Defects in heart-looping, valve formation, and contractile performance were detected in β-lapachone-treated embryos. A: Embryos at 24 hours post-fertilization (hpf) were treated with DMSO or β-lapachone for 4 h and fixed at 48 and 72 hpf for nppa and cmlc2 hybridization (a-h). A Q-RT-PCR analysis indicated nppa expression levels in β-lapachone-treated 48- and 72-hpf embryos (i). B: Images of respective hearts with ventricles at either end-diastolic volume of DMSO (a), β-lapachone-treated embryo containing few erythrocytes (b), and β-lapachone-treated embryo containing no erythrocytes (c), or at end-systolic volume of DMSO (a'), β-lapachone-treated embryo containing few erythrocytes (b'), and β-lapachone-treated embryo containing no erythrocytes (c') are shown. Fractional shortening (FS) of the atrial and ventricular chamber of DMSO or β-lapachone-treated 52-hpf embryos was measured and calculated according to the formula, FS = (ED - ES)/ED × 100%, where ED is the end-diastolic diameter and ES is the end-systolic diameter of either the atrial or ventricular chambers (d). In β-lapachone-treated embryos, embryos containing few or no erythrocytes were recorded. Error bars indicate the standard error. Student's t-test was used to compare DMSO- and β-lapachone-treated embryos. * p < 0.001. C: DMSO- and β-lapachone-treated 48- and 72-hpf embryos were fixed for bmp4, versican, and nfatc1 hybridization. D: Paraffin sectioning and H&E staining of hearts of respective DMSO- and β-lapachone-treated 72- and 96-hpf embryos are shown. Arrows indicate the positions of the cardiac cushion (a) and valve (b). A, atrium; V, ventricle; VB, ventriculobulbal junction; AV, atrioventricular junction. Scale bars represent 100 μm.