Jau-Nian Chen, Ph.D. - US grants

DESCRIPTION: (Applicant's Abstract)Organ formation requires many steps to assemble cells from different origins with specific fates to form a functional unit. How to make a properly formed organ and maintain its normal function is a vital task for complex organisms. Abnormalities in genes that are involved in the patterning and/or function of organs often cause embryonic lethality, or induce severe health problems later in life, as evident in congenital heart failure, arrhythmia, leukemia and diabetes. Understanding how the organs are patterned, which genes are involved in organ patterning and/or function, and how these genes interact with each other are important issues for studying vertebrate organ formation and function. In-depth knowledge of these issues not only will facilitate further studies on the basic mechanisms of organogenesis, but also will lead to the development of early diagnosis and treatment for human diseases. To understand how vertebrate organs are formed and to identify genes and pathways that are involved in vertebrate organogenesis. We will take a combination of genetic, embryological and genomic approach using zebrafish as a model system. Zebrafish is the vertebrate model organism that can facilitate large-scale phenotype-based genetic screen. Recent advances in zebrafish genomic resources allow rapid mapping and positional cloning of the mutated genes. Furthermore, the zebrafish organ systems are the simplest forms of all vertebrates. The transparent nature of the zebrafish embryos makes it suitable for the application of the GFP technology. We will generate a transgenic line in which six organ primordia are marked by GFP expression. We will use these fish to construct a 4-dimensional fate map for these organs. This information will provide foundation for future studies of vertebrate organ formation. We will mutagenize these fish with ENU and screen for mutations affecting zebrafish organ formation and function. We will also develop reagents and technologies to facilitate a retroviral insertional gain-of-function screen. Mutations identified from this screen will provide entry points to dissect genetic and molecular pathways involved in vertebrate organogenesis.

DESCRIPTION (provided by applicant): Abnormalities in the formation and/or function of the embryonic heart often lead to embryonic lethality or severe health problems later in life. Understanding the fundamental mechanisms underlying early heart development at the cellular and molecular level is not only scientifically challenging but is also clinically relevant. We choose the zebrafish as our model organism to study early cardiac patterning because it is amenable to genetic, embryological and molecular manipulation. Furthermore, the embryonic hearts of all vertebrate species undergo similar morphogenic processes and are regulated by conserved genetic circuits. Information obtained from one model organism may apply to other vertebrates, including humans. In this project, we take three independent approaches to study the cellular and molecular mechanisms governing primitive heart tube morphogenesis. Our recent study on the zebrafish heart and mind mutation uncovered an unexpected role of Na,K-ATPase a1B1 in the elongation of the primitive heart tube. We will investigate the impact of loss of function of Na,K-ATPase a1 B1 on the cellular architecture of cardiomyocytes. We will also investigate the interaction of Na,K-ATPase a1 B1 other genes critical for the morphogenesis of the primitive heart tube in zebrafish at the cellular and genetic level (Aim 1). Furthermore we will investigate the molecular mechanism by which the Na,K-ATPase a1 B1 regulates the elongation of the primitive heart tube in the zebrafish and in cultured cells, as the first step toward understanding genetic networks involved in the primitive heart tube formation (Specific Aim 2). Finally, from an ongoing zebrafish genetic screen, we identified a new mutation affecting the patterning of the primitive heart tube. We will characterize the phenotypes of this mutant and identify the molecular lesion causing this mutant phenotype (Aim 3). The combination of cellular, molecular and genetic studies proposed in this project will provide new and in-depth insight into mechanisms of primitive heart tube morphogenesis.

DESCRIPTION (provided by applicant): A well-patterned and functioning heart is required for the growth and survival of embryos. While many genes critical for early cardiogenesis have been identified by previous genetic studies, genetic networks required for establishing and maintaining embryonic cardiac function remain to be explored. We have previously shown that calcium homeostasis has an important role in maintaining embryonic cardiac rhythmicity in zebrafish and that loss of function of NCX1h, one of the primary molecules responsible for calcium extrusion in the heart, abolishes synchronized cardiac contraction and leads to chaotic cardiac movements known as cardiac fibrillation. Consistent with the role of NCX1 in calcium homeostasis, we observed abnormal calcium transients in NCX1h null zebrafish embryonic hearts. These observations suggest that the NCX1h mutant zebrafish can serve as a tool for studying the calcium- regulatory networks important for embryonic cardiac function. From a chemical-based suppression screen on the zebrafish tremblor/NCX1h genetic model, we identified a critical component of the gene network governing embryonic cardiac function. We discovered that OK-F7, a novel small molecule suppresses cardiac fibrillation in the tremblor/NCX1 null genetic background, and our biochemical study indicated that the mitochondrial protein VDAC2 is the protein target of OK-F7. Furthermore, over expression of VDAC2 restores rhythmic cardiac contractions in embryos lacking NCX1h activity, suggesting a critical role for VDAC2 and mitochondria in calcium regulation and embryonic cardiac rhythmicity. As the first step toward understanding the role for VDAC2 in embryonic cardiac rhythmicity, we propose to evaluate the requirement of VDAC2 in cardiac development by both gain-of-function and loss-of-function approaches (Aim1). Second, to understand how the interaction of OK-F7 and VDAC modulates calcium homeostasis, we propose to evaluate whether OK-F7 treatment changes VDAC2 channel activity. We will also investigate the impact of OK-F7 on mitochondrial calcium influx. Information obtained from this line of study will provide insight into the mechanism by which OK-F7 and VDAC2 suppress cardiac fibrillation (Aim2). Finally, we will investigate whether OK-F7 treatment can restore rhythmic calcium waves in tremblor and other zebrafish embryos that have calcium-handling defects. We will also determine whether forced expression of other VDAC proteins can restore rhythmic cardiac contractions in embryos lacking NCX1h activity. The success of this line of study will further our understanding of the role for VDAC proteins in embryonic cardiac rhythmicity at the molecular level (Aim3). Our overall goal of this research program is to gain insight into gene networks important for calcium homeostasis and embryonic cardiac rhythmicity through multi-disciplinary studies. Information obtained from this research program will reveal previously unrecognized roles for VDAC and mitochondria in embryonic cardiac function.

DESCRIPTION (provided by applicant): Research in vascular biology has been responsible for remarkable changes in how we prevent, monitor and treat cardiovascular disease. The last fifty years have witnessed a transformation in patient care, increase in life expectancy and improvement in the quality of lives of those afflicted with vascular problems. It is through training of the next generation that we have made these achievements and it is through training that we will continue to make additional improvements in prevention and health care. The present application request funds to continue the interdisciplinary training of graduate students and post-doctoral fellows in vascular biology. In particular we aim at developing scientists who can: (1) speak various languages (metabolomics, pathology, molecular biology, genomics, and biomathematics), (2) integrate information and think towards (3) solving real clinical problems. To achieve these goals we have developed a multi-mentorship approach, novel didactic components and incorporated an interactive exposure to medicine into the structure of the training. UCLA houses a tremendous resource of interdisciplinary groups whose research focuses in vascular biology. The group includes 27 laboratories that currently offer training to 124 graduate students and post-doctoral fellows. It is this community that constitutes the pillars of a unique training program for the next generation of investigators in vascular biology. Being the only Vascular Biology Training grant in Los Angeles and one in four in California, we have trained 28 graduate students and post-doctoral fellows since 2002. These have produced 101 peer-reviewed publications while in the program and 9 have progressed to develop independent research groups in industry and universities across the nation. Furthermore, the activities associated with the training program have catalyzed interactions between groups intensifying collaborative activities. On the average 10 peer reviewed publications show the participation of two or more laboratories each year. Here, we request funding for 7 pre and 3 post-doctoral fellows / year, in which 2-3 new graduate students and either 1 or 2 post-doctoral fellows will enter the program each year. It is also our goal to actively seek and promote training and engagement of underrepresented minority groups (since 2002 we have a 21% minority trainee representation). It is an important objective of this program to be at the forefront of innovation in vascular biology education with strong emphasis in research integrity and ethics. In this current renewal we proposed the implementation of several new strategies to attain these goals and further improve the participation of minorities and the quality of training in vascular research. The success of this training program has engendered enthusiasm by the School of Medicine, College of Letters and Sciences and the Graduate Division at UCLA all of which have committed to provide additional institutional support towards activities developed by the VBTP.

? DESCRIPTION (provided by applicant): Understanding molecular mechanisms driving a pluripotent progenitor cell to differentiate into a specific cell type is germane to the study of organogenesis and has important significance in regenerative medicine. While the roles of key cardiac-specific transcription factors in heart development have been carefully studied, the importance for protein complexes involved in epigenetic regulation or transcription elongation in cardiac cell formation is just being revealed. In this proposal, we aim to investigate molecular mechanisms by which the RNA Polymerase II- Associated Factor 1 Complex (PAF1C) controls heart development. Biochemical and genetic studies in multiple model organisms suggest that PAF1C functions as a transcription platform required for critical signaling events. However, its roles in heart formation are not known. Our recent genetic studies discovered a novel and essential role for Rtf1, a component of the PAF1C, in the formation and proliferation of cardiomyocytes. We found that zebrafish rtf1 deficient embryos lack the entire population of cardiac progenitor cells, demonstrating that Rtf1 is absolutely required for heart development. Our preliminary data obtained from structure-function analysis and loss- and gain-of- function studies lead us to hypothesize that Rtf1 controls cardiac gene expression by PAF1C- associated epigenetic modification and PAF1C-independent transcription regulation. We will examine this hypothesis in zebrafish and evaluate how these mechanisms influence the formation of cardiac progenitor cells and the proliferation of cardiomyocytes (Aim 1). We showed that Rtf1 promotes cardiac differentiation from embryonic mesoderm in zebrafish. We will examine whether this mechanism is conserved in mammals using mouse ES cells as an in vitro differentiation model. We will also create cardiac-specific conditional knockout mice to assess the role of Rtf1 in mouse heart development (Aim 2). Finally, many developmentally regulated genes are reutilized during heart regeneration and Tbx20, an Rtf1 downstream transcription factor, is upregulated after ventricular resection. We thus propose to examine whether the Rtf1-Tbx20 pathway is involved in heart regeneration using both adult zebrafish and neonatal mouse heart regeneration models (Aim 3). Successful completion of the proposed projects will provide new mechanistic insights into the regulation of cardiac progenitor cell formation and cardiomyocyte proliferation during development and in regeneration.

DESCRIPTION (provided by applicant): Tightly regulated Ca2+ homeostasis is essential for establishment of regular cardiac rhythm. Even a slight increase in cytosolic Ca2+ concentration can trigger fibrillation-like contraction and cardiac standstill in vivo and in vitro. Through zebrafish phenotype-based suppressor screening, we have recently identified a small molecule that restores fibrillation-like contraction of the heart. Biochemical analyses suggest that this compound binds to and potentiates a mitochondrial Ca2+ transporter, VDAC2. These data suggest a novel function of mitochondria as a fine-tuner of Ca2+ handling in the regulation of cardiac rhythmicity. Encouraged by the success of zebrafish screening, we propose to establish high- throughput screening system for forward chemogenetics approach to identify novel genetic pathways critical for the regulation of human cardiac Ca2+ homeostasis and rhythmicity using newly developed human embryonic stem cell- derived cardiomyocytes (hESC-CMs) differentiation protocol. Bioengineering approach, zebrafish in vivo heart and isolated adult mouse cardiomyocytes will be used as models to validate the hit compounds. We specifically propose to; 1. Assay development: We will optimize modalities to induce fibrillation, timing of the compound treatment, and readout for HTS. Efsevin will serve as a positive control. 2. Pilot screening: Pilot screening will be conducted with hESC-CM. 3. Hit validation: Once hits are identified, they will be tested on three model system. The long-term goal of this project is to establish a novel mechanism and treatment of atrial fibrillation, which affects 1% of total population older than 60 and 5% of 75 and older, and poses a high risk to stroke and other systemic emboli.

Project Summary Understanding the molecular mechanisms that drive a pluripotent progenitor cell to differentiate into a specific cell type is germane to Developmental Biology and has important significance in regenerative medicine. Past studies have illustrated that dynamic cardiac transcription programs (CTPs) guide cardiogenesis during development and support cardiac structure and function in homeostasis. While the contributions of key cardiac-specific transcription factors and cardiogenic signaling pathways have been clearly delineated, molecular mechanisms that coordinate the expression of cardiogenic genes to drive myocardial cell specification and direct the maturation of cardiomyocytes remain elusive. Our recent studies using both zebrafish and mouse models indicate that the multi-functional protein Rtf1 is a transcription regulator that orchestrates cardiac gene programs responsible for myocardial specification and differentiation. Knockdown of rtf1 in mouse embryonic stem cells inhibits the cardiac gene program and prevents cardiac differentiation. In vivo, Rtf1 deficient zebrafish and mouse embryos lack myocardial progenitor cells and cannot develop a heart. We also found that Rtf1 deficiency in committed cardiomyocytes impairs cardiac gene program. Collectively, these findings demonstrate a need for Rtf1 activity in myocardial cells at multiple developmental stages. Insights into how Rtf1 regulates dynamic CTPs come from our structure-function analysis showing differential requirements for Rtf1's Plus3 and HMD domains in two temporally distinct cardiogenic events; the Plus3 domain is required for the activation of the CTP and myocardial specification whereas the HMD domain influences histone modifications and directs heart tube morphogenesis. These exciting findings lead to our overarching hypothesis that Rtf1 controls dynamic transcriptional programs in myocardial cells during development by CTP activation and epigenetic modulation. We will employ a set of new transgenic zebrafish lines to define the transcriptional networks of Rtf1 in myocardial progenitors and committed cardiomyocytes. We will investigate how Rtf1's Plus3 domain coordinates the expression of cardiogenic genes to drive the multi-potent mesodermal cells to a myocardial fate (Aim1). We will also interrogate the hypothesis that the Rtf1's HMD domain controls heart tube morphogenesis by influencing the propagation and/or maturation of newly differentiated cardiomyocytes via epigenetic modulation (Aim 2). Successful completion of the proposed projects will provide new mechanistic insights into the transcriptional regulation of myocardial specification and differentiation and will pave the way for the development of novel therapeutic strategies to treat heart diseases.

Project Summary Heart failure is a major cause of death in the US, contributing significantly to the burden of the healthcare system every year. Despite the heterogeneity of the causes of heart failure, the heart undergoes gene expression changes during failure resulting in structural and functional defects. Our long-term goal is to understand the transcriptional regulatory mechanisms that sustain the structure and function of the heart in homeostasis and that can induce cardiac protective effects or promote cardiac repair upon injury. In this application, we will use the transcription regulator Rtf1 as a point of entry to address this critical question in cardiac biology. Critical roles for transcription elongation in cellular RNA biogenesis have gained increasing attention in recent years, but how they contribute to the maintenance of cardiac homeostasis and how modulating transcription elongation might promote cardiac repair in damaged hearts remain elusive. Using both zebrafish and mouse genetics, we have previously shown that Rtf1 activity is essential for myocardial development. Rtf1 depletion destabilizes promoter-proximal pausing of RNA Pol II, blocks activation of the myocardial gene program and prevents myocardial progenitor cell formation resulting in a heartless embryo. In preliminary data leading to this proposal, we have found that Rtf1 plays important roles in normal and stressed adult hearts. Ablation of Rtf1 activity in adult cardiomyocytes leads to rapid heart failure with dysregulated cardiac gene expression and a loss of contractility. In stressed hearts, we observed elevated Rtf1 expression within cardiomyocytes after injury, suggesting a role for Rtf1 in the cardiac stress response. Overexpression of Rtf1 also promotes cardiomyocyte proliferation in a zebrafish ventricular resection model. The dysregulated cardiac gene expression and reduction of epigenetic marks of active transcription in Rtf1-deficient failing hearts suggest that Rtf1 functions as a key transcriptional regulator for cardiomyocytes. These findings lead to our central hypothesis that Rtf1 modulates transcriptional pausing and co-transcriptional histone modification to facilitate efficient mRNA synthesis in cardiomyocytes and thereby sustains cardiac structure and function in normal and stressed hearts. We have delineated three Aims to interrogate this hypothesis. Specifically, we will investigate Rtf1-dependent gene expression in cardiomyocytes and decipher the progressive molecular, cellular, physiological and metabolomic changes occurring during heart failure (Aim 1). We will use an array of molecular approaches to uncover the molecular basis by which Rtf1 impacts the transcriptome in cardiomyocytes (Aim 2). We will also investigate how Rtf1 responds to cardiac damage and the potential of manipulating Rtf1 activity to promote cardiac repair (Aim 3). Accomplishing these aims will not only provide significant new insights into the regulatory network of cardiac gene expression but also a possible therapeutic target to promote cardiac health and post-injury repair.