Korean Circ J.  2017 Nov;47(6):960-969. 10.4070/kcj.2017.0080.

Zebrafish Larvae Model of Dilated Cardiomyopathy Induced by Terfenadine

• Gu G
• Na Y
• Chung H
• Seok SH
• Lee HY

• KMID: 2396489
• DOI: http://doi.org/10.4070/kcj.2017.0080

Abstract

BACKGROUND AND OBJECTIVES
Dilated cardiomyopathy can be the end-stage of severe cardiac disorders and directly affects the cardiac muscle, inducing cardiomegaly and heart failure (HF). Although a wide variety of animal models are available to study dilated cardiomyopathy, there is no model to assess dilated cardiomyopathy with non-invasive, simple, and large screening methods.
METHODS
We developed a dilated cardiomyopathy model in zebrafish larvae using short duration terfenadine, a known cardiotoxic drug that induces ventricular size dilation. Fractional shortening of zebrafish hearts was calculated.
RESULTS
We treated zebrafish with 5 to 10 µM terfenadine for 24 hours. In terfenadine-treated zebrafish, blood frequently pooled and clotted in the chamber, and circulation was remarkably reduced. Atria and ventricles were swollen, and fluid was deposited around the heart, mimicking edema. Cardiac contractility was significantly reduced, and ventricular area was significantly enlarged. Heart rate was markedly reduced even after terfenadine withdrawal. Acridine orange staining also showed that terfenadine increased cardiomyocyte apoptosis. A significant increase of natriuretic peptide B (NPPB) mRNA was found in terfenadine-treated zebrafish. A low dose of terfenadine (5-10 µM) did not show mortality in short-term treatment (24 hours). However, moderate dose (35-45 µM) terfenadine treatment reduced zebrafish survival within 1 hour.
CONCLUSION
With advantages of rapid sample preparation procedure and transparent observation of the live heart, this model can potentially be applied to large-scale drug screening and toxicity assays for non-ischemic HF.

Keyword

Zebrafish; Heart failure; Cardiomyopathies

MeSH Terms

Acridine Orange
Apoptosis
Cardiomegaly
Cardiomyopathies
Cardiomyopathy, Dilated*
Drug Evaluation, Preclinical
Edema
Heart
Heart Failure
Heart Rate
Larva*
Mass Screening
Models, Animal
Mortality
Myocardium
Myocytes, Cardiac
RNA, Messenger
Terfenadine*
Zebrafish*
Acridine Orange
RNA, Messenger
Terfenadine

Figure

• 

  Figure 1 Morphology of 5 dpf zebrafish larvae after H&E staining. dpf = days post fertilization; H&E = hematoxylin and eosin.

• 

  Figure 2 Transient terfenadine treatment reduced heart rate, inducing blood stagnation. (A) Representative image of control zebrafish larvae treated with 0.001% DMSO for 24 hours. (B, C) Representative images of zebrafish larvae treated with 5 µM (B) and 10 µM (C) terfenadine for 24 hours. Terfenadine-treated zebrafish showed enlarged heart size (black arrow) and venous congestion (hollow arrow). (D) Heart rates in control and terfenadine-treated zebrafish larvae (n=20 zebrafish/group). DMSO = dimethyl sulfoxide. *p<0.050.

• 

  Figure 3 Atrioventricular dyssynchrony was induced after terfenadine treatment. (A) Representative image of cmlc-2:GFP transgenic zebrafish larvae. (B) Images taken from in vivo video recording at 4 dpf in the control (0.001% DMSO) and 24-hr terfenadine (20 μM)-treated zebrafish larvae. The interval between images in the montage is 0.196 seconds. Outlines of the atrium are shown with white dashed lines, and outlines of ventricles with red lines. (C) Arrhythmia rate of the control (0.001% DMSO) and 24-hr terfenadine (20 μM)-treated zebrafish larvae (n=20 zebrafish/group). (D) Atrial size of the control (0.001% DMSO) and 24-hr terfenadine (20 μM)-treated zebrafish larvae (n=20 zebrafish/group). A = atrium; DMSO = dimethyl sulfoxide; dpf = days post fertilization; FV/FA = fetal ventricle/fatal atrium; V = ventricle. *p<0.050.

• 

  Figure 4 Transient terfenadine treatment impaired cardiac contraction, resulting in HF. (A) Representative images of control (0.001% DMSO) or terfenadine (20 μM)-treated zebrafish larva. (B) Lateral view of zebrafish larvae at 4 dpf. The control zebrafish (0.001% DMSO) exhibited normal cardiac morphology, whereas terfenadine (20 μM)-treated zebrafish larvae showed pericardial edema (blue circle) and venous congestion (green circle). (C) Quantification of ventricular FS in control (0.001% DMSO) and terfenadine (20 μM)-treated zebrafish larvae. Ventricle size of control (0.001% DMSO) and terfenadine (20 μM)-treated zebrafish larvae after a 24 hours treatment. n=20 zebrafish/group, scale bar=0.5 mm. a = Atrium; DMSO = dimethyl sulfoxide; dpf = days post fertilization; FS = fractional shortening; HF = heart failure; V = ventricle. *p<0.050; †p<0.010.

• 

  Figure 5 Terfenadine treatment induced apoptosis in cardiomyocytes. (A) H&E staining of longitudinal sections showed normal cardiomyocyte morphology of control (0.001% DMSO) zebrafish larvae. (B) Terfenadine (20 μM)-treated zebrafish larvae for 24 hours showed elongated and thin cardiomyocytes. (C) Detection of apoptotic cardiomyocytes by acridine orange staining in the control group. (D) Detection of apoptotic cardiomyocytes by acridine orange staining in terfenadine-treated zebrafish. Outlines of ventricles are shown with red lines. Apoptotic cells are indicated by red arrows. Scale bar=0.5 μm. DMSO = dimethyl sulfoxide; dpf = days post fertilization; H&E = hematoxylin and eosin.

• 

  Figure 6 Terfenadine treatment induced HF and impaired survival in zebrafish larvae. (A) Quantitative RT-PCR of NPPB was increased in terfenadine-treated zebrafish larvae. (B) Survival rate of zebrafish treated with control and 35 to 45 µM of terfenadine. n=20 zebrafish/group. HF = heart failure; NPPB = natriuretic peptide B; RT-PCR = real-time polymerase chain reaction. *p<0.050.

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