The distribution and characterization of HNK-1 antigens in the developing avian heart

The heart originates from splanchnic mesoderm and to a lesser extent from neural crest cells. The HNK-1 monoclonal antibody is a marker for early migrating neural crest cells, but reacts also with structures which are not derived from the neural crest. We investigated whether heart structures are HNK-1 positive before neural crest cells colonize these target tissues. To that end, we determined the HNK-1 antigen expression in the developing avian heart on immunohistochemical sections and on Western blots. The HNK-1 immunoreactivity in the developing chick heart is compared with data from literature cm the localization of neural crest cells in chick/quail chimeras. Structures with neural crest contribution, including parts of the early outflow tract and the related endocardial cushions, the primordia of the semilunar valve leaflets and the aorticopulmonary septum were HNK-1 positive. Furthermore, other structures were HNK-1 positive, such as the atrioventricular cushions, the wall of the sinus venosus at stage HH 15 through 21, parts of the endocardium at E3, parts of the myocardium at E6, and the extracellular matrix in the myocardial base of the semilunar valves at E14. HNK-1 expression was particularly observed in morphologically dynamic regions such as the developing valves, the outflow tract cushion, the developing conduction system and the autonomie nervous system of the heart. We observed that atrioventricular endocardial cushions are HNK-1 positive. We conclude that: a HNK-1 immunoreactivity does not always coincide with the presence of neural crest cells or their derivatives; (2) the outflow tract cushions and atrioventricular endocardial cushions are HNK-1 positive before neural crest cells are expected (stage HH 19) to enter the endocardial cushions of the outflow tract; (3) the observed spatio-temporal HNK-1 patterns observed in the developing heart correspond with various HNK-1 antigens. Apart from a constant pattern of HNK-1 antigens during development, stage-dependent HNK-1 antigens were also found

Acutely altered hemodynamics following venous obstruction in the early chick embryo

In the venous clip model specific cardiac malformations are induced in the chick embryo by obstructing the right lateral vitelline vein with a microclip. Clipping alters venous return and intracardiac laminar blood flow patterns, with secondary effects on the mechanical load of the embryonic myocardium. We investigated the instantaneous effects of clipping the right lateral vitelline vein on hemodynamics in the stage-17 chick embryo. 32 chick embryos HH 17 were subdivided into venous clipped (N=16) and matched control embryos (N=16). Dorsal aortic blood flow velocity was measured with a 20 MHz pulsed Doppler meter. A time series of eight successive measurements per embryo was made starting just before clipping and ending 5h after clipping. Heart rate, peak systolic velocity, time-averaged velocity, peak blood flow, mean blood flow, peak acceleration and stroke volume were determined. All hemodynamic parameters decreased acutely after venous clipping and only three out of seven parameters (heart rate, time-averaged velocity and mean blood flow) showed a recovery to baseline values during the 5h study period. We conclude that the experimental alteration of venous return has major acute effects on hemodynamics in the chick embryo. These effects may be responsible for the observed cardiac malformations after clipping

Ventricular diastolic filling characteristics in stage-24 chick embryos after extra-embryonic venous obstruction

Alteration of extra-embryonic venous blood flow in stage-17 chick embryos results in well-defined cardiovascular malformations. We hypothesize that the decreased dorsal aortic blood volume flow observed after venous obstruction results in altered ventricular diastolic function in stage-24 chick embryos. A microclip was placed at the right lateral vitelline vein in a stage-17 (52-64 h of incubation) chick embryo. At stage 24 (4.5 days of incubation), we measured simultaneously dorsal aortic and atrioventricular blood flow velocities with a 20-MHz pulsed-Doppler velocity meter. The fraction of passive and active filling was integrated and multiplied by dorsal aortic blood flow to obtain the relative passive and active ventricular filling volumes. Data were summarized as means +/- S.E.M. and analyzed by t-test. At similar cycle lengths ranging from 557 ms to 635 ms (P>0.60), dorsal aortic blood flow and stroke volume measured in the dorsal aorta were similar in stage-24 clipped and normal embryos. Passive filling volume (0.07+/-0.01 mm(3)) was decreased, and active filling volume (0.40+/-0.02 mm(3)) was increased in the clipped embryo when compared with the normal embryo (0.15+/-0.01 mm(3), 0.30+/-0.01 mm(3), respectively) (P<0.003). In the clipped embryos, the passive/active ratio was decreased compared with that in normal embryos (P<0.001). Ventricular filling components changed after partially obstructing the extra-embryonic venous circulation. These results suggest that material properties of the embryonic ventricle are modified after temporarily reduced hemodynamic load

Histopathology of aortic complications in bicuspid aortic valve versus Marfan syndrome: relevance for therapy?

Patients with bicuspid aortic valve (BAV) and patients with Marfan syndrome (MFS) are more prone to develop aortic dilation and dissection compared to persons with a tricuspid aortic valve (TAV). To elucidate potential common and distinct pathways of clinical relevance, we compared the histopathological substrates of aortopathy. Ascending aortic wall biopsies were divided in five groups: BAV (n = 36) and TAV (n = 23) without and with dilation and non-dilated MFS (n = 8). General histologic features, apoptosis, the expr

Epicardium-derived cells are important for correct development of the Purkinje fibers in the avian heart

During embryonic development, the proepicardial organ (PEO) grows out over the heart surface to form the epicardium. Following epithelial-mesenchymal transformation, epicardium-derived cells (EPDCs) migrate into the heart and contribute to the developing coronary arteries, to the valves, and to the myocardium. The peripheral Purkinje fiber network develops from differentiating cardiomyocytes in the ventricular myocardium. Intrigued by the close spatial relationship between the final destinations of migrating EPDCs and Purkinje fiber differentiation in the avian heart, that is, surrounding the coronary arteries and at subendocardial sites, we investigated whether inhibition of epicardial outgrowth would disturb cardiomyocyte differentiation into Purkinje fibers. To this end, epicardial development was inhibited mechanically with a membrane, or genetically, by suppressing epicardial epithelial-to-mesenchymal transformation with antisense retroviral vectors affecting Ets transcription factor levels (n = 4, HH39-41). In both epicardial inhibition models, we evaluated Purkinje fiber development by EAP-300 immunohistochemistry and found that restraints on EPDC development resulted in morphologically aberrant differentiation of Purkinje fibers. Purkinje fiber hypoplasia was observed both periarterially and at subendocardial positions. Furthermore, the cells were morphologically abnormal and not aligned in orderly Purkinje fibers. We conclude that EPDCs are instrumental in Purkinje fiber differentiation, and we hypothesize that they coo

Coding of coronary arterial origin and branching in congenital heart disease: The modified Leiden Convention

Objectives: Variations in coronary anatomy are common and may relate to the position of the coronary ostium relative to the aortic sinus, the angle of coronary take-off, or the course of the coronary arterial branches. Several classification systems have been proposed. However, they all lack a simple rationale that is applicable irrespective of the relative position of the great arteries, as well as in bicuspid aortic valves. We present a modification of a relatively simple system introduced in the early 1980s, designated the “Leiden Convention.” Methods: The first step of the Leiden Convention is that the clinician takes position in the nonfacing sinus of the aorta looking toward the pulmonary orifice. The right-hand facing sinus is sinus 1, and the left-hand facing sinus is sinus 2. The coronary branches arising from sinus 1 are annotated proceeding in a counterclockwise fashion toward sinus 2. “Usual” (normal) coronary anatomy would be 1R-2LCx. Given their clinical relevance, single sinus coronary arteries are discussed separately. Results: This system was originally designed and highly applicable in hearts with an altered great artery relationship, such as in the var