br Introduction Cardiovascular diseases rank the first in

Introduction Cardiovascular diseases rank the first in cause of death, which account for ∼30% of all deaths worldwide (Mozaffarian et al., 2015). Unlike robust cardiac regeneration observed in adult zebrafish and embryonic and neonatal mice (Porrello et al., 2011; Poss et al., 2002; Sturzu et al., 2015), adult mammalian hearts are proposed to be postmitotic (Rumiantsev and Carlson, 1991) and possess extremely low renewal potency (no more than 1%–2% per year) (van Berlo and Molkentin, 2014). Thus, the human heart is scarcely able to compensate the lost cardiomyocytes after heart failure (Bergmann et al., 2015; Laflamme and Murry, 2011). Promising therapeutic strategies to cure the injured heart include activation of endogenous cardiac progenitor/stem cell differentiation, stimulation of pre-existing cardiomyocyte proliferation via administration of chemical compounds, modified mRNAs, genes, and recombinant proteins (Eulalio et al., 2012; Sahara et al., 2015; Zangi et al., 2013), and transplantation of cardiac progenitor/stem cells/cardiomyocytes (Sahara et al., 2015). For the application of cardiomyocyte-based therapy to humans in the clinic, it is essential to clearly understand molecular mechanisms of differentiation from various cell sources to functional cardiomyocytes. Several approaches have been established in mouse and human to obtain functional cardiomyocytes, including directed reprogramming of fibroblasts to cardiomyocytes using developmental transcription factors, such as GATA4, MEF2C, and TBX5, in mice (Ieda et al., 2010), activating resident stem or progenitor HOBt by WT1/THYMOSIN to induce the proliferation of endogenous cardiomyocytes in mice (Smart et al., 2011), and inducing differentiation of cardiomyocytes from embryonic stem cells in humans (Chong et al., 2014) or induced pluripotent stem cells (PSCs) from human fibroblasts and patients with heart failure (Burridge et al., 2014; Feaster et al., 2015; Zwi-Dantsis et al., 2013). Nevertheless, there are many factors and pathways, including genetic and epigenetic regulations (Burridge et al., 2015), which affect regenerative function of these cells. Establishment of novel differentiation models will undoubtedly help our understanding of molecular mechanisms of cardiomyocyte differentiation. Zebrafish hearts can rapidly regenerate without scarring after 20% ventricular resection (Poss et al., 2002). Thus, zebrafish cardiac muscle differentiation is an ideal model for the interpretation of molecular mechanisms of cardiac regeneration in the mammalian heart. Recently, several factors and pathways in the regulation of cardiomyocyte regeneration have been identified in zebrafish, such as Caveolin-1, Cdk9, nuclear factor κB, Telomerase, Neuregulin 1 (Nrg1), microRNA-101a, and bone morphogenetic protein signaling (Beauchemin et al., 2015; Bednarek et al., 2015; Cao et al., 2016; Gemberling et al., 2015; Karra et al., 2015; Matrone et al., 2015; Uygur and Lee, 2016; Wu et al., 2016). Contractile cells were first observed under spontaneous differentiation conditions in vitro using late-gastrula cells (Huang et al., 2012). Heart aggregates were generated spontaneously, in vitro, from larval zebrafish 3 days post fertilization with an efficiency of 0.4 heart aggregate per larval fish (Grunow et al., 2015). Furthermore, several questions remain open, such as which conditions are required for efficient in vitro cardiomyocyte differentiation and whether cardiomyocyte-like cells produced in vitro are functional. Therefore, systematic modeling of cardiomyocyte differentiation with high efficiency is especially necessary in zebrafish. In this study, we established a rapid and efficient method for cardiomyocyte differentiation from zebrafish primary embryonic cells after determination of their pluripotency timing at different developmental stages. Crucial factors affecting in vitro generation of cardiomyocytes were then characterized. Contractile kinetics and sarcomere formation were also investigated. Lastly, functional electrophysiological features of beating cell clusters (BCCs) were identified. These findings are valuable for the development of high-throughput strategic screening of agents for drug discovery, disease modeling, and assessment of cardiotoxic agents, in addition to dissecting the molecular mechanisms of heart development and regeneration.