Cardiac transcriptome and dilated cardiomyopathy genes in zebrafish

Abstract

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.

Keywords: MYH6/7; cardiomyopathy, dilated; fetal gene program; transcriptome; zebrafish.

Figure 1

Expressional Analysis of MYH Homologues in Zebrafish. A, Heat map of the average expression in log₁₀(reads per kilobase per million reads [RPKM]) for myh6/amhc, vmhc, vmhcl, myh7ba, myh7bb, and myhc4 (muscle-specific myh) in embryo heart, adult heart, and adult muscle. B, Validation of the RNA sequencing data in (A) showing expression levels using semiquantitative reverse transcriptase polymerase chain reaction (semi-qPCR). gapdh was used as control. C, Expression of myh6/amhc, vmhc, vmhcl, myh7ba, and myh7bb in adult atrium and ventricle as a percentage of total expression level from qPCR, normalized to the expression of gapdh. D, Schematics of genomic region for MYH6/MYH7 homologues in human, mice, and zebrafish. Chr indicates chromosome.

Figure 2

Transcripts of vmhc and vmhcl Respond to Doxorubicin. Adult zebrafish were injected with doxorubicin (20 mg/g), and gene expression in ventricle was determined using qPCR at 1 day, 3 days, 1 week, 2 weeks, and 3 weeks after injection. The expression level was normalized to that of actb2. Shown are expression levels of vmhc and vmhcl (A), nppa and nppb (B), and pln, ryr2b, atp2a2, and slc8a1a (C). In A, the ratio of vmhc to vmhcl was increased compared with that in an uninjected fish heart (green line), right scale. dpi indicates days post injection; wpi, weeks post injection. N=3 in each experimental group.