Emulation of Conduction System Functions in the Hearts of Early Mammalian Embryos

Abstract

Embry o culture. Whole embryo culture was performed as previously described (5). Wistar rat embryos were explanted on gestational d 9.5, d 0 being the day of positive vaginal smear.Embryos were explanted in Tyrode's solution, the decidual mass was removed, and Reichert's membrane was opened. Embryos were then placed in roller bottles containing heat-inactivated rat serum and cultured under an atmosphere of 90% N z , 5% Oz, and 5% COz as described by New (6). At explanation, embryos contain up to three somites and the primitive heart tube has not yet formed. Embryos were studied either at the five-to sevensomite stage (24 h in culture), at which stage the heart tube has formed but has not begun to loop, or at the 13-to IS-somite stage (36 h in culture), after cardiac looping is complete but before development of the atrial or ventricular septa.Wall motion recording. Embryos with prelooped or looped hearts were examined in Tyrode's solution using a Wild M5 microscope. Embryos were positioned and immobilized as shown in Figure I in a depression made in 9% agar. Embryos were maintained at 37.5 ± OSC by a thermostatically-controlled stage warming/cooling device (Sensortek, Clifton, NJ). Video-tape recordings of the embryonic hearts (at lOOx and 200 x magnification) were made using high-resolution closed circuit video and electronic processing to produce monochrome images of maximum contrast. For wall motion recording, these videotape recordings were played back on a 19-inch monitor. Fiber optic cables were placed over the video images of the proximal atrium, distal atrium, proximal ventricle, and proximal bulbus cordis as indicated in Figure I. The fiber optic cables were connected to photocell amplifiers that converted the changes in light intensity of the video image caused by wall motion to voltage signals, which were then recorded by a multichannel recorder at a paper speed of 50 mm /s. This apparatus has been described previously (7). Because of the nature of the opt ical system , the moment of peak wall motion is the most reliably detected point. Peak wall motion is also less affected by motion of adjacent structures than earliest motion would be, so position of the fiber optic cables was adjusted to optimize recording of peak wall motion. Cycle length was measured from the most proximal site recorded. Intervals between peak wall motion in the proximal and distal atrium (intra-atrial delay), distal atrium and proximal ventricle (atrioventricular delay), and proximal ventricle and proximal bulbus cordis (intraventricular delay) were measured to the nearest 5 ms as shown in Figure 2. Each measurement reported was the mean of five sequential contractions. Linear distance between recording sites was measured on the video screen and converted to JIm by reference to micrometer calibration recorded on each videotape at the same magnifications.Stat istical analysis. Intervals between maximum contraction within the atrium and ventricle and atrioventricular delay in prelooped and looped hearts were analyzed by analysis o...

Immunolabelling patterns of gap junction connexins in the developing and mature rat heart

“…1986;Lloyd and Baldwin 1990). The onset of synchronised contraction in the ventricles of the looping rat heart (Lloyd and Baldwin 1990) matches our initial detection of gap junctions in the right and left ventricular segments (ie. at 10M2 days post-coitus).…”

“…at 10M2 days post-coitus). The paucity of cardiac gap junctions in the atrioventricular junction of 12-day embryos is correlated with delayed conduction through this zone, and the establishment of sequential contraction of the atria and ventricles within comparable staged cultured embryonic rat hearts (Lloyd and Baldwin 1990). A similar delay in electrical conduction through the atrioventricular junction has been reported from studies of avian embryonic hearts (Lieberman and Paes de Carvalho 1965;Arguello et al 1986).…”

“…Although we were unable to detect anti-HJ immunoreactivity in the atrioventricular junction of the embryonic rat heart, it is likely that myocytes in this region are coupled by gap junctions, as it shows coordinated propagation of contractility (Lloyd and Baldwin 1990). In addition to the larger gap-junctional aggregates readily identifiable by confocal microscopy, freeze fracture studies of mammalian developing (Gros et al 1978;Shibata et al 1980;reviewed Larsen 1977) and adult conducting tissues (Masson-Pevet et al 1979;Sugi and Hirakow 1986;Skepper and Navaratnam 1986) reveal that a significant proportion of the gap junctions occur as diminutive (< 0.1 gm diameter), linear and circular aggregates of particles.…”

“…On the contrary, from early in cardiac organogenesis the ventricular trabeculae probably constitute a fast peripheral conduction system whose function appears to relate to the appearance of synchronised contraction of the ventricles (e.g. Lloyd and Baldwin 1990). Furthermore, the co-expression of supposed phenotypes for conducting (i.e.…”

Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart.

“…Although no experimental data are available for mammalian embryos, such a conclusion would agree with data available for comparable stages of chick embryos.45 Furthermore, we have observed in the chick that, indeed, the inside of the ventricular free wall is activated earlier than the outside.5 On the other hand, myocardial zones in which we have not been able to detect binding of antibodies directed against connexin43 are characterized by the persistence of a very low conduction velocity in embryonic hearts. [1][2][3][4][5]40 Therefore, the surface density of large gap junctions appears to qualify as a parameter that can reveal regional differences in conduction velocity in the embryonic heart. In this respect, it is interesting that an inverse relation has been observed between gap junction conductance and voltage sensitivity in developing chick hearts,27,4' suggesting that the gap junctions that we can observe immunohistochemically are no longer voltage sensitive.…”