Xu, Xiaolei

Rationale: Anthracycline-induced cardiotoxicity (AIC) is a major side effect that limits the use of anthracyclines as effective chemotherapeutics. No mechanism-based therapy is available when cardiac function deteriorates. Objective: We aim to elucidate the dynamic autophagic defects in AIC and to identify a mechanism-based therapy via both genetic and pharmacological studies. Method and Results: Through phenotyping an adult AIC zebrafish model, we detected a biphasic response in autophagy: activation in the early stage and suppression in the later phase that is characterized by a decline in cardiac function. We conducted conditional genetic studies with atg7 , which encodes a rate-limiting autophagy core protein, and found that atg7 overexpression leads to therapeutic effects in the late phase but deleterious effects in the early phase of adult AIC. We then assessed the therapeutic effects of 37 Food and Drug Administration–approved autophagy activators using an embryonic AIC zebrafish model and identified spironolactone, pravastatin, and minoxidil as top-ranking drugs. We demonstrated the therapeutic efficacy of these Food and Drug Administration–approved autophagy activators in the adult AIC model and confirmed that these drugs exert therapeutic effects in the late phase but not in the early AIC phase. Finally, we demonstrated that the time-dependent therapeutic effects are conserved in a mouse AIC model and that spironolactone and rapamycin activated autophagy in an Atg7-dependent fashion. Conclusions: Our findings suggest that atg7-based autophagy activation is an effective therapeutic avenue to reversing the decline in cardiac function in AIC, highlighting the time-dependent nature of autophagy-based therapy.

Background: TITIN (TTN) has more than 300 exons and encodes a gigantic protein that is crucial for heart and muscle development. Mutations in TTN caused a variety of human diseases including cardiomyopathy and muscular dystrophy. Recently, dilated cardiomyopathy-associated mutations on TTN have been found more frequently in exons encoding A-band domains but less in exons encoding the N-terminal Z-disc domains, suggesting that mutations in different exons of TTN cause distinct consequences. To elucidate the underlying mechanisms, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to introduce truncating mutations in different exons of ttn, and then study their effects on heart and somites. Results: We generated truncational mutations in different exons of zebrafish titins encoding Z-disc, N2B, Novex-3, and A domains, respectively. Because zebrafish contains two titin homologues, ttna and ttnb, we introduced mutations in both genes at the corresponding loci. While both Z-disc and A band mutations on ttna disrupted sarcomere assembly in heart and somites, Z-disc or A band mutations on ttnb only affect somites without affecting the heart. Interestingly, a Z-disc mutation on ttna resulted in milder phenotypes than an A-band mutation, while a Z-disc mutation on ttnb generated severer phenotypes than an A-band mutation. No phenotype was observed in the homozygous fish in either ttna-novex-3 or ttnb-N2B mutant fish. Conclusions: A spectrum of truncational mutations in ttna and ttnb has been generated in zebrafish using the TALEN technology. Mutations in different exons result in different phenotypes. Detailed characterization of these mutants and double mutants will be presented, which shall elicit distinct contribution of alternative splicing and exon skipping as two candidate mechanisms during pathogenesis of Titinopathies.

Zebrafish ( Danio rerio ) is an efficient vertebrate model of human cardiomyopathy which is amenable to the medium throughput screening approaches opening opportunities to search new genetic modifiers via mutagenesis screening and assessing compound-based therapies at larger scale. The advent of genome editing technology enables the generation of a panel of genetic models of cardiomyopathy with mutations in leading cardiomyopathy genes. However, one of the major bottlenecks for adult zebrafish as a cardiomyopathy model is the lack of appropriate cardiac functional assays. Due to small heart size, in vivo methods such as those based on echocardiography, are limited by their insufficient resolution. Here, we report the development of an ex vivo approach aimed to facilitate phenotyping in adult zebrafish. We show that our method is able to quantify parameters of pump function of the heart, including end-diastolic/systolic length/volumes, ejection and shortening fractions, and velocities of contraction/relaxation. We defined the basic parameters of these indices using different wild-type strains, age, and sex, and then demonstrated that our method can be useful in definition of progression of pathogenesis of both acquired (doxorubicin-injected) and inherited cardiomyopathy models. We conclude that our novel approach shall facilitate cardiac phenotyping in adult zebrafish models of heart diseases.

We recently developed a forward mutagenesis screening strategy in adult zebrafish to screen gene-breaking transposon (GBT) mutants, and identified four genetic modifiers for doxorubicin-induced cardiomyopathy. However, it remains unclear whether these genetic modifiers identified from an acquired cardiomyopathy model exert similar modifying effects on inherited cardiomyopathy models. To address this question, we generated BCL2-associated athanogene 3 (bag3) gene knockout in adult zebrafish, using the transcription activator-like effector nucleases (TALEN) genome editing technology. In the bag3-/- fish, progressive cardiac phenotypes reminiscent of human cardiomyopathy such as fetal gene activation, myofibril loss and cardiac dysfunction were detected. At the single myofibril level, reduced active contractility was observed, supporting the dilated cardiomyopathy (DCM)-like phenotype. Based on the ejection fraction index quantified using a newly developed ex vivo assay, different pathogenesis stages including pre-DCM, early-DCM and late DCM were defined. Next, we assessed the potential modifying effects of the four DIC-modifying mutants on bag3 -based cardiomyopathy model. Different from the other three GBT mutants, GBT0411+/- , which tags the long isoform of dnajb6 b gene, dramatically accelerated the cardiac dysfunction and fish mortality in the bag3-/- fish, suggesting dnajb6b as a sensitive genetic modifier for bag3-based cardiomyopathy. Mechanistically, we showed that Bag3 physically interacts with Dnajb6, and we hypothesize that impaired autophagy and/or endoplasmic reticulum stress convey the synergistic cardiac dysfunction and fish mortality phenotypes in the GBT0411+/-;bag3-/- double mutants. In summary, this study demonstrates that an inherited cardiomyopathy model can be established in an adult zebrafish, which can be utilized to search genetic modifiers. Future studies employing this simple vertebrate model amenable to forward mutagenesis screening promise systematic identification of genetic modifiers for different types of cardiomyopathies, a foundation for individualized medicine.

Background: Rapid advance of genome technologies accelerate the discovery of genetic basis of cardiomyopathy and heart failure and also enable system biology studies to pinpoint underlying mechanism. However, the limited throughput of mammalian models restricted the number of genes that can be studied in a particular lab. Adult zebrafish has been recently pursued as a new model with higher throughput. However, as a non-mammalian model, its conservation is not tested. Objective: To assess the conservation of zebrafish for genetic studies of human dilated cardiomyopathy (DCM) via transcriptome analysis of 51 known DCM-causative genes. Methods and Results: By conducting RNA-sequencing (RNA-seq) analysis of larva and adult zebrafish, we identified genes with high expression level in the heart and fetal gene program using differential expression between embryonic and adult stages. We then searched zebrafish orthologues for 51 reported human DCM-causative or associated genes and identified zebrafish orthologues for 49 of them. While 30 genes have a single orthologue, 14 genes have two homologues and the remaining 5 genes have more than three. We then applied the transcriptome data to prioritize these homologues for the 19 DCM causative genes with more than one homologue. Based on the cardiac abundance and cardiac enrichment hypothesis, we are able to recommend a single zebrafish homologue of high priority for 12 out of 19 DCM genes, 2 zebrafish homologues of high priority for ACTC1. Interestingly, our expressional data suggested zebrafish othologues for human MYH6 and MYH7 , respectively. Similar to that in mammals, these two zebrafish othologues are oppositely expressed during zebrafish embryonic and adult stage. Conclusions: Orthologues for the majority of DCM causative genes can be found in Zebrafish, supporting its usage as a conserved vertebrate model for studying DCM. The definition of cardiac transcriptome in zebrafish will facilitate the future system biology studies. This vertebrate model with higher throughput can be further leveraged to validate the novel variants identified from human patients, to understand underlying signaling pathways and to develop novel therapeutics.

The human genome encodes about 20,000 genes and only a fraction of them are expressed in the heart. We hypothesized that annotation of this category of genes may facilitate our understanding of cardiac development and function. Recently, we reported a pilot insertional mutagenesis screening in zebrafish to annotate cardiac gene functions using a gene-break transposon (GBT). It demonstrated the feasibility of generation of a Zebrafish Insertional Cardiac (ZIC) mutant collection by screening gene expression in the embryonic heart. Here, we report an alternative strategy to identify cardiac mutants based on adult heart expression, and explore the application of expanded ZIC lines for understanding cardiac biology. From 203 GBT lines, we identified 34 lines with detectable expression in the dissected adult hearts. Various expression levels of the tagged genes were detected in embryonic hearts. Based on their tissue-specific expression, the candidate ZIC lines can be grouped into myocardium lines, endocardium lines, or both. Based on their subcellular expression, myocardium ZIC lines can be further categorized into sarcomeric lines, mitochondrial lines, and/or nucleic lines. Phenotypic analysis is being conducted to identify recessive mutants including embryonic lethal lines. We will present the current status of our ZIC collection, and discuss its application as a genomic resource platform for elucidating cardiac biology. Our data suggest that ~15% genes in the whole genome might have detectable cardiac expression. The sensitivity of screening for cardiac genes at adult stage is higher than that at embryonic stage (34/203 versus 18/322). We plan to expand the ZIC collection by screening 1,000 GBT lines that are being generated at Mayo Clinic. It is expected that this living mutant collection of cardiac genes will open doors to systematic annotation of novel cardiac genes and forward cardiac mutagenesis screening.

Background: Titin, the largest protein found in nature, has been found to be crucial for heart and muscle development and mutations on titin caused human diseases. Especially, truncated mutations on TITIN were found to be a major causative gene (more than 25%) for familiar dilated cardiomyopathy (DCM) but the mechanism is lacking. Due to the gigantic size and number of exons, genetic study of titin has been challenging. To facilitate genetic analysis of titin biology, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to edit the titin gene directly. Results: We generated TALEN pairs targeting different domains of zebrafish titin, including Z-disc domain, N2B domain, and A domains. Because zebrafish contain two titin homologues, ttna and ttnb , TALEN pairs targeting the corresponding loci for both homologues were made. The mutations are efficiently introduced on the titin using TALENs. In addition to these truncated mutants, TALEN technology can also create internal deletions. The titin mutant fish were identified and further phenotypic analysis has been carried out. Conclusions: Combined with TALEN technology, zebrafish can provide a convenient vertebrate model for genetic studies of titin biology. Moreover, this gene-editing technology will facilitate the use of the zebrafish model as a powerful research tool for modeling human diseases and investigating their pathogenesis.

Rationale: The generation of animal models for human cardiomyopathy creates opportunities to uncover mechanisms of diseases’ pathogenesis and to identify genes that modify the progression of pathogenesis. In contrast to candidate gene-based approaches, the phenotype-based forward genetic screening approach is less effectively employed in a vertebrate, mainly due to the prohibitively enormous colony management efforts. Objective: To identify genetic modifiers of cardiomyopathy through developing an unbiased forward genetic approach in the vertebrate Danio rerio. Methods and Results: Using an expression-based insertional mutagenesis screening strategy, we identified 49 Zebrafish Insertional Cardiac (ZIC) mutants from a collection of ~300 gene break-transposon (GBT) lines. By applying doxorubicin (DOX) stress to these heterozygous ZIC mutants, we initially identified four GBT lines that either enhanced or suppressed DOX-induced cardiomyopathy phenotypes. Here, we focus on the GBT0411 line which significantly enhances DOX-induced cardiomyopathy phenotypes. We cloned the gene and identified a single transposon insertion that disrupts the long nuclei isoform of dnajb6b in this GBT0411 mutant. Consistent with its cardiomyopathy modifier function, we detected a predominant expression of dnajb6b in the heart. While mutations affecting the cytoplasmic functions of DNAJB6 were recently reported to cause limb-girdle muscular dystrophy type 1D, our study suggested that the impaired balance between Dnajb6b long (nuclei) and short (cytoplasmic) isoform accounted for the cardiomyopathy modifier effect. Conclusions: Our results demonstrated the feasibility of identifying genetic modifiers of cardiomyopathy using a forward genetic approach in a vertebrate. Specifically, our data implicate DNAJB6 as a new genetic risk factor for DOX-induced cardiomyopathy. Without any presumption, this forward genetic strategy opens the door to systematically identify genetic factors that modify the progression of cardiomyopathy, which will facilitate the practice of personalized medicine and the development of novel therapies.

Background: Cardiomyopathy-like phenotype has been recently reported in adult zebrafish stressed by either anemia or doxorubicin. However, it still remains unclear whether gene-based manipulation in this emerging animal model can faithfully recapitulate cardiomyopathy phenotypes in mammals. Here, we aim to address this question by generating the first transgenic model for cardiomyopathy in zebrafish. Results: We generated a panel of three transgenic fish lines, including Tg(cmlc2:Tnnt2) that overexpress wild type Tnnt2 in the whole heart, Tg(cmlc2:Tnnt2c) that drive a C-terminal amino acid truncated Tnnt2 (Tnnt2C) in the whole heart, and Tg(vmhc:Tnnt2c) that drives Tnnt2c expression only in the ventricle. Upon overexpression of the Tnnt2C, but not the full-length control, in the heart caused reduced ventricular size but enlarged atrium. A same morphological phenotype was noted in Tg(vmhc:Tnnt2c). At the cellular level, we found significantly reduced size of cardiomyocyte and increased cell proliferation. The morphological remodeling is accompanied by hallmarks of cardiomyopathy including reduced cardiac function and myofibril disarray. Conclusion: Transgenic overexpression of Tnnt2C in zebrafish faithfully recapitulates unique cardiomyopathy phenotypes noted in both human patients harboring the similar mutation and mice transgenic models, underscoring zebrafish as a conserved vertebrate model for cardiomyopathy. Our study further suggested that the dysfunction of Tnnt2C in ventricle is primarily responsible for cardiac phenotypes, while the enlarged atrium is a consequence of a defective ventricle.

Background: Cardiomyopathy and heart failure affect millions of people worldwide. Because genetic modifiers contribute in large part to the highly variable phenotypic expression of cardiomyopathy in patients even with identical disease-causing mutations, the identification of modifier genes for this disease will greatly improve risk stratification, prognostic test development, and personalized therapy. However, only a rather limited number of modifier genes for cardiomyopathy have been identified sporadically. Objective: To identify genetic modifiers for cardiomyopathy using a novel insertional mutagenesis screening approach in adult zebrafish. Methods and Results: We screened 476 gene break-transposon (GBT) lines and isolated 44 zebrafish insertional cardiac (ZIC) mutants. Employing doxorubicin (DOX) stress to these ZIC mutants, we identified four candidate GBT lines that modified the progression of DOX-induced cardiomyopathy. Here, we report the detailed study of the GBT0411 mutant that exacerbated DOX-induced cardiomyopathy. GBT0411 mutant was tagged to the dnajb6b gene. Mutations in the short (sarcomeric) isoform of its human homologue gene DNAJB6 was recently reported to cause limb-girdle muscular dystrophy type 1D. Interestingly, our data showed that long (nuclei) isoform (dnajb6b[L]) was the major isoform expressed in the heart, and loss-of-function of which deteriorated the progression of DOX-induced cardiomyopathy. We further found that a cardiomyocyte-specific dnajb6b(L) transgene reverted the deleterious modifying effect of GBT0411 mutant, and exerted a cardioprotective function on chronic anemia induced cardiomyopathy. Mechanistically, Dnajb6b(L) could partially localize to endoplasmic reticulum (ER) upon ER stress, and function as an ER stress suppressor. Indeed, inhibition of ER stress by using a chemical chaperon mimics the cardioprotective effect of dnajb6b(L) transgene. Conclusions: By conducting an unbiased mutagenesis screening in adult zebrafish, we identified dnajb6b as a novel genetic modifier for cardiomyopathy. A cardioprotective function was identified by overexpressing its long isoform in cardiomyocytes, which might be conveyed by inhibition of ER stress response.