Modeling GATAD1-Associated Dilated Cardiomyopathy in Adult Zebrafish

Abstract

Animal models have played a critical role in validating human dilated cardiomyopathy (DCM) genes, particularly those that implicate novel mechanisms for heart failure. However, the disease phenotype may be delayed due to age-dependent penetrance. For this reason, we generated an adult zebrafish model, which is a simpler vertebrate model with higher throughput than rodents. Specifically, we studied the zebrafish homologue of GATAD1, a recently identified gene for adult-onset autosomal recessive DCM. We showed cardiac expression of gatad1 transcripts, by whole mount in situ hybridization in zebrafish embryos, and demonstrated nuclear and sarcomeric I-band subcellular localization of Gatad1 protein in cardiomyocytes, by injecting a Tol2 plasmid encoding fluorescently-tagged Gatad1. We next generated gatad1 knock-out fish lines by TALEN technology and a transgenic fish line that expresses the human DCM GATAD1-S102P mutation in cardiomyocytes. Under stress conditions, longitudinal studies uncovered heart failure (HF)-like phenotypes in stable KO mutants and a tendency toward HF phenotypes in transgenic lines. Based on these efforts of studying a gene-based inherited cardiomyopathy model, we discuss the strengths and bottlenecks of adult zebrafish as a new vertebrate model for assessing candidate cardiomyopathy genes.

Phenotyping cardiomyopathy in adult zebrafish

“…A transgenic erbb2 zebrafish heart manifests signs of the DCM phenotype, such as enlarged ventricle and reduced trabeculation (Reischauer et al, 2014). An enlarged heart was also noted in transgenic fish harboring a DCM-causative mutation in gatad1 (Yang et al, 2016). Besides transgenic models, cardiac phenotypes in adult fish containing mutations in sarcomeric genes have been defined.…”

“…Cardiomyocyte hypertrophy can be validated by culturing isolated single cardiomyocytes and then measuring the cell size directly (Ding et al, 2011). Cardiomyocyte proliferation can be quantified by staining sectioned heart with proliferating cell nuclear antigen; cardiomyocyte apoptosis can be quantified by staining sectioned heart with TUNEL (terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling) (Ding et al, 2016; Ding et al, 2011; Hoage et al, 2011; Sun et al, 2009; Yang et al, 2016). These assays are particularly important in zebrafish, because both hyperplasia (muscle growth by cell division) and hypertrophy (muscle growth by cell size) occur during adulthood (Palstra et al, 2010).…”

Modeling Inherited Cardiomyopathies in Adult Zebrafish for Precision Medicine

“…Because many sequence variants are missense mutations, loss-of-function knockout mutants could result in negative conclusions or provide misleading information. As shown by recent studies of sequence variants from GATAD1, KCNJ8, and DNAJB6(L) genes (Ding et al, 2016;Yang et al, 2016;Tessadori et al, 2018), knock-in or transgenic technology can be used to precisely model missense mutations.…”

A Langendorff-like system to quantify cardiac pump function in adult zebrafish

“…Several acquired cardiomyopathy models have been generated, including anemia-induced cardiomyopathy ( Hoage et al, 2011 ; Sun et al, 2009 ), doxorubicin (DOX)-induced cardiomyopathy (DIC) ( Ding et al, 2011 ; Packard et al, 2017 ) and hyperglycemia-induced cardiomyopathy ( Sun et al, 2017 ). Recently, several studies provided proof-of-principle evidence for the value of zebrafish models for assessing the functional effects of human genetic variants ( Asimaki et al, 2014 ; Huttner et al, 2013 ; Reischauer et al, 2014 ; Yang et al, 2016 ). Of 51 genes known to cause dilated cardiomyopathy, 49 (96%) have been shown to have corresponding zebrafish homologs ( Shih et al, 2015 ), and morpholino-mediated knockdown of many of those genes causes dilated cardiomyopathy ( Vogel et al, 2009 ).…”

“…The transparency of embryonic zebrafish is lost with age, and a major factor that has limited adult zebrafish studies has been the lack of effective cardiac phenotyping tools ( Yang et al, 2016 ). In mammalian animal models, cardiac function is frequently quantified in vivo using ultrasound-based echocardiography.…”