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In vivo cardiac imaging system of zebrafish using a fluorescent
dye for the assessment of anticancer drug-induced cardiotoxicity.
Saki Ito1, Noriko Umemoto1, Kana Okamori1, Yuhei Nishimura1,2,3,4, Miko Kawabata1,5, Zi Zhang1, Beibei Zhang1, Junya Kuroyanagi1,
Yasuhito Shimada1,2,3,4, Toshio Tanaka1,2,3,4
1Dept. Mol. and Cell. Pharmacol., Pharmacogeno. and Pharmacoinfor., Mie Univ. Grad. Sch. Med, 2Mie Univ. Med. Zebra. Res. Cent, 3Dept.
Bioinfo. Mie Univ. Life Sci. Res. Cent, 4Dept. Omics Med., Mie Univ. Indu. Tech. Innov. Inst, 5Dept. Clin. Anesthe. Mie Univ. Sch. Med
Introduction
Result
1. Assessment of cardiac function
In vivo cardiac fluorescent imaging system of zebrafish
98 hpf
Doxorubicin induced-cardiotoxicity
Zebrafish were administered doxorubicin 5uM from 58hpf to 108hpf.
We assessed cardiac function at both 98hpf and 108hpf and analyzed gene
expression files in the heart at 108hpf.
58hpf
98hpf 108hpf
*
60
30
VDd
Left : in vivo cardiac image of
bright-field
Right : in vivo cardiac image
using Bodipy-ceramide
①Measure
cardiac
parameters
M-mode image
↑Dorsal
VDd
long
axis
80
0.3
60
0.2
40
20
VDs
0
EDV
long axis
ESV
0
SV
FS
EF
0.4
0μM
5μM
120
**
90
*
**
60
30
100
0μM
5μM
80
0.3
60
0.2
**
**
VDd
VDs
short axis
VDd
40
0.1
20
0
0
0
VDs
0μM
5μM
%
150
EDV
long axis
ESV
SV
FS
EF
At 108 hpf, VDs, VDd, EDV and SV of zebrafish treated with
doxorubicin were significantly decreased compared to those of control
zebrafish.
HR
200
150
0μM
100
5μM
50
0
98hpf
To observe cardiac lumen clearly, we applied
Bodipy-ceramide which is a commercial
fluorence dye.
VDd
0μM
5μM
108 hpf
Heat rate(time/min)
Because of their greater transparency, poorly
pigmented nacre mutants of zebrafish were used
in this study.
Short
axis
100
At 98 hpf, VDs, but not other parameters of zebrafish treated with
doxorubicin were significantly decreased compared to those of control
zebrafish.
72hpf 96hpf 120hpf
2. Cardiac fluorescent imaging system
VDs
short axis
250
Cardiac
fluorescent
imaging
0μM
5μM
0.1
Doxorubicin
administration
(Lister J, et al. Development 126: 3757-3767, 1999)
volume(nl)
90
volume(nl)
1. Experimental design
Asessment of
cardiac function
Gene expression
analysis
120
VDs
diameter(um)
Method
24hpf 48hpf
0.4
0μM
5μM
0
The risk of cardiotoxicity is the most serious drawback to the clinical usefull of
anticancer drug including doxorubicin. Doxorubicin(Dox) is effective in a wide
range of cancers, including both hematological and solid tumors. Dox has
accumulated action and is associated with a dose dependent-cardiotoxicity that can
eventuate into heart failure. Dox-induced cardiotoxicity might be associated with
inhibition of synthesis of DNA and RNA, generation of reactive oxygen species
(ROS),direct membrane effects and so on. However, The mechanism is not
completely elucidated.
0hpf
*<0.05, **<0.01
%
150
diameter(um)
Zebrafish has emerged as a model organism for cardiac research. Because of the
transparency, observation of the heart rhythm as well as the vasculature and
circulation in zebrafish is possible. However, it is difficult to clearly detect the
boundary between the ventricular wall and the lumen in bright field images. To
visualize the cardiac lumen, we have developed in vivo cardiac imaging of
zebrafish using Bodipy-ceramide. We applied this method to assess anticancer
drug-induced cardiotoxicity.
At 98-108hpf, HR didn’t shown significant
change.
108hpf
Zebrafish cardiotoxicity was detected by the in vivo cardiac imaging .
2. Gene expression analysis
We analysed gene expression profiles of doxorubicin induced-cardiotoxicity
using zebrafish cardiotoxicity model.
We identified 7 downregulated genes and 39 upregulated genes(RP・SAM,
FDR<0.3).
MAP2k1 and PKC were detected as the upstream regulator of gene altering
expression by gene network analysis.
↓Ventral
M-mode image
VDd
VDs
←Aorta
②Calculate
ventricular
volumes and
other parameters
Apex→
EDV(end-diastolic volume), ESV(end-systolic volume)
= (shortVDd,VDs/2)2×longVDd,VDs/2×π×4/3×10-6
SV(stroke volume) = EDV－ESV
FS(Fractional Shortening)
= (shortDd－shortDs)/shortDd×102
EF(Ejection Fraction) = SV/EDV×102
green: upatream regulator detected by pathway analysis
red: up regulation detected by microarray
blue: down regulation detected by microarray
3. Gene expression analysis
We analysed gene expression files of their heart at 108hpf.
Cardiac isolation
Microarray
cut
Gene network analysis
Conclusion
This study revealed that both VDs and VDd of zebrafish treated with
doxorubicin were significantly decreased compared to those of control zebrafish,
suggesting that the in vivo cardiac imaging can be used for the assessment of
cardiotoxicity of both preclinical and clinically-used drugs.