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Clozapine is one of the antipsychotic drugs for treating schizophrenia, but its cardiotoxicity was the primary obstacle for its clinical use, due to the unknown mechanism of clozapine-induced cardiotoxicity. In this study, we studied the cardiotoxicity of clozapine by employing zebrafish embryos. Acute clozapine exposure showed dose-dependent mortality with the LC50 at 59.36 μmol L−1 and 49.60 μmol L−1 when determined at 48 and 72 h post exposure, respectively. Morphological abnormalities like pericardial edema, incompletely heart looping, and bradycardia were detected after clozapine exposure in a time- and dose-dependent manner. Clozapine treatment also resulted in a slower heart rate and disturbed rhythm in zebrafish embryos. Also, oxidative stress was observed after clozapine exposure by measurement of ROS (reactive oxygen species), MDA (a lipid peroxidation marker), antioxidant enzyme activities, and oxidative stress-related gene expression. The elevation of inflammation coincided with oxidative stress by the assay of inflammation-related genes expression accompanied by clozapine incubation. Collectively, the data indicate that clozapine might achieve cardiotoxic effect in zebrafish larva through increasing oxidative stress, attenuation in antioxidant defense, and up-regulation of inflammatory cytokines. The data could provide experimental explanations for myocarditis and pericarditis induced by clozapine in clinics, and help find an effective solution to reduce its cardiotoxicity.

BackgroundThe cardiotoxicity of isoniazid on zebrafish embryos and its underlying mechanism is unclear.MethodsHere, we exposed zebrafish embryos at 4 h post-fertilization to different levels of isoniazid and recorded the morphology and number of malformed and dead embryos under the microscope.ResultsThe high concentration of isoniazid group showed more malformed and dead embryos than the low concentration of isoniazid group and control group. The morphology of the heart and its alteration were visualized using transgenic zebrafish (cmlc2: GFP) and confirmed by in situ hybridization. The negative effects of isoniazid on the developing heart were characterized by lower heart rate and more heart looping disorders. Mechanistically, PCR showed decreased expression of heart-specific transcription factors when exposed to isoniazid. Oxidative stress was induced by isoniazid in cardiomyocytes, mediated by decreased activities of catalase and superoxide dismutase, which were rescued by scavengers of reactive oxygen species.ConclusionIn conclusion, this study demonstrated that isoniazid led to heart looping disturbance by the downregulation of cardiac-specific transcription factors and induction of cardiomyocyte apoptosis.

Dextrocardia, a term used to describe all varieties of developmental malformations resulting in the positioning of the heart in the right hemithorax, is linked to a number of highly significant cardiac disorders. Current estimates vary tremendously in the literature. Only about 10 % of patients with diagnosed dextroversion show no substantial cardiac pathology; however, the incidence of congenital heart defects associated with dextrocardia is close to 100 %. The majority of studies previously reported include dextrocardia associated with situs inversus and cases of Kartagener syndrome. There is complex embryology and pathogenesis that results in dextrocardia. Physical examinations of the heart, such as percussion and palpation during routine exams, are vitally important initial diagnostic instruments. X-ray, CT scan, echocardiography (ECHO), and MRI are all invaluable imaging modalities to confirm and classify the diagnosis of dextrocardia. In summary, heart malposition is a group of complex pathologic associations within the human body, rather than just a single congenital defect. Clinicians such as anesthesiologists have unique challenges managing patients with dextrocardia. An appreciation of associated pathogenesis, appropriate diagnosis, and management is paramount in ensuring the best outcome for these patients perioperatively.

Zebrafish tbx5 expresses in the heart, pectoral fins and eyes of zebrafish during embryonic development. In zebrafish, injection of tbx5 morpholino antisense RNA caused changes of heart conformation, defect of heart looping, pericardium effusion, dropsy of ventral position and decreased heart rate. We suggested that cardiac myogenesis genes might be responsible for this phenomenon. Morpholino antisense RNA which against the initiation site of tbx5 gene was designed in order to knockdown the expression of tbx5, and the results were analyzed by whole-mount in situ hybridization and quantitative real-time PCR. Expression of cardiac myogenesis genes amhc, vmhc and cmlc2 were expressed constantly at the early embryonic development and reached its highest rate right before cardiac looping initiated. These cardiac myogenesis genes showed insufficient expressions within different heart defect embryos. Moreover, vmhc showed ectopic expression in addition to heart looping defect in heart defective embryos at 36 hpf. Our data suggests that the heart failure caused by the knockdown of tbx5 gene might result from the down-regulation of cardiac myogenesis genes.

Vertebrates have characteristic and conserved left-right (L-R) visceral asymmetries, for example the left-sided heart. In humans, alterations of L-R development can have serious clinical implications, including cardiac defects. Although little is known about how the embryonic L-R axis is established, a recent study in the chick embryo revealed L-R asymmetric expression of several previously cloned genes, including Cnr-1 (for chicken nodal-related-1), and indicated how this L-R molecular asymmetry might be important for subsequent visceral morphogenesis. Here we show that nodal is asymmetrically expressed in mice at similar stages, as is Xnr-1 (for Xenopus nodal related-1) in frogs. We also examine nodal expression in two mouse mutations that perturb L-R development, namely situs inversus viscerum (iv), in which assignment of L-R asymmetry is apparently random and individuals develop either normally or are mirror-image-reversed (situs inversus), and inversion of embryonic turning (inv), in which all individuals develop with situs inversus. In both, nodal expression is strikingly affected, being reversed or converted to symmetry. These results further support a key role for nodal and nodal-related genes in interpreting and relaying L-R patterning information in vertebrates. To our knowledge, our results provide the first direct evidence that iv and inv normally function well before the appearance of morphological L-R asymmetry.

Background β-Lapachone has antitumor and wound healing-promoting activities. To address the potential influences of various chemicals on heart development of zebrafish embryos, we previously treated zebrafish embryos with chemicals from a Sigma LOPAC1280™ library and found several chemicals including β-lapachone that affected heart morphogenesis. In this study, we further evaluated the effects of β-lapachone on zebrafish embryonic heart development. Methods Embryos were treated with β-lapachone or dimethyl sulfoxide (DMSO) at 24 or 48 hours post fertilization (hpf) for 4 h at 28°C. Heart looping and valve development was analyzed by whole-mount in situ hybridization and histological analysis. For fractional shortening and wall shear stress analyses, AB and Tg (gata1:DsRed) embryos were recorded for their heart pumping and blood cell circulations via time-lapse fluorescence microscopy. Dextran rhodamine dye injection into the tail reticular cells was used to analyze circulation. Reactive oxygen species (ROS) was analyzed by incubating embryos in 5-(and 6-)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate (CM-H2DCFDA) and recorded using fluorescence microscopy. o-Dianisidine (ODA) staining and whole mount in situ hybridization were used to analyze erythrocytes. TUNEL assay was used to examine DNA fragmentation. Results We observed a linear arrangement of the ventricle and atrium, bradycardia arrhythmia, reduced fractional shortening, circulation with a few or no erythrocytes, and pericardial edema in β-lapachone-treated 52-hpf embryos. Abnormal expression patterns of cmlc2, nppa, BMP4, versican, and nfatc1, and histological analyses showed defects in heart-looping and valve development of β-lapachone-treated embryos. ROS production was observed in erythrocytes and DNA fragmentation was detected in both erythrocytes and endocardium of β-lapachone-treated embryos. Reduction in wall shear stress was uncovered in β-lapachone-treated embryos. Co-treatment with the NQO1 inhibitor, dicoumarol, or the calcium chelator, BAPTA-AM, rescued the erythrocyte-deficiency in circulation and heart-looping defect phenotypes in β-lapachone-treated embryos. These results suggest that the induction of apoptosis of endocardium and erythrocytes by β-lapachone is mediated through an NQO1- and calcium-dependent pathway. Conclusions The novel finding of this study is that β-lapachone affects heart morphogenesis and function through the induction of apoptosis of endocardium and erythrocytes. In addition, this study further demonstrates the importance of endocardium and hemodynamic forces on heart morphogenesis and contractile performance.

Grafts of mesoderm taken from the precardiac region of quail embryos of stages 5-7 were inserted into the precardiac mesoderm of chick embryos of stages 5-7. The experiments were of four types and were code named to indicate the origin and the destination of the graft. QACP: tissue from the anterior end of the quail precardiac area was inserted into the posterior end of the chick precardiac mesoderm; QPCA: tissue from the posterior end of the quail precardiac area was inserted into the anterior end of the chick precardiac mesoderm; QACA: tissue from the anterior end of the quail precardiac area was inserted into the anterior end of the chick precardiac mesoderm; QPCP: tissue from the posterior end of the quail precardiac area was inserted into the posterior end of the chick precardiac mesoderm. In no case was precardiac tissue removed from the host. Three main-types of anomaly were obtained: inverted hearts, in which looping took place to the left rather than to the right; compact hearts, in which no looping occurred, and hearts in which extra tissues or regions were apparent. The incidence of compact hearts was significantly greater with QPCA than with any other category of experiment. When older donors were used (stages 8-9), the incidence of compact hearts fell. No variations in the origin of the graft, nor in its ultimate destination in the host, were found to affect the frequency of any of the anomalies. Sections showed that quail hearts tended to have thicker walls than chick hearts; although quail tissues were often incorporated into the host chick hearts, they retained the histological characteristics of the donors. The fact that no compact hearts resulted from the experiment QACA, or from the mock operations, leads us to conclude that failure to loop in the compact hearts was not due to mechanical trauma caused by the operation, but to some specific difference between grafts taken from the anterior and posterior precardiac mesoderm. The fact that compact hearts were obtained when chick donors were used instead of quails, shows that the effect is not species-specific. We propose that a morphogen is secreted by the posterior end of the precardiac mesoderm and this plays a role in controlling the cessation of looping.

Caffeine is a naturally occurring product that acts as a mild central nervous system stimulant. In humans the major sources of caffeine are coffee, tea, and soft drinks, as well as cocoa, chocolate, and certain medications. Caffeine is metabolized more slowly in pregnant women and due to the hydrophobic properties of caffeine it can cross the placenta and the brain-blood barrier. Studies in human and animal models have shown that caffeine exposure during pregnancy affects the perinatal cardiovascular system as well as central nervous system and can result in intrauterine growth retardation and stillbirth. Recent studies show that caffeine intake increases risk of first-trimester spontaneous abortion in human. However, the extent and the mechanism by which maternal caffeine intake influences embryonic cardiovascular function during early morphogenesis is not known. We hypothesized that caffeine ingestion during early pregnancy impairs embryonic cardiac function by delaying the onset of heart beat and alters the normal increase in heart rate (HR) resulting in growth delay and first-trimester spontaneous abortion. Eight to 12 week-old pregnant CD-1 mice and 81embryos were studied under an approved IACUC protocol. Caffeine was dissolve in distilled water and administered daily by gavage at a dose of 120mg/kg from gestational days 0.5 to 10.5. We monitored embryonic heart rate (HR) from gestational days 8.5 to 10.5 at 24 hour intervals using a 40MHz ultrasound biomicroscope. At gestational day 10.5, embryos were fixed and somite number and external morphology was assessed. This period of gestational includes the onset of heart beat of the primitive heart tube through the completion of heart looping. Onset of heart beat was significantly delayed in caffeine group at gestational day 8.5 (heart beat was detected in 41% of caffeine treated embryos versus 79% of sham treated embryos). HR increase was higher in caffeine group at gestational days 9.5 (127±4 in caffeine vs. 112±5 in sham, respectively, p

During the first 20 days of development, the human embryo has no cardiovascular structure. Over the next month, the heart and great vessels complete their development and look very much like they will at full gestation. This amazing process transforms isolated angiogenic cell islets into a complex, four-chambered structure. During this transformation, the single heart tube begins to beat at 23 days of development and by 30 days blood circulates through the embryo.