Xiaolei Xu, Ph.D. - US grants

Dilated cardiomyopathy (DCM) is a disorder with genetic heterogeneity and variable phenotypes. TITIN truncating variants (TTNtvs)- nonsense, frameshift, and essential splice site, has been found to be the most common genetic factor for DCM. However, TTNtvs are also found in reference populations and it remains unclear why pathogenic TTNtvs were mainly found in exons encoding A-band domains, while TTNtvs in other exons such as those affecting the N-terminal Z-disc domain are likely benign. Moreover, patients with the same TTN mutation can exhibit highly variable disease phenotypes, presumably because of different genetic background in personal genomes. To address these two bottlenecks on TTN-based DCM, we leveraged efficient zebrafish genetics. To fill the first knowledge gap on allelic heterogeneity, we utilized genome-editing technology and generated more than 10 ttntvs affecting either Z-disc or A-band exons in zebrafish. To fill the second knowledge gap on variable phenotypes, we established a novel mutagenesis screening-based strategy and successfully identified 4 genetic modifiers for DOX-induced cardiomyopathy. We premise that some of these modifiers and related therapies for DOX-induced cardiomyopathy might also be applicable to TTN-based DCM. We propose to conduct comprehensive genetic studies of these ttntvs and candidate genetic modifiers to elucidate mechanisms and to seek therapeutic strategies for TTNtv-based DCM. In our specific Aim 1, we aim to define phenotypic traits for ttn-based DCM, and to discern toxic peptide, exon usage and cronos hypotheses for allelic heterogeneity of ttn-based DCM. In our specific Aim 2, we will elucidate novel functions of the short ttn-novex3 isoform, and test the hypothesis that N-terminal ttntvs affecting the ttn-novex3 isoform are cardiomyopathy modifiers. In our specific Aim 3, we will determine whether the 4 modifying mutants for DOX-induced cardiomyopathy also exert similar modifying effects on ttn-based DCM, and which gene can be a therapeutic target. Completion of the proposal will establish zebrafish as an efficient vertebrate model that facilitates the prognosis, diagnosis and therapeutic development for cardiomyopathies including TTN-based DCM.

DESCRIPTION (provided by applicant): Cardiomyopathy and related heart failure affect millions of Americans. Therefore, pathway-based therapies are highly desirable. The goal of this proposal is to leverage unique genetic tools in zebrafish to elucidate cardioprotective functions of target of rapamycin (TOR) signaling and develop novel therapeutic compounds via modifier screens. There are two obstacles prohibiting modifier screens from being conducted in adult zebrafish: the lack of adult assays to analyze cardiomyopathy-like phenotypes, and the difficulty to track adult mutant fish. To address the former challenge, we established and characterized first adult fish models of cardiomyopathy, including a model induced by Doxorubicin (DOX). To address the latter challenge, we adapted a transposon-based insertional mutagenesis strategy that facilitates the identification of all mutants by a RFP tag. We have conducted both phenotype-based insertional mutagenesis screens and chemical screens, and identified gene modifiers and compound modifiers of TOR signaling that sequentially affect DOX-induced cardiomyopathy. Preliminary studies using these genetic resources suggested that activated autophagy conveys the cardioprotective function of TOR inhibition. In this proposal, we will continue to leverage zebrafish genetics to test our central hypothesis that the cardioprotective functions of TOR inhibition are conferred by the activated autophagy, which can be harnessed by modifier screens in adult zebrafish to identify novel genes and therapeutics for treating cardiomyopathy. In Specific Aim 1, we will determine the conservation of adult zebrafish as a model organism for cardiomyopathy. In Specific Aim 2, we will discern functions of autophagy and pS6K subpathways in cardiomyopathy via phenotyping two modifier mutants that differentially affect TOR signaling. In Specific Aim 3, we plan to discover novel compound modifiers of TOR signaling that could be of greater therapeutic value for cardiomyopathy. At the end of the project, we expect to validate a cardioprotective function of TOR inhibition, to define autophagy as the major downstream signaling branch that confers this cardioprotective function, and to identify compounds that specifically interfere with the TOR-autophagy subpathway that might be of better therapeutic value than rapamycin. Our studies will establish adult zebrafish as a conservative animal model to identify novel modifiers of cardiomyopathy, as well as a complete in vivo model organism to facilitate drug discovery. PUBLIC HEALTH RELEVANCE: This proposal leverages the unique tools in zebrafish to seek TOR signaling-based therapies for cardiomyopathy and heart failure. We aim to use this unique animal model to identify gene modifiers of cardiomyopathy that will elucidate cardioprotective functions of TOR inhibition, and compounds in this pathway with therapeutic value for cardiomyopathy.

? DESCRIPTION (provided by applicant): Cardiomyopathy is a group of disorder with genetic heterogeneity and variable expression. To develop effective therapy, it is vital to identify both causative genes and genetic modifiers. This proposal aims to establish a novel adult zebrafish-based screening approach that allows systematically identification of genetic modifiers of cardiomyopathy. Having generated a Doxorubicin-induced cardiomyopathy model and developed an efficient method to generate cardiac mutants, we tested a forward genetic screening approach for this purpose. A pilot screen of >500 gene-breaking transposon (GBT) mutants identified four mutant lines that modified DOX-induced cardiomyopathy. Three affected genes, including sorbin and SH3 domain containing 2b (sorbs2b), anoctamin 5a (ano5a) and retinoid X receptor, alpha a (rxraa), have been previously linked to cardiomyopathy; while the fourth gene, Dnaj (Hsp40) homolog, subfamily B, member b (dnajb6b), is a new cardiac gene, demonstrating the capacity of our screening method to discover both known and new cardiomyopathy genes. Before we scale up the screen, we consider it as pivotal to determine whether and how these zebrafish mutants facilitate our understanding of human cardiomyopathy. By focusing on dnajb6b, the new cardiac gene, and sorbs2b, the gene with newly defined non-cardiomyocyte expression, we propose to test the central hypothesis of this proposal predicting that novel cardiomyopathy modifying genes can be identified via a mutagenesis screen approach, which can be further studied to profile their modifying effects on different genetic types of cardiomyopathy and to develop targeted therapy. The proposal is divided into the following three specific aims. In Specific Aim 1, we will determine the modifying effects of dnajb6b and sorbs2b on different types of heritable cardiomyopathy. For this purpose, we have generated three heritable cardiomyopathy models including bag3 KO, mBAG3 OE and lmna KO. In Specific Aim 2, we will elucidate the modifying effects of dnajb6b and sorbs2b via mechanistic studies and to seek therapeutics. We will examine whether UPR inhibition ameliorates the modifying effects of dnajb6b and/or sorbs2b. In specific Aim 3, we will validate the modifying effects of human DNAJB6 and SORBS2 variants and the targeted therapeutics in both fish and mouse models. Completion of these aims are expected to demonstrate the feasibility of zebrafish to model different types of cardiomyopathy, to identify modifying genes, t elucidate the modifying effects of these genes on different type of cardiomyopathies, and to discover targeted therapeutics. If successful, our strategy shall greatly advance prognostic test development, risk stratification, and personalized therapy for cardiomyopathy.

ABSTRACT Heart failure remains the leading cause of morbidity and mortality in the US, afflicting nearly 5 million people. Recently, adult Zebrafish (Danio rerio) have been utilized to model different types of heart failure, and to search for genetic modifiers via mutagenesis screening. However, the small size of the zebrafish heart hinders precise electrical and mechanical assessments following genetic modifications. During the previous funding cycle, we integrated a flexible micro-electrode array with high-frequency ultrasonic transducers to demonstrate that early regenerating cardiomyocytes lack the electrical phenotypes needed to integrate into injured hearts. We further showed that the pressure gradient across the atrioventricular valve is greater than that across the ventriculobulbar valve following ventricular cryo-injury. However, the initial rise and subsequent normalization of ventricular passive (E) and active (A) filling waves (E/A ratios) indicate recovery of diastolic function. In the next funding cycle, we will combine our micro-sensing capacity with novel genetic models of cardiomyopathy to elucidate electromechanical coupling following chemotherapy-induced injury and genetic models of cardiomyopathy. Our multi-disciplinary team established an adult zebrafish model of doxorubicin (Dox)-induced cardiomyopathy (CM) as a conserved vertebrate model to investigate myocardial injury and regeneration in response to the breast cancer chemotherapy targeting ErbB2 (HER2)/NEU. Our team has further developed three murine genetic models of CM; namely, bag3 knockout (KO), mBAG3 overexpression (OE), and Imna KO. We have further developed a forward-genetic approach to identify genetic modifiers of Dox-induced CM. A pilot screen of >500 gene-breaking transposon (GBT) mutants has identified four GBT lines, of which GBT419/rxraa (retinoid X receptor alpha a) resembles mTOR to improve zebrafish survival following Dox-induced CM. Our goal is to integrate micro-sensors with advanced imaging to study electrical conduction and mechanical function of the injured myocardium in response to Dox-induced and 3 genetic models of CM. Our hypothesis is that genetic modifiers such as GBT419/rxraa promotes electromechanical coupling in Dox-induced and genetic models of CM to restore contractile function. To test our hypothesis, we have three aims: In Aim 1, we will determine electrical conduction in our Dox-induced and genetic models. In Aim 2, we will demonstrate mechanical function in our Dox-induced and genetic models. In Aim 3, we will assess electromechanical coupling following treatments with CM modifying genes. Overall, these aims will provide new insights into electromechanical coupling in cardiomyopathy using forward-genetics to discover therapeutic modifiers capable of restoring heart function.

Project Summary Cardiomyopathy is a disorder with high heterogeneity: >100 genes have been linked to different types of cardiomyopathy, such as hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM); patients with the same mutation can manifest highly variable disease onset and severity, presumably because of different genetic and environmental factors. However, the identity of genetic modifiers remains largely unknown. To address the need, our team has been leading the development of adult zebrafish as a vertebrate model for cardiomyopathy. During the previous 2 funding cycles, we developed a novel forward genetic screening strategy for discovering modifier genes for doxorubicin (DOX)?induced cardiomyopathy (DIC). Here, we plan to extend this approach to inherited cardiomyopathies. We have generated substantial preliminary data to prove the feasibility of the proposal, including the identification of 5 original DIC modifier genes and 4 additional candidate DIC modifier genes, the generation of a zebrafish model of BAG3 cardiomyopathy, the identification of mtor as a therapeutic modifier gene, and the establishment of an embryonic fish-adult fish- mouse drug assessment platform. Together, these data prompted us to test the central hypothesis of this proposal, which predicts that modifier genes for an inherited cardiomyopathy model can be identified via a forward genetic strategy in zebrafish, from which therapeutic target genes and related compounds can be rapidly discovered by efficient zebrafish genetics. The proposal is organized into 2 specific aims. In Specific Aim 1, we will test the hypothesis that a forward genetics-based approach is extendable to bag3 cardiomyopathy to identify therapeutic modifiers. We will determine phenotypic progression and variation in zebrafish bag3 KO, assess modifying effects of 9 DIC modifiers on bag3 cardiomyopathies, and then identify therapeutic modifiers for bag3 cardiomyopathy. We will elucidate underlying mechanism by transcriptome analysis. In Specific Aim 2, we will elucidate underlying mechanisms of the therapeutic effects of mTOR inhibition, prove autophagy-based therapy for bag3 cardiomyopathy, and repurpose FDA-approved autophagy- activating drugs to treat bag3 cardiomyopathy. It is anticipated that the novel strategy developed by this proposal will significantly advance prognostic test development, risk stratification, and personalized therapy for cardiomyopathies.