Citations

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.

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.

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.

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.

Titin-truncating variants (TTNtvs) are the major cause of dilated cardiomyopathy (DCM); however, allelic heterogeneity (TTNtvs in different exons) results in variable phenotypes, and remains a major hurdle for disease diagnosis and therapy. Here, we generated a panel of ttn mutants in zebrafish. Four single deletion mutants in ttn.2 or ttn.1 resulted in four phenotypes and three double ttn.2/ttn.1 mutants exhibited more severe phenotypes in somites. Protein analysis identified ttnxu071 as a near-null mutant and the other six mutants as hypomorphic alleles. Studies of ttnxu071 uncovered a function of titin in guiding the assembly of nascent myofibrils from premyofibrils. By contrast, sarcomeres were assembled in the hypomorphic ttn mutants but either became susceptible to biomechanical stresses such as contraction or degenerated during development. Further genetic studies indicated that the exon usage hypothesis, but not the toxic peptide or the Cronos hypothesis, could account for these exon-dependent effects. In conclusion, we modeled TTNtv allelic heterogeneity during development and paved the way for future studies to decipher allelic heterogeneity in adult DCM.

Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b ( dnajb6b(L) ) was identified as a CM susceptibility gene, supported by identification of rare variants in its human ortholog DNAJB6 from CM patients. Mechanistic studies indicated that the deleterious, loss-of-function modifying effects of dnajb6b(L) can be ameliorated by inhibition of ER stress. In contrast, overexpression of dnajb6(L) exerts cardioprotective effects on both fish and mouse CM models. Together, our findings establish a mutagenesis screening strategy that is scalable for systematic identification of genetic modifiers of CM, feasible to suggest therapeutic targets, and expandable to other major human diseases.

Background— Genetic studies of cardiomyopathy and heart failure have limited throughput in mammalian models. Adult zebrafish have been recently pursued as a vertebrate model with higher throughput, but genetic conservation must be tested. Methods and Results— We conducted transcriptome analysis of zebrafish heart and searched for fish homologues of 51 known human dilated cardiomyopathy–associated genes. We also identified genes with high cardiac expression and genes with differential expression between embryonic and adult stages. Among tested genes, 30 had a single zebrafish orthologue, 14 had 2 homologues, and 5 had ≥3 homologues. By analyzing the expression data on the basis of cardiac abundance and enrichment hypotheses, we identified a single zebrafish gene for 14 of 19 multiple-homologue genes and 2 zebrafish homologues of high priority for ACTC1 . Of note, our data suggested vmhc and vmhcl as functional zebrafish orthologues for human genes MYH6 and MYH7 , respectively, which are established molecular markers for cardiac remodeling. Conclusions— Most known genes for human dilated cardiomyopathy have a corresponding zebrafish orthologue, which supports the use of zebrafish as a conserved vertebrate model. Definition of the cardiac transcriptome and fetal gene program will facilitate systems biology studies of dilated cardiomyopathy in zebrafish.

ABSTRACTMutations in sarcomere genes have been found in many inheritable human diseases, including hypertrophic cardiomyopathy. Elucidating the molecular mechanisms of sarcomere assembly shall facilitate understanding of the pathogenesis of sarcomere‐based cardiac disease. Recently, biochemical and genomic studies have identified many new genes encoding proteins that localize to the sarcomere. However, their precise functions in sarcomere assembly and sarcomere‐based cardiac disease are unknown. Here, we review zebrafish as an emerging vertebrate model for these studies. We summarize the techniques offered by this animal model to manipulate genes of interest, annotate gene expression, and describe the resulting phenotypes. We survey the sarcomere genes that have been investigated in zebrafish and discuss the potential of applying this in vivo model for larger‐scale genetic studies. Anat Rec, 297:1681–1693, 2014. © 2014 Wiley Periodicals, Inc.