Generation of a Triadin KnockOut Syndrome Zebrafish Model

Abstract

Different forms of sudden cardiac death have been described, including a recently identified form of genetic arrhythmogenic disorder, named "Triadin KnockOut Syndrome" (TKOS). TKOS is associated with recessive mutations in the TRDN gene, encoding for TRIADIN, but the pathogenic mechanism underlying the malignant phenotype has yet to be completely defined. Moreover, patients with TKOS are often refractory to conventional treatment, substantiating the need to identify new therapeutic strategies in order to prevent or treat cardiac events. The zebrafish (Danio rerio) heart is highly comparable to the human heart in terms of functions, signal pathways and ion channels, representing a good model to study cardiac disorders. In this work, we generated the first zebrafish model for trdn loss-of-function, by means of trdn morpholino injections, and characterized its phenotype. Although we did not observe any gross cardiac morphological defect between trdn loss-of-function embryos and controls, we found altered cardiac rhythm that was recovered by the administration of arrhythmic drugs. Our model will provide a suitable platform to study the effect of TRDN mutations and to perform drug screening to identify new pharmacological strategies for patients carrying TRDN mutations.

Keywords: Triadin KnockOut Syndrome; arrhythmic drugs; heart defects; zebrafish.

Figure 1

Expression analysis of trdn in zebrafish embryo. (A) RT-PCR performed on RNA isolated from zebrafish embryos at different developmental stages. β-actin was used as the loading control. (B–F) WISH analyses with a trdn-specific probe in zebrafish embryos. trdn is expressed in skeletal muscles and heart. (B–D) Lateral view of a 13 somite (B, anterior to the left), 24 hpf (C) and 48 hpf (D) embryos. (E,F) Dorsal (E) and frontal (F) view of a 48 hpf embryo. Scale bars indicate 100 μm. sk, skeletal muscle; h, heart.

Figure 2

Phenotypical analysis of embryos with trdn loss-of-function. (A) Schematic representation of Triadin interactions network and trdn knockdown strategy. RyR: ryanodine receptor; DHPR: dihydropyridine receptor; SERCA: sarco-endoplasmic reticulum calcium ATPase (B–E) Representative bright field images of the phenotype of 48 hpf embryos injected with std-MO and trdn-MOs. (F) Classification of the phenotypes obtained with trdn-MOs injection. Values indicate the number of embryos for each phenotype. Scale bars indicate 100 μm. e, edema; ctt, curved trunk/tail.

Figure 3

Skeletal muscle morphology evaluation. (A–C) Representative images of WISH analysis using a ckma probe in std-MO (A), class 1 wild-type-like (B) and class 3 affected (C) trdn-MO-injected embryos at 48 hpf. (D–F) Representative birefringence images of std-MO (D), class 1 wild-type-like (E) and class 3 affected (F) trdn-MO-injected embryos at 3 dpf. (G–I) Representative MF20 immunofluorescence images of std-MO (G), class 1 wild-type-like (H) and class 3 affected (I) trdn-MO-injected embryos. (A–F) Scale bars indicate 100 µm. (G–I) Scale bars indicate 50 μm.

Figure 4

Heart morphology and function evaluation. (A,B) Frontal view of 48 hpf std-MO and trdn-MOs embryos hybridized with cmlc2 probe using WISH technique. Scale bars indicate 100 μm. (C,D) Semithin section of std-MO and trdn-MOs embryos in the heart region. Scale bars indicate 100 μm. (E) Quantification of heartrate in embryos at 48 hpf injected with std-MO and trdn-MOs. (F) Percentage of embryos with a heartbeat superior to the mean of the heartbeat (bpm) of trdn-MOs. (G) Heartbeat count in 3 dpf std-MO and trdn-MOs untreated or adrenaline-, isoprenaline- and atropine-treated embryos. (H) Heartbeat count in 3 dpf std-MO and trdn-MOs untreated or treated with metoprolol or flecainide. (F–H) At least 30 embryos were analyzed for each group. Values are expressed as mean ± SEM. *** p < 0.001, ** p < 0.01, n.s. = not significant, Mann–Whitney test (E) and one-way ANOVA followed by Tukey post-hoc correction (G,H). Adr, adrenaline; Iso, isoprenaline; Atr, atropine.