Citations

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

ABSTRACT Modifier genes contribute significantly to our understanding of pathophysiology in human diseases; however, effective approaches to identify modifier genes are still lacking. Here, we aim to develop a rapid F0-based genetic assay in adult zebrafish using the bag3 gene knockout (bag3e2/e2) cardiomyopathy model as a paradigm. First, by utilizing a classic genetic breeding approach, we identified dnajb6b as a deleterious modifier gene for bag3 cardiomyopathy. Next, we established an F0-based genetic assay in adult zebrafish through injection of predicted microhomology-mediated end joining (MMEJ)-inducing single guide RNA/Cas9 protein complex. We showed that effective gene knockdown is maintained in F0 adult fish, enabling recapitulation of both salutary modifying effects of the mtor haploinsufficiency and deleterious modifying effects of the dnajb6b gene on bag3 cardiomyopathy. We finally deployed the F0-based genetic assay to screen differentially expressed genes in the bag3 cardiomyopathy model. As a result, myh9b was identified as a novel modifier gene for bag3 cardiomyopathy. Together, these data prove the feasibility of an F0 adult zebrafish-based genetic assay that can be effectively used to discover modifier genes for inherited cardiomyopathy.

To sense and to respond to mechanical stimuli is a fundamental process of cardiomyocytes during both heart development and diseases. However, how mechanical stimuli control cardiomyocyte size remains elusive. Here, we report a novel inhibitory mechanotransductive response suggested by loss-of-functions studies of α-Actinin2 (Actn2), a predominant sarcomeric Z-disc protein. During zebrafish cardiogenesis, depletion of Actn2 results in a defective Z-disc, while other sarcomeric substructures remain normal. As a consequence, ventricle chamber size is severely reduced. Interestingly, this chamber size reduction can be rescued by the cessation of heart contraction, suggesting the involvement of an inhibitory mechanotransductive reponse. At the molecular level, both tcapa and tcapb, two zebrafish tcap homologues that are transcriptionally responsive to stretch, are transcriptionally activated in actn2 knockdown embryos while activation of these gene results in smaller cardiomyocytes. Depletion of either gene rescues the reduced cardiomyocyte size in actn2 knockdown embryos. Consistent to embryonic studies, tcaps are transcriptionally activated during pathogenesis of anemia-induced cardiomyopathy, while over expression of tcapa via transgenics exerts a cardioprotective effect. Together, we conclude that transcriptional regulation of tcaps conveys a novel inhibitory mechanotransductive response that modulates cardiomyocyte size during both zebrafish cardiogenesis and adult cardiac remodeling.

Myosin heavy chain 7 (MYH7) is a major causative gene for hypertrophic cardiomyopathy, but the affected signaling pathways and therapeutics remain elusive. In this research, we identified ventricle myosin heavy chain like ( vmhcl ) as a zebrafish homolog of human MYH7 , and we generated vmhcl frameshift mutants. We noted vmhcl -based embryonic cardiac dysfunction (VEC) in the vmhcl homozygous mutants and vmhcl -based adult cardiomyopathy (VAC) phenotypes in the vmhcl heterozygous mutants. Using the VEC model, we assessed 7 known cardiomyopathy signaling pathways pharmacologically and 11 candidate genes genetically via CRISPR/Cas9 genome editing technology based on microhomology-mediated end joining (MMEJ). Both studies converged on therapeutic benefits of mTOR or mitogen-activated protein kinase (MAPK) inhibition of VEC. While mTOR inhibition rescued the enlarged nuclear size of cardiomyocytes, MAPK inhibition restored the prolonged cell shape in the VEC model. The therapeutic effects of mTOR and MAPK inhibition were later validated in the VAC model. Together, vmhcl/myh7 loss of function is sufficient to induce cardiomyopathy in zebrafish. The VEC and VAC models in zebrafish are amenable to both efficient genetic and chemical genetic tools, offering a rapid in vivo platform for discovering candidate signaling pathways of MYH7 cardiomyopathy.

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.