Genetic Basis for Human Congenital Heart Disease? A gene discovery approach with mouse chemical mutagenesis was launched in 2002 to recover novel mutations causing congenital heart defects. We developed the use of mouse fetal echocardiography as a high throughput noninvasive method for cardiovascular phenotyping fetal mice in utero. Over the course of 3 years, we scanned nearly 14,000 fetuse from nearly 500 families (defined by the G1 male), with an estimate of 10,000 genes scanned (500 X 20 genes/family), or approximately 0.5 genome equivalent. We recovered mutants with phenotypes that include all of the major congenital heart defects seen clinically. The mutations in 15 families have been mapped, with 11 mutations identified. Surprisingly, 7 of the 14 mutants with structural heart defects show heart and limb anomalies, with 4 exhibiting defects in left-right patterning. 7 of the 11 mutations identified were entirely novel genes not previously known to be associated with structural heart defects. The collection of mutations we have recovered suggest a key pathway in congenital heart defects involves the cilia and planar cell polarity signaling. Thus, the focal point for much of the present research in my laboratory revolves around understanding the role of the cilia in orchestrating events in cardiovascular development, and how it transduces and regulate signaling through the cilia.? ? High Frequency Ultrasound Cardiovascular Phenotyping? My laboratory has a strong research interest in developing the use of ultra high frequency ultrasound imaging for mouse fetal cardiovascular assessments. In our ENU mouse mutagenesis screen, we utilized a clinical ultrasound system, the Acuson Sequoia for in utero fetal mouse imaging with a 15 MHz transducer. From the data obtained from scanning nearly 13,000 fetuses, a large database of normative values was established for cardiac structure and hemodynamic function in embryos from E14.5 to E19.5 (term) (Yu et al., 2008). Using the Visualsonics Biomicroscope, we quantitatively assessed adult cardiac function, utilizing the recently developed ECG gated imaging algorithm known as EKV. EKV simulates a frame rate of 1000 frames/second, a temporal resolution that is unmatched by any clinical ultrasound system. This high frame rate allows accurate 2D visualization of the adult heart through its entire cardiac cycle the adult mouse heart usually beats at a rate of 450-550 beats per minute. Using EKV, we did a detailed study of a new mutant mouse model recovered in our ENU screen with hypertrophic cardiomyopathy (Leatherbury et al., 2008). ? ? Primary Ciliary Dyskinesia in Human Complex Congenital Heart Disease? A clinical project has been initiated in February 2008 involving the investigation of the role of primary ciliary dyskinesia (PCD) in complex congenital heart disease associated with heterotaxy. Primary ciliary dyskinesia refers to the dyskinetic movement or immotility of cilia in the airway epithelia, which normally beat in rapid synchrony to facilitate mucociliary clearance. Our ENU mutagenesis screened recovered a mutation in Dnahc5, a gene well established to cause PCD in human patients. Analysis our mouse mutant showed it is a bona fide model of PCD. Surprisingly, these mice exhibited an unexpectedly high incidence (40%) of complex congenital heart disease together with heterotaxy, indicating heterotaxy and complex structural heart defects can arise from mutations causing PCD. Heterotaxy patients with complex congenital heart disease often must undergo risky cardiac surgeries to correct or repair the structural heart defects, and not infrequently have complicated course with high mortality, with a subset becoming ventilator dependent for unknown causes. We hypothesize that the latter complications might be related to an underlying undiagnosed disease involving PCD. More broadly, the association between PCD and heterotaxy and complex congenital heart disease could signify a ciliary basis for a wide spectrum of congenital heart disease previously thought to have no unifying etiology. With this underlying hypothesis, we initiated a human study protocol to evaluate for evidence of PCD in patients undergoing high risk cardiac surgeries at Childrens National Medical Center to correct for complex structural heart defects associated with heterotaxy. Since February 2008, 29 patients have been recruited to our study, and surprisingly, nearly 25-30% of these patients show evidence indicative of PCD based on the analysis of ciliary motion and/or nasal NO measurements. Ultrastructural EM analysis are underway to examine for ciliary ultrastural defects, as well as sequencing for mutations in DNAH5, the human homolog of Dnahc5. It should be noted that PCD is otherwise relatively uncommon in the human population, with an incidence of 0.005%. These findings suggest a significant association of undiagnosed PCD with complex congenital heart disease. The validation of these findings will suggest a change in the standard of care, such that cardiac surgery patients may benefit from presurgical evaluation for PCD, with more aggressive pre and post-surgical pulmonary management for patients with evidence of PCD. This may improve outcomes with improvements in cardiopulmonary status and the reduction in long term dependency on ventilator-assisted breathing.? ? Atlas of Mouse and Human Cardiovascular Development? To facilitate analysis of complex structural heart defects in our mutant mouse models, we developed instrumentation and methodology for episcopic fluorescence image capture (EFIC). EFIC is a histological method where tissue autofluorescence is used to image the block face as paraffin embedded tissue is sectioned. This generates 2D image stacks in perfect registration (Rosenthal et al., 2003), thereby allowing digital resectioning of the specimen in any plane. Moreover, high resolution 3D reconstructions can be generated with ease. Using EFIC, we have created a Mouse Cardiovascular Development Atlas containing 2D images and 3D volumes of the embryonic and fetal mouse heart from E9.5 to term. We also constructed an Atlas of the Human Embryo from Carnegie Stage 13 to stage 23. Using data obtained form the Human Embryo Atlas, we have constructed a detailed analysis of human cardiovascular development from Carnegie stages 13 to 23. These studies complements our mouse mutagenesis screen, allowing further mouse/human comparisons that may yield novel insights into the etiology of human congenital heart disease. The Human Atlas study, as well as the study of human cardiac development has been submitted for publication and are now under review.? ? Connexin43 and the Modulation of Cardiovascular Development? ? My laboratory has a long standing interest in understanding the role of connexins in cardiovascular development. Our focus is on the gap junction gene, connexin43, as connexin43 knockout mice are known to die at birth due to outflow tract obstruction. Our recent studies showed this entails an unexpected role for connexin43 in modulating the dynamic organization of the actin and tubulin cytoskeleton (Xu et al., 2006). Studies are under way using various connexin43-GFP fusion constructs and connexin43 deficient mouse embryonic fibroblasts together with TIRF microscopy, to identify the protein domains important in modulating crosstalk between connexin43 and the actin/tubulin cytoskeleton. Our studies indicate an essential integration of connexin43 with the dynamic regulation of the cytoskeleton. These studies have been submitted as two manuscripts for publication.
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