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.