The force of the spontaneously contracting zebrafish heart, in the assessment of cardiovascular toxicity: Application on adriamycin Triantafyllia Orfanidou a , Chrisovalantis Papaefthimiou a,⇑ , Efthimia Antonopoulou a , Ioannis Leonardos b , George Theophilidis a a Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece b Department of Biological Applications and Technologies, School of Sciences and Technologies, University of Ioannina, GR-45110 Ioannina, Greece article info Article history: Received 2 November 2012 Accepted 8 March 2013 Available online 21 March 2013 Keywords: Zebrafish Heart Ventricle Contraction Force Adriamycin abstract The heart of the zebrafish has been used extensively to assess the cardiotoxic effect of compounds, using the frequency of heart contractions as the main index of cardiac response to drugs. In this study, the force and the frequency generated by the spontaneously contracting zebrafish heart, isolated in saline, were found to be 0.87 ± 0.05 mN and 1.54 ± 0.03 Hz (n = 6) respectively within the first hour of recording. Both values of force and frequency remained constant for over 8 h. The advantage of prolonged vitality in the assessment of cardiovascular toxicity was shown using the well-known anticancer drug adriamycin, which has severe cardiotoxic side effects. At 10.0 lM there was a 21.05 ± 4.42% (p = 0.02, n = 4) decrease in the force of contraction, while the frequency was not affected after 3 h treatment (p > 0.05). At 50.0 and 100.0 lM there was a 33.24 ± 3.0 and 46.6 ± 4.80% irreversible decrease in force (p < 0.05, n = 4), while a 18.02 ± 4.07% and 16.16 ± 4.07% reversible increase was observed in the frequency (p = 0.02, n = 4). These contradictory positive chronotropic and negative inotropic responses indicate the strong inhibitory effect of adriamycin on ventricular cardiomyocytes and its excitatory effects on auto-rhythmical pacemaker cells. If heart frequency was the only parameter used to assess the cardiotoxic effect of adriamycin, at the above range of concentrations, this compound would have been classified as non-cardiotoxic. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The pharmacological responses of the zebrafish heart to well- characterized cardiotoxins are strikingly similar to responses in humans (McGrath and Li, 2008). For this reason, a number of in vivo and in vitro cardiotoxicological studies have been per- formed, both in zebrafish embryos and adults. In most cases, the main parameter used to assess the cardiotoxic effect has been the modulation of heart frequency (Schwerte and Fritsche, 2003), which can be measured using a number of methodologies, such as direct measurement with visual observation, video-edge detec- tion systems and by recording electrocardiograms (Milan et al., 2006; Arnaout et al., 2007; Denvir et al., 2008; Rana et al., 2010; Tsai et al., 2011; Kitambi et al., 2012). However, in similar ex vivo studies, using hearts from larger animals, the simultaneous mea- surement of both the frequency and force of heart contraction, are necessary for a better and more conclusive evaluation of the cardiotoxic effects of a compound (Aho and Vornanen, 1999; Shiels et al., 2002; Tryfonos et al., 2009). The need to monitor the force generated by the spontaneous contraction of the zebrafish heart is obvious, but the measurements which have been made so far used optical methods, where the con- traction was expressed in peak acceleration units in cm/s 2 , or ven- tricular wall motion amplitude in lm, or arbitrary muscle displacement units (Malone et al., 2007; Denvir et al., 2008; Munte- an et al., 2010). In the case of Hecker et al. (2008), an attempt was made to measure the real force of the contraction of the zebrafish ventricle, but they were able to record for only 1.5 h, while they claimed that ‘‘future studies are needed to determine a time course of the functional properties of zebrafish hearts after their isolation’’. The purpose of the method presented in this study is therefore: (1) to accurately measure the force generated by the spontaneous contracting zebrafish heart isolated in saline and (2) to demon- strate the potential of the method in the assessment of cardiovas- cular-induced toxicity using adriamycin. Adriamycin, was chosen as the pilot compound because it is known to induce cardiomyop- athy in humans (Octavia et al., 2012), and also affects cardiac con- tractility in zebrafish (Ding et al., 2011). 2. Materials and methods 2.1. The zebrafish heart preparation Adult zebrafish (Danio rerio) of the wild type AB, 4–6 months of both sexes were used. The study was mainly focused on the female, 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.03.004 ⇑ Corresponding author. Tel.: +30 2310 998275; fax: +30 2310 998269. E-mail address: cpapaef@bio.auth.gr (C. Papaefthimiou). Toxicology in Vitro 27 (2013) 1440–1444 Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit