Abstract 240: Differential Impact of RBFox Family Proteins in Adult Murine Heart

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Chen Gao ◽  
Shuxun Ren ◽  
Yibin Wang
Keyword(s):  
Heart Failure ◽  
Alternative Splicing ◽  
Rna Splicing ◽  
Adult Mouse ◽  
Regulatory Elements ◽  
Global Changes ◽  
Mouse Heart ◽  
Cardiac Physiology ◽  
Differential Impact ◽  
Adult Heart

Background: The complexity of cardiac transcriptome and proteome is significantly contributed by alternative splicing of mRNA. Alternative splicing is regulated by the cis-regulatory elements located in pre-mRNA together with the trans-activating factors guiding the assembling and function of the spliceosome. In our earlier study, we have observed global changes of alternative splicing events during pressure-overload induced heart failure, and identified RBFox1 as a key regulator for cardiac RNA splicing regulation during postnatal development and pathological remodeling. Both RBFox1 and RBFox2 are highly enriched in cardiomyocytes, and their expression are both significantly repressed in response to pathological stress. Loss-of-function studies for RBFox1 and RBFox2 are achieved using cardiac specific but constitutively active Cre. Therefore, the isoform specific contribution of RBFox1 vs. RBFox2 in maintaining cardiac physiology and homeostasis in adult heart is unknown. Methods and Results: We generated mouse models of cardiac specific and inducible knockout of RBFox1 and RBFox2 individually in adult hearts by breeding the individual floxed alleles with the αMHC-Mer-Cre-Mer mice. At baseline, inactivating RBFox1 in adult heart caused a slight but significant decrease of cardiac function without activating hypertrophy gene expression. However, following myocardial infarction, the RBFox1 deficient hearts showed enhanced global fibrosis in non-infarcted areas comparing to the control animals. In contrast, inactivating RBFox2 in adult mouse heart caused overt heart failure associated with chamber dilation without external stress as early as 2 weeks post tamoxifen administration. Conclusion: We have identified differential impact of RBFox1 and RBFox2 deficiency in adult mouse heart. Our in vivo study illustrate the functional importance of the RBFox family RNA splicing regulators in normal physiology of adult heart, and support the pathogenic contribution of loss of RBFox expression to heart failure. Further analysis focusing on the underlying molecular mechanisms for their differential impact would yield new insights on transcriptome regulation and complexity in cardiac physiology and diseases.

Abstract 235: Global RNA Splicing and Regulation in Cardiac Maturation and Diseases

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
CHEN GAO ◽  
Vincent Ren ◽  
Jae-Hyung Lee ◽  
Xinshu (Grace) Xiao ◽  
Jau-nian Chen ◽  
...  
Keyword(s):  
Heart Failure ◽  
Alternative Splicing ◽  
Cardiac Function ◽  
Rna Splicing ◽  
Cardiac Development ◽  
Mrna Splicing ◽  
Pcr Analysis ◽  
Mouse Heart ◽  
Global Pattern ◽  
Alternative Mrna Splicing

Background: The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, the global pattern of alternative mRNA splicing during cardiac development and diseases is unknown, and the regulatory mechanisms remain unexplored. Methods and Results: Using deep RNA-Sequencing, we have identified global alternative splicing changes associated with both cardiac development and pathological remodeling in mouse heart following pressure-overload induced heart failure. The alternative RNA splicing events observed in failing hearts mimics the profile in fetal hearts, suggesting a fetal-like RNA splicing program induced in diseased hearts. Using RNA-Seq database and real-time PCR analysis, we examined the expression profile of a large number of known alternative splicing regulators. Among them, we identified Fox1 as a significantly induced regulator during cardiac development in zebrafish, mouse and human, and down-regulated in both mouse and human failing hearts. Morpholino mediated Fox1 knockdown in zebrafish embryos led to lethal phenotype associated with reduced cardiac function and defects in chamber specificity. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene, suggesting a highly conserved cardiac function of Fox1 for normal cardiac development and function in vertebrates. Conclusion: Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified Fox1 as a key regulator for alternative RNA splicing in heart. This study expands our current understanding to the complexity of cardiac transcriptome, and reveals the functional importance of RNA-splicing in cardiac development and diseases.

Abstract 124: Fetal-Like RNA Splicing Remodeling in Failing Heart Revealed by Total Transcriptome Analysis

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Chen Gao ◽  
Vincent Ren ◽  
Grace (Xinshu) Xiao ◽  
Jaunian Chen ◽  
Yibin Wang
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Cardiac Development ◽  
Heart Diseases ◽  
Expression Profiles ◽  
Mrna Splicing ◽  
Mouse Heart ◽  
Failing Heart ◽  
Splicing Regulators ◽  
A Genome

The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, little knowledge is available about the scope of alternative splicing at whole genome level in heart diseases and even less about the mechanisms underlying the regulation of mRNA splicing in response to pathological injury in heart. Using a genome-wide RNA-Seq analysis, we have identified global alternative splicing changes associated with both development and pathological remodeling in mouse heart. Most significantly, the alternative RNA splicing events observed in failing heart mimicked the splicing profile in fetal hearts, suggesting a fetal like RNA splicing remodeling in failing hearts. After examining the expression profiles of splicing regulators in neonatal, normal adult, and failing adult hearts, Fox-1 was identified as one to be significantly down regulated in the failing and fetal hearts. Morpholino mediated Fox-1 knock-down in zebrafish embryos led to lethal phenotype associated with impaired cardiac development and function. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene. Therefore, our established functional significance of Fox1 mediated RNA alternative splicing serves as a key molecular player in transcriptome remodeling during cardiac development and pathology.

Abstract 47: GATA Factors Caught at a Crossroad of Gene Duplication with Functional Divergence

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Jop H van Berlo ◽  
Jeffery D Molkentin
Keyword(s):  
Heart Failure ◽  
Functional Divergence ◽  
Severe Heart Failure ◽  
Pressure Overload ◽  
Mouse Heart ◽  
Adult Heart ◽  
Functional Roles ◽  
Hypertrophic Response ◽  
Gata Factors ◽  
Cardiac Gene

Background Six individual members comprise the GATA family of Zn finger-containing transcription factors that play major roles in the hematopoietic system and many mesoderm and endoderm derived tissues. The adult heart expresses both GATA4 and GATA6. Here, we examined the overlapping and diverging functional roles of GATA4 and GATA6 in the adult heart, both at baseline and under stress. Results Pressure overload by transverse aortic constriction (TAC) caused a blunted hypertrophic response when GATA4 was deleted from the adult heart, with severe heart failure ensuing after 4 weeks. Similarly, deletion of GATA6 from the mouse heart showed a blunted hypertrophic response and heart failure. Next, we deleted 1 allele of GATA4 and 1 allele of GATA6 from the adult heart, also resulting in blunted hypertrophy and cardiac dysfunction. Deletion of all four alleles of GATA4 and 6 resulted in spontaneous heart failure and death by 3 months of age. These results suggested functional overlap or synergistic activation. To address this concept more directly we deleted GATA6 from the adult heart and overexpressed either GATA4 or GATA6 in a cardiac-specific manner. As expected, we were able to completely revert the phenotype to wild type when GATA6 was overexpressed in mice that had GATA6 genetically deleted. Surprisingly, overexpression of GATA4 was unable to rescue the absence of GATA6 and actually worsened cardiac function in response to pressure overload. Possible explanations for this functional divergence were suggested by an observed rarefaction in capillaries of the heart in absence of GATA4, but enhanced angiogenesis in absence of GATA6. Moreover, when we induced cardiac hypertrophy through MAPK activation, we observed a critical necessity for GATA4, while GATA6 was dispensable. Conclusion Although GATA4 and 6 may be functionally complementary for cardiac gene expression and hypertrophy, they evolved some specific roles in the heart, such as angiogenesis and stress activation. We are currently unraveling how GATA4 and 6 may differentially regulate genes.

scholarly journals Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

Circulation ◽  
2011 ◽  
Vol 123 (5) ◽  
pp. 504-514 ◽  
Author(s):  
Mortimer Korf-Klingebiel ◽  
Tibor Kempf ◽  
Klaus-Dieter Schlüter ◽  
Christian Willenbockel ◽  
Torben Brod ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Heart Failure ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Adult Mouse ◽  
Mouse Heart ◽  
Fibroblast Growth ◽  
Transgenic Expression

scholarly journals Evidence for minimal cardiogenic potential of Sca-1 positive cells in the adult mouse heart

2018 ◽  
Author(s):  
Lauren E. Neidig ◽  
Florian Weinberger ◽  
Nathan J. Palpant ◽  
John Mignone ◽  
Amy M. Martinson ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Heart Failure ◽  
Mouse Model ◽  
Lineage Tracing ◽  
Mouse Heart ◽  
Genetic Lineage ◽  
Adult Heart ◽  
New Therapeutic Approaches ◽  
Genetic Lineage Tracing

ABSTRACTBackgroundDespite modern pharmacotherapy, heart failure remains a major medical burden. The heart has a limited regenerative capacity, and bolstering regeneration might represent new therapeutic approaches for heart failure patients. Various progenitor cells in the heart have been proposed to have cardiomyogenic properties, but this evidence is based mostly on cell culture and transplantation studies. One population of interest is characterized by the expression of Stem Cell Antigen-1 (Sca-1). Here we tested the hypothesis that Sca-1+cells are endogenous progenitors for cardiomyocytes in the adult heart.MethodsWe evaluated the innate cardiogenic potential of Sca-1+cellsin vivoby generating a novel mouse model to genetically lineage-trace the fate of Sca-1 expressing cells. This was accomplished by introducing a tamoxifen-inducible Cre-recombinase into the Sca-1 locus (Sca-1mCm/+). Crossing this mouse line to a Cre-dependent tdTomato reporter line allowed for genetic lineage-tracing of endogenous Sca-1+cells (Sca-1mCmR26tdTomato). The frequency of Sca-1+cardiomyocytes was quantified from dispersed cell preparations and confirmed by in situ histology.ResultsWe validated the genetic lineage tracing mouse model in bone marrow and heart. Unlike previous publications suggesting significant cardiogenic potential, we found that less than 0.02% of cardiomyocytes per year were derived from Sca-1+cells in the adult heart under homeostatic conditions. At six months after myocardial infarction, we found less than 0.01% of cardiomyocytes were derived from Sca-1+cells.ConclusionOur results show that Sca-1+cells in the adult heart have minimal cardiogenic potential under homeostatic conditions or in response to myocardial infarction.

Abstract 137: Ctcf is Essential for Transcriptome Regulation in the Healthy Heart

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Manuel Rosa-Garrido ◽  
Todd Kimball ◽  
Douglas J Chapski ◽  
Tsai-Ting Shih ◽  
Elizabeth Soehalim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Severe Heart Failure ◽  
Surgical Techniques ◽  
Ctcf Binding ◽  
Global Changes ◽  
Mouse Heart ◽  
Precise Control ◽  
Transcriptome Regulation ◽  
Heart Size

Heart failure is a syndrome resulting from complex genetic predisposition and multiple environmental factors. It is well established that global changes in gene expression accompany the transition through hypertrophy and on to overt failure producing deterioration of function at the organ level. Unknown, however, are the global changes in chromatin structure that allows for pathological gene expression. The epigenetic factor CTCF is a ubiquitous protein thought to be involved in maintenance of chromatin microenvironments. We found that hypertrophic stimulation down-regulates CTCF in the heart, and CTCF expression is significantly correlated with heart size in disease across genetic backgrounds. To test this relationship further, we have generated an inducible cardiac-specific CTCF knockout animal. These animals quickly develop severe heart failure characterized by a dilated left ventricle, decreased ejection fraction, and reactivation of the fetal gene program. Our analysis of published CTCF ChIP-seq data performed in mouse heart has also shown CTCF binding peaks flanking the fetal genes. Furthermore, we have found that CTCF abundance at these sites changes after hypertrophic agonist isoproterenol treatment, suggesting that CTCF protects from the development of cardiac disease by regulating gene expression. We also used RNA-seq to compare the transcriptome of adult myocytes isolated from control, CTCF KO mice, and mice that underwent transverse aortic constriction (TAC) to induce heart failure. These analyses showed that CTCF depletion remodels gene expression in a manner that mimics aspects of the pathological transcriptome in heart failure. Our data demonstrate that precise control of CTCF levels is required for normal transcriptome regulation in the adult heart and that alterations in CTCF levels (either via genetic, pharmacologic or surgical techniques) lead to cardiac pathology.

scholarly journals Systems proteomics of cardiac chromatin identifies nucleolin as a regulator of growth and cellular plasticity in cardiomyocytes

2013 ◽  
Vol 305 (11) ◽  
pp. H1624-H1638 ◽  
Author(s):  
Emma Monte ◽  
Kevin Mouillesseaux ◽  
Haodong Chen ◽  
Todd Kimball ◽  
Shuxun Ren ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Chromatin Structure ◽  
Functional Characterization ◽  
Strong Acid ◽  
Global Changes ◽  
Mouse Heart ◽  
Morphological Development ◽  
Altered Expression ◽  
Associated Proteins

Myocyte hypertrophy antecedent to heart failure involves changes in global gene expression, although the preceding mechanisms to coordinate DNA accessibility on a genomic scale are unknown. Chromatin-associated proteins alter chromatin structure by changing their association with DNA, thereby altering the gene expression profile. Little is known about the global changes in chromatin subproteomes that accompany heart failure, and the mechanisms by which these proteins alter chromatin structure. The present study tests the fundamental hypothesis that cardiac growth and plasticity in the setting of disease recapitulates conserved developmental chromatin remodeling events. We used quantitative proteomics to identify chromatin-associated proteins extracted via detergent and to quantify changes in their abundance during disease. Our study identified 321 proteins in this subproteome, demonstrating it to have modest conservation (37%) with that revealed using strong acid. Of these proteins, 176 exhibited altered expression during cardiac hypertrophy and failure; we conducted extensive functional characterization of one of these proteins, Nucleolin. Morpholino-based knockdown of nucleolin nearly abolished protein expression but surprisingly had little impact on gross morphological development. However, hearts of fish lacking Nucleolin displayed severe developmental impairment, abnormal chamber patterning and functional deficits, ostensibly due to defects in cardiac looping and myocyte differentiation. The mechanisms underlying these defects involve perturbed bone morphogenetic protein 4 expression, decreased rRNA transcription, and a shift to more heterochromatic chromatin. This study reports the quantitative analysis of a new chromatin subproteome in the normal and diseased mouse heart. Validation studies in the complementary model system of zebrafish examine the role of Nucleolin to orchestrate genomic reprogramming events shared between development and disease.

Abstract 345: Maturation of Cardiomyocytes via Rbfox1

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Josh Z Lee ◽  
Chen Gao ◽  
Ivan Pushkarsky ◽  
Chaowei Hu ◽  
Haodong Xu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Regulatory Networks ◽  
Disease Modeling ◽  
Ectopic Expression ◽  
Mouse Heart ◽  
Valuable Insight ◽  
Cardiomyocyte Differentiation ◽  
Mature Adult ◽  
Cell Based Therapy

Background: Cardiovascular disease is the leading cause of death in the world with a dearth of effective therapies. Heart undergoes a complex differentiation and maturation process throughout embryonic and post-natal stages. Intense efforts have been made in the study of cardiomyocyte differentiation, maturation and pathological remodeling. With the aid of stem cells, investigators are able to recapitulate events in early cardiac development and gain valuable insight in transcriptional regulatory networks directing early cardiomyocyte differentiation. However, the molecular network that determine myocyte maturation in postnatal development, which are especially important for understanding diseases and developing cell based clinical applications, are far less well characterized. Human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CMs) are lineage committed but remain immature and fetal-like in molecular, morphological and functional characteristics. Their application in cell based therapy or serving as a disease model for heart failure is limited, in part due to the lack of sufficient insight to promote their maturation into adult myocytes. Results: We find from previous studies that postnatal cardiomyocyte development is marked by global alternative splicing (AS) programming. In mouse heart, we find RNA splicing regulator RBFox1 is markedly induced in heart only in post-natal period and functions as a key regulator to post-natal global RNA alternative splicing reprogramming during cardiomyocyte maturation. Transcriptome analysis revealed that exogenous expression of RBFox1 promotes gene expression in neonatal ventricular myocytes reminiscent of mature adult heart, and results in cellular maturation based on sarcomere organization and calcium cycling characteristics. Furthermore, ectopic expression of rodent RBFox1 in human iPSC derived CMs resulted in similar maturation effects, implicating a conserved mechanism in human and rodent myocytes. Conclusion: RBFox1 mediated RNA splicing is an important contributor to post-natal myocyte maturation and it can be manipulated to promote cardiomyocyte maturation for cell-based therapy or disease modeling.

Abstract 123: Erythropoietin Enhances Abundance of Cardiomyogenic Precursors in vitro and in vivo

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Maria Patapia Zafeiriou ◽  
Claudia Noack ◽  
Bernhard Unsoeld ◽  
Michael Didie ◽  
Ali El-Armouche ◽  
...  
Keyword(s):  
Adult Mouse ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Mouse Heart ◽  
Rat Cardiomyocytes ◽  
Adult Heart ◽  
Cell Pool ◽  
Epor Expression

Recent data suggest that erythropoietin (Epo) can improve cardiac function following ischemia reperfusion injury. We found EpoR to be prominently expressed in embryonic rather than adult mouse heart. In the latter EpoR expression was confined to interstitial cells. We isolated the non-myocyte fraction of adult mouse hearts by enzymatic digestion and straining (pore size: 30 µm) and found a 4-fold higher EpoR expression compared to unselected cardiac cells (n=3). Flow cytometry (FC) revealed that 24±3% of this heterogeneous cell pool expressed EpoR (n=3) and 11±2% co-expressed αMHC. Half of the αMHC+ cells stained positive for Ki67, consistent with FC showing that 49±7% αMHC+/EpoR+ cells were in S and G2 phase (n=3; vs. 13±3% in αMHC-/EpoR-; n=3). Interestingly, co-culture with neonatal rat cardiomyocytes yielded EGFP+ cardiomyocyte-like cells. Since αMHC+ proliferated and differentiated in vitro we termed them cardiomyogenic precursors (CMPs). Epo treatment of the non-myocyte pool enhanced Akt phosphorylation (n=6/group) and increased CMP abundance in vitro (2-fold; n=3). Cell cycle arrest abrogated the aforementioned effect, suggesting that Epo influenced CMP proliferation. Finally, we tested the potential of Epo to protect against ischemia by enhancing CMP numbers in a model of myocardial infarction (MI). Following MI mice were treated twice with 2,000 U/kg Epo (i.p.). Ten weeks post MI echocardiography revealed blunted myocardial deterioration in mice receiving Epo (ΔEF=-11.18%, n=5) compared to control (ΔEF=-20.82%, n=6). FC revealed an enhanced αMHC+/cTnT+ cell pool in the Epo treated group (20.3±1.9% vs. 10.6±2.3% in control, n=8/6) 4 weeks post MI. In conclusion, we found that EpoR is expressed in a putative cardiomyogenic precursor cell pool in the adult heart. CMPs appear to proliferate in vitro and in vivo; with further enhanced proliferation under Epo administration. This may contribute to Epo mediated protection after MI and highlights the therapeutic effect of Epo upon ischemic cardiac injury. Whether these cells remain from embryogenesis or just constitute an immature myocyte population in the adult heart deriving from cardiomyocyte dedifferentiation needs to be further investigated.

scholarly journals Role of Alternative Splicing in Regulating Host Response to Viral Infection

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1720
Author(s):  
Kuo-Chieh Liao ◽  
Mariano A. Garcia-Blanco
Keyword(s):  
Innate Immunity ◽  
Alternative Splicing ◽  
Immune Responses ◽  
Viral Infection ◽  
Rna Splicing ◽  
Type I ◽  
Host Immune Responses ◽  
Transcriptional Regulatory Mechanisms ◽  
Host Virus Interactions

The importance of transcriptional regulation of host genes in innate immunity against viral infection has been widely recognized. More recently, post-transcriptional regulatory mechanisms have gained appreciation as an additional and important layer of regulation to fine-tune host immune responses. Here, we review the functional significance of alternative splicing in innate immune responses to viral infection. We describe how several central components of the Type I and III interferon pathways encode spliced isoforms to regulate IFN activation and function. Additionally, the functional roles of splicing factors and modulators in antiviral immunity are discussed. Lastly, we discuss how cell death pathways are regulated by alternative splicing as well as the potential role of this regulation on host immunity and viral infection. Altogether, these studies highlight the importance of RNA splicing in regulating host–virus interactions and suggest a role in downregulating antiviral innate immunity; this may be critical to prevent pathological inflammation.

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