Korean Circ J. 2023;53(1):34-46.

Development of a Zebrafish Larvae Model for Diabetic Heart Failure With Reduced Ejection Fraction.

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제1저자 김인호 · 교신저자 석승혁, 이해영

PMID 36627738 PMC9834558 DOI 10.4070/kcj.2022.0210 IF 3.4 순환기내과

소속 (4)

Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea.
Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Korea.
Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea. lamseok@snu.ac.kr.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea. hylee612@snu.ac.kr.

Abstract

BACKGROUND AND OBJECTIVES Diabetes mellitus (DM)-associated heart failure (HF) causes high morbidity and mortality. In this study, we established a zebrafish larvae model for in vivo research on diabetic HF.

METHODS DM-like phenotypes were induced by treating zebrafish larvae with a combination of D-glucose (GLU) and streptozotocin (STZ). HF was induced by treatment with terfenadine (TER), a potassium channel blocker. Additionally, myocardial contractility, motility, and viability were evaluated.

RESULTS The zebrafish larvae treated with a combination of GLU and STZ showed significantly higher whole-body glucose concentrations, lower insulin levels, and higher phosphoenolpyruvate carboxykinase levels, which are markers of abnormal glucose homeostasis, than the group treated with only GLU, with no effect on viability. When treated with TER, DM zebrafish showed significantly less myocardial fractional shortening and more irregular contractions than the non-DM zebrafish. Furthermore, in DM-HF with reduced ejection fraction (rEF) zebrafish, a significant increase in the levels of natriuretic peptide B, a HF biomarker, markedly reduced motility, and reduced survival rates were observed.

CONCLUSIONS We established a DM-HFrEF zebrafish model by sequentially treating zebrafish larvae with GLU, STZ, and TER. Our findings indicate the potential utility of the developed zebrafish larvae model not only in screening studies of new drug candidates for DM-HFrEF but also in mechanistic studies to understand the pathophysiology of DM-HFrEF.

Keywords

Diabetes mellitusHeart failureZebrafish

Figures

**Figure 1** Effects of various concentrations of GLU and/or STZ on the survival rates and motility of zebrafish larvae. (A) Schematic of the study design. (B) Kaplan-Meier survival analysis of zebrafish larvae after treatment with various concentrations of GLU (n = 48 per group) and (C) a combination of 40 mM GLU and various concentrations of STZ. (n = 48 per group). GLU = D-glucose; STZ = streptozotocin. ^****p<0.0001 vs. (B) control group or (C) GLU 40 mM group.

**Figure 2** Induction of diabetes mellitus-like phenotypes in zebrafish larvae via combined treatment with GLU and STZ. (A) Relative mRNA expression of *ins* (n = 8 per group) and (B) Whole-body insulin level (n = 6 per group). (C) Relative mRNA expression of *pck1* (n = 8 per group). (D) Whole-body glucose concentration (n = 6–8 per group). (E) Changes in whole-body glucose concentration after high-GLU challenge and (F) the corresponding AUC (n = 5 per group). (G) Relative gene expression of *Ucn3* and (H) *Slc30a8* (n = 8–11 per group). (I) Representative fluorescence microscopic image of pancreatic β-cells of the Tg (*ins:EGFP*) zebrafish larvae. Each group had 6–11 samples, with (A, B, and D-H) 10 or (C) 30 larvae per sample. Data are presented as mean ± standard deviation and each dot represents the value of each sample. AUC = area under the curve; GLU = D-glucose; MAN = D-mannitol; STZ = streptozotocin; Tg = transgenic; EGFP = enhanced green fluorescent protein. ^*p<0.05, ^**p<0.01, ^***p<0.001, ^****p<0.0001 vs. indicated group.

**Figure 3** HF phenotype induced by TER in the DM zebrafish model. (A) Representative fluorescent microscopic images of the Tg (*myl7:EGFP*) zebrafish heart with a GFP at 7 dpf. (B) vFS (n = 21–26 per group). (C) Representative blood pulse graphs. (D) SD of the beat-to-beat interval (n = 21–28 per group). (E) Relative mRNA expression of *nppb* (n = 8 per group). Each group had 8 samples, with 10 larvae per sample. Data are presented as mean ± SD and each dot represents the value of each sample. DM = diabetes mellitus; dpf = days post-fertilization; EGFP = enhanced green fluorescent protein; HF = heart failure; myl7 = myosin light chain 7; SD = standard deviation; TER = terfenadine; Tg = transgenic; VD = ventricular diastole; VEH = vehicle; VS = ventricular systole. ^*p<0.05, ^**p<0.01, ^****p<0.0001 vs. indicated group.

**Figure 4** Reduced motility and viability of the diabetic HF zebrafish model. (A) Representative images of motility tracking for 5 minutes in zebrafish larvae. (B) Average moved distance and (C) movement duration per minute (n = 24 per group). Data are presented as mean ± SD and each dot represents the value of each sample. (D) Kaplan-Meier survival analysis (n = 72 per group). DM = diabetes mellitus; TER = terfenadine; VEH = vehicle. ^*p<0.05, ^***p<0.001, ^****p<0.0001 vs. indicated group.

그림 출처: PubMed Central 공개 논문 (PMC9834558)

Conflict of interest statement

The authors have no financial conflicts of interest.

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