNA20 overexpressed N-iCMC showed an increased expression of cardiac-specific genes (Fig. C) when compared to the N-iCMCs. These studies clearly indicate that scaRNA1 and scaRNA20 plays an important role in cardiomyogenesis. Further studies are underway to explore the mechanisms of these scaRNAs in regulation of cardiac genes. Conclusions: Our findings indicate that scaRNA1 and scaRNA20 are involved in mRNA splicing and maturation of cardiac genes. Moreover, these scaRNAs are dysfunctional in NS patient’s iCMCs and targeting these molecules will have a therapeutic potential for a patient with NS.

Abstract 254: Profiling Transcriptional Changes and Identifying Novel Molecular Markers During the Dedifferentiation of Cardiomyocyte

The loss of cardiomyocytes (CMs) during heart failure (HF) cannot be replaced by new CMs due to their limited proliferative capacity. Regenerating the failing heart by promoting CM cell-cycle re-entry has thus become a possible solution rigorously pursued. Some genes have been proven to promote endogenous CM proliferation, nearly always preceded by CM dedifferentiation, wherein terminally-differentiated CMs are reversed back initially to a less-matured status, preceding cell division. However, very little else is known about CM dedifferentiation, including the lack of robust molecular markers, the mechanisms driving it, whether it necessarily leads to CM proliferation, and the effect of CM dedifferentiation alone on cardiac function. Therefore, investigating CM dedifferentiation is of great significance. Here, we profiled genome-wide transcriptional changes and identified novel molecular markers during CM dedifferentiation. We used the established in vitro model where adult mouse CMs dedifferentiated and re-differentiated (DR) upon 2-week long culture, without detectable cell division. DR was evident by breakdown and rebuilding of sarcomeres, and differential regulation of cardiac fetal genes. Tracking the transcriptome change pointed to gene pathways involved in this process and potential markers of CM dedifferentiation. Another assumption is that transient induction of Yamanaka factors ( Oct4, Sox2, Klf4, Myc, OSKM ) induces CM dedifferentiation of CMs in vivo , since the four factors are themselves sufficient to induce pluripotency of human and mouse somatic cells. Our preliminary results show that CM-specific overexpression of OSKM using adeno-associated virus serotype 9 (AAV9) induced fetal-like hallmarks, down-regulation of sarcomeric genes, re-activation of cell cycle, reduced fatty acid utilization, and associated loss of cardiac function . Putative markers identified in the in vitro model was also validated to be upregulated in OSKM -overexpressed CMs. In conclusion, through 2 models of CM dedifferentiation, we have screened out potential novel markers and regulators of CM dedifferentiation which now requires further validation.

ATF6β is an Adaptive Transcription Factor in Cardiac Myocyte.

RationaleATF6 is an endoplasmic reticulum (ER) transmembrane protein, which is activated in response to the accumulation of misfolded proteins in the ER, a phenomenon known as ER stress. During ER stress, ATF6 is activated through proteolytic cleavage in the Golgi, which releases the N‐terminal active form that will translocate to the nucleus and act as a transcription factor, regulating a widespan of adaptive genes in an attempt to restore proteostasis in the ER, thus resolving ER stress and promoting cell survival. If ER proteostasis is terminally impaired, a maladaptive response will be activated and cell death will occur. There exist two isoforms of ATF6, ATF6α and ATF6β, which have high sequential and structural homology in their ER transmembrane and DNA binding domains. Our previous studies have shown that acute activation of ATF6α is protective in cardiac myocytes under different simulated pathologies in the heart, such as ischemic injury and pressure overload. On the other hand, the precise role of ATF6β in cardiac myocyte gene regulation and viability has yet to be determined.HypothesisATF6β is an adaptive transcription factor that is critical to cardiac myocyte viability.MethodWe used cultured primary neonatal rat ventricular myocytes (NRVM) as a model to study the role of ATF6β, in a loss‐of‐function or gain‐of‐function approach. To study the impact of ATF6β loss‐of‐function, we used small interfering RNA targeted to ATF6β to knockdown its expression. To study the impact of ATF6β gain‐of‐function, we overexpressed ATF6β by infection of NRVMs with ATF6β‐encoding adenovirus. Thereafter, in both models, we assessed cell viability by MTT assay over a period of four days after treatment with or without the chemical ER‐stress inducer tunicamycin (TM). We also determined the adaptive and maladaptive gene profiles of NRVMs in both loss‐of‐function and gain‐of‐function models using real‐time quantitative PCR.ResultsOur results showed that ATF6β knockdown significantly decreased cardiac myocyte viability over time without TM, and TM‐induced ER‐stress exacerbated this loss in viability. ATF6β overexpression maintained cell viability throughout the time course compared to cells treated with control adenovirus, both with and without TM. Gene expression analysis in the ATF6β loss‐of‐function model showed a downregulation of adaptive genes, such as the chaperone Hsp90b1 and anti‐apoptotic gene Bcl2, and an upregulation of the maladaptive, pro‐apoptotic gene Bbc3. Consistent with the loss‐of‐function model, in the gain‐of‐function model, gene expression analysis showed an upregulation of chaperone gene Hspa5 and a downregulation of Bbc3.ConclusionThe effect of ATF6β loss‐ and gain‐of‐function on cell viability and Bbc3 gene expression demonstrate that ATF6β has an adaptive role in cardiac myocyte viability and suggest a regulatory role of ATF6β on Bbc3 gene expression to achieve its effect on viability.Support or Funding InformationAmerican Heart Association, National Institute of Health

Genetic and Epigenetic Control of Heart Development.

A transcriptional program implemented by transcription factors and epigenetic regulators governs cardiac development and disease. Mutations in these factors are important causes of congenital heart disease. Here, we review selected recent advances in our understanding of the transcriptional and epigenetic control of heart development, including determinants of cardiac transcription factor chromatin occupancy, the gene regulatory network that regulates atrial septation, the chromatin landscape and cardiac gene regulation, and the role of Brg/Brahma-associated factor (BAF), nucleosome remodeling and histone deacetylation (NuRD), and Polycomb epigenetic regulatory complexes in heart development.

Transcriptional and Epigenetic Regulation of Cardiac Electrophysiology

Spatiotemporal gene expression during cardiac development is a highly regulated process. Activation of key signaling pathways involved in electrophysiological programming, such as Notch and Wnt signaling, occurs in early cardiovascular development and these pathways are reactivated during pathologic remodeling. Direct targets of these signaling pathways have also been associated with inherited arrhythmias such as Brugada syndrome and arrhythmogenic cardiomyopathy. In addition, evidence is emerging from animal models that reactivation of Notch and Wnt signaling during cardiac pathology may predispose to acquired arrhythmias, underscoring the importance of elucidating the transcriptional and epigenetic effects on cardiac gene regulation. Here, we highlight specific examples where gene expression dictates electrophysiological properties in both normal and diseased hearts.

Direct visualization of cardiac transcription factories reveals regulatory principles of nuclear architecture during pathological remodeling

Heart failure is associated with hypertrophying of cardiomyocytes and changes in transcriptional activity. Studies from rapidly dividing cells in culture have suggested that transcription may be compartmentalized into factories within the nucleus, but this phenomenon has not been tested in vivo and the role of nuclear architecture in cardiac gene regulation is unknown. While alterations to transcription have been linked to disease, little is known about the regulation of the spatial organization of transcription and its properties in the pathological setting. In the present study, we investigate the structural features of endogenous transcription factories in the heart and determine the principles connecting chromatin structure to transcriptional regulation in vivo. Super-resolution imaging of endogenous RNA polymerase II clusters in neonatal and adult cardiomyocytes revealed distinct properties of transcription factories in response to pathological stress: neonatal nuclei demonstrated changes in number of clusters, with parallel increases in nuclear area, while the adult nuclei underwent changes in size and intensity of RNA polymerase II foci. Fluorescence in situ hybridization-based labeling of genes revealed locus-specific relationships between expression change and anatomical localization—with respect to nuclear periphery and heterochromatin regions, both sites associated with gene silencing—in the nuclei of cardiomyocytes in hearts (but not liver hepatocytes) of mice subjected to pathologic stimuli that induce heart failure. These findings demonstrate a role for chromatin organization and rearrangement of nuclear architecture for cell type-specific transcription in vivo during disease. RNA polymerase II ChIP and chromatin conformation capture studies in the same model system demonstrate formation and reorganization of distinct nuclear compartments regulating gene expression. These findings reveal locus-specific compartmentalization of stress-activated, housekeeping and silenced genes in the anatomical context of the endogenous nucleus, revealing basic principles of global chromatin structure and nuclear architecture in the regulation of gene expression in healthy and diseased conditions.

Competent for commitment: you've got to have heart!

The mature heart is composed primarily of four different cell types: cardiac myocytes, endothelium, smooth muscle, and fibroblasts. These cell types derive from pluripotent progenitors that become progressively restricted with regard to lineage potential, giving rise to multipotent cardiac progenitor cells and, ultimately, the differentiated cell types of the heart. Recent studies have begun to shed light on the defining characteristics of the intermediary cell types that exist transiently during this developmental process and the extrinsic and cell-autonomous factors that influence cardiac lineage decisions and cellular competence. This information will shape our understanding of congenital and adult cardiac disease and guide regenerative therapeutic approaches. In addition, cardiac progenitor specification can serve as a model for understanding basic mechanisms regulating the acquisition of cellular identity. In this review, we present the concept of "chromatin competence" that describes the potential for three-dimensional chromatin organization to function as the molecular underpinning of the ability of a progenitor cell to respond to inductive lineage cues and summarize recent studies advancing our understanding of cardiac cell specification, gene regulation, and chromatin organization and how they impact cardiac development.

Loss of microRNA-22 prevents high-fat diet induced dyslipidemia and increases energy expenditure without affecting cardiac hypertrophy.

Obesity is associated with development of diverse diseases, including cardiovascular diseases and dyslipidemia. MiRNA-22 (miR-22) is a critical regulator of cardiac function and targets genes involved in metabolic processes. Previously, we generated miR-22 null mice and we showed that loss of miR-22 blunted cardiac hypertrophy induced by mechanohormornal stress. In the present study, we examined the role of miR-22 in the cardiac and metabolic alterations promoted by high-fat (HF) diet. We found that loss of miR-22 attenuated the gain of fat mass and prevented dyslipidemia induced by HF diet, although the body weight gain, or glucose intolerance and insulin resistance did not seem to be affected. Mechanistically, loss of miR-22 attenuated the increased expression of genes involved in lipogenesis and inflammation mediated by HF diet. Similarly, we found that miR-22 mediates metabolic alterations and inflammation induced by obesity in the liver. However, loss of miR-22 did not appear to alter HF diet induced cardiac hypertrophy or fibrosis in the heart. Our study therefore establishes miR-22 as an important regulator of dyslipidemia and suggests it may serve as a potential candidate in the treatment of dyslipidemia associated with obesity.

HBL1 Is a Human Long Noncoding RNA that Modulates Cardiomyocyte Development from Pluripotent Stem Cells by Counteracting MIR1

Cardiogenesis processes in human and animals have differential dynamics, suggesting the existence of species-specific regulators during heart development. However, it remains a challenge to discover the human-specific cardiac regulatory genes, given that most coding genes are conserved. Here, we report the identification of a human-specific long noncoding RNA, Heart Brake LncRNA 1 (HBL1), which regulates cardiomyocyte development from human induced pluripotent stem cells (hiPSCs). Overexpression of HBL1 repressed, whereas knockdown and knockout of HBL1 increased, cardiomyocyte differentiation from hiPSCs. HBL1 physically interacted with MIR1 in an AGO2 complex. Disruption of MIR1 binding sites in HBL1 showed an effect similar to that of HBL1 knockout. SOX2 bound to HBL1 promoter and activated its transcription. Knockdown of SOX2 in hiPSCs led to decreased HBL1 expression and increased cardiomyocyte differentiation efficiency. Thus, HBL1 plays a modulatory role in fine-tuning human-specific cardiomyocyte development by forming a regulatory network with SOX2 and MIR1.

Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera.

Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG’s were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes.

Gerald W. Dorn II: Thinker, Teacher, Tinkerer.

Cardiologist and molecular and cell biologist Gerald Dorn, MD, has built his research career crafting interesting questions and teaching himself techniques needed to find answers. Using approaches including genetic and physiological manipulation in animal models, development of mini-mouse technology to elucidate cardiac mysteries, and human genetics/genomics, Dorn is helping to unravel cellular and molecular mechanisms underlying cardiomyopathy and heart failure.1,2 Gerald W. Dorn II Dorn, 57, is founding director of the Center for Pharmacogenomics at the Washington University in St. Louis School of Medicine, where he joined the faculty in 2008. As a teenager Dorn sped through high school, starting college two years early. He earned his medical degree and trained in internal medicine, pharmacology, and clinical and interventional cardiology at the Medical University of South Carolina (MUSC) in Charleston, where two key mentors sparked in him a passion for patient care and research. After a couple of years at the University of Texas Health Science Center at San Antonio, Dorn moved to the University of Cincinnati, where an American Heart Association Established Investigator Award kick-started his research program. From 1990 to 2008 Dorn rose through the ranks at Cincinnati, capitalizing on the institution’s strength in mouse cardiac transgenesis. Dorn’s current interests include molecular mechanisms of cell death,3 microRNA regulation of cardiac genes,4 and mitochondrial dynamism5—work that has expanded to Parkinson’s disease. Building on an upbringing that valued lifelong growth, and drawing from his own broad experience, Dorn now leverages the holism of systems biology to fill in what he calls his field’s “infinitely large shades of gray.” My father was a career naval aviator. Whenever he moved, we moved. So I’m not really from anywhere. We spent a lot of time on both coasts. As a child I lived, in addition to …

Role of Histone Demethylases in Cardiomyocytes Induced to Hypertrophy.

Epigenetic changes induced by histone demethylases play an important role in differentiation and pathological changes in cardiac cells. However, the role of the jumonji family of demethylases in the development of cardiac hypertrophy remains elusive. In this study, the presence of different histone demethylases in cardiac cells was evaluated after hypertrophy was induced with neurohormones. A cell line from rat cardiomyocytes was used as a biological model. The phenotypic profiles of the cells, as well as the expression of histone demethylases, were studied through immunofluorescence, transient transfection, western blot, and qRT-PCR analysis after inducing hypertrophy by angiotensin II and endothelin-1. An increase in fetal gene expression (ANP, BNP, and β-MHC) was observed in cardiomyocytes after treatment with angiotensin II and endothelin-1. A significant increase in JMJD2A expression, but not in UTX or JMJD2C expression, was observed. When JMJD2A was overexpressed in cardiomyocytes through transient transfection, the effect of neurohormones on fetal cardiac gene expression was increased. We conclude that JMJD2A plays a principal role in the regulation of fetal cardiac genes, which increase in expression during the pathological hypertrophic process.

Sirt1 mediates the effects of a short-term high-fat diet on the heart

High-fat diet leads to development of cardiac dysfunction through molecular mechanisms poorly known. The aim of this study is to elucidate the early events in cardiac dysfunction caused by a high-fat diet, before massive alterations due to obesity and indirect mechanisms of heart damage take place. Moreover, we analyzed the role of Sirt1, a major mediator of cardiac gene regulation, in these effects. Short-term high-fat feeding (5 weeks) caused a similar mild increase in body weight and triglyceridaemia in wild-type (wt) and Sirt1+/− mice. The high-fat diet suppressed the expression of lipid catabolism (PPARα target) gene expression in the hearts of wt mice, but not Sirt1+/− mice. Pro-inflammatory genes were induced and estrogen-related receptor-alpha (ERRα) target genes was suppressed in the hearts of wt fed the high-fat diet, but not in Sirt1+/− mice. We found the formation of a complex between PPARα and Sirt1 in wt mice under high-fat diet conditions which might account for suppression of the ERRα pathway. Sirt1 haploinsufficiency impairs the formation of this complex and promotes the binding of PPARα to the p65 subunit of NF-κB, thereby mediating inhibition of pro-inflammatory pathways and induction of PPARα target genes. Short-term high-fat diet causes metabolic and inflammatory alterations in heart, and Sirt1 is critical for mediating these cardiac alterations. The capacity of Sirt1 to interact with transcriptional regulators such as NF-κB and PPARα appears to be involved in the cardiac responsiveness to a high-fat diet.