Forward genetic screen using a gene-breaking trap approach identifies a novel role of grin2bb-associated RNA transcript (grin2bbART) in zebrafish heart function

Abstract

LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish (Danio rerio) for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it “bigheart.” Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript grin2bb associated RNA transcript (grin2bbART). Using in situ hybridization, we confirmed localized grin2bbART expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored grin2bbART expression. Morpholino-mediated antisense downregulation of grin2bbART in wild-type zebrafish embryos mimicked bigheart mutants; this suggests grin2bbART is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of grin2bbART, a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish.

Zebrafish as a Model for Cardiovascular and Metabolic Disease: The Future of Precision Medicine

“…P, Q, R, S & T represent regular ECG waves. The image in the figure were adapted from the author’s publication [ 119 ].…”

“…Several zebrafish lines with ion channel mutations are now being used as vertebrate models for understanding long-or short-QT syndrome [105]. [119] Towards this end, a transgenic zebrafish arrhythmia model with the human SCN5A D1275N (cardiac sodium channel) mutation was created. The mutations in SCN5A-D1275N are associated with various cardiac phenotypes, including atrial and ventricular arrhythmias in humans and mice.…”

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“…Zebrafish experiments were carried out according to the standard protocols approved by Institutional Animal Ethics Committee (IAEC) of CSIR-Institute of Genomics and Integrative Biology. Zebrafish lines used in this study are Tubingen (TU), Tg(NBT:dsRed) (45), Tg(myl7:RFP) (46) and Tg(sox10:eGFP) (47).…”