Tony L. Creazzo

Targeted inactivation of the sodium-calcium exchanger (Ncx1) results in the lack of a heartbeat and abnormal myofibrillar organization

Contraction of cardiac muscle is triggered by an intracellular buildup of Ca2+ during excitation‐contraction (E‐C) coupling. The Na+/Ca2+ exchanger (Ncx1) is highly expressed in cardiomyocytes and is thought to serve a housekeeping function by maintaining a low intracellular Ca2+ concentration. However, its role in E‐C coupling is controversial. To determine the precise role of Na+/Ca2+ exchange in development of the mammalian heart, we used gene targeting to delete Ncx1. Heterozygous mice are normal and fertile, whereas Ncx1‐null embryos are growth‐retarded and survive to 11.0 days postcoitum but lack a spontaneously beating heart. Moreover, normal heart morphogenesis (specification, looping, and chamber formation) occurred relatively normally within Ncx1‐null embryos. In addition, Ncx1‐nulls displayed relatively normal transient Ca2+ signals when electrically stimulated. This suggests that the Ca2+ delivery mechanism was fundamentally intact, and that Ncx1‐null cardiomyocytes can regulate intracellular Ca2+ concentrations despite the absence of Ncx1. However, ultrastructural analysis revealed that Ncx1‐null cardiomyocytes have a complete lack of organized myofibrils and Z‐lines when compared with normal littermates. These data demonstrate that Ncx‐1 is a Ca2+‐ gene that is essential for normal cardiomyocyte development and function and may serve as an animal model for functionally related human congenital heart defects.

A NOVEL ROLE FOR CARDIAC NEURAL CREST IN HEART DEVELOPMENT

Ablation of premigratory cardiac neural crest results in defective development of the cardiac outflow tract. The purpose of the present study was to correlate the earliest functional and morphological changes in heart development after cardiac neural crest ablation. Within 24 hours after neural crest ablation, the external morphology of the hearts showed straight outflow limbs, tighter heart loops, and variable dilations. Incorporation of bromodeoxyuridine in myocytes, an indication of proliferation, was doubled after cardiac neural crest ablation. The myocardial calcium transients, which are a measure of excitation-contraction coupling, were depressed by 50% in both the inflow and outflow portions of the looped heart tube. The myocardial transients could be rescued by replacing the cardiac neural crest. The cardiac jelly produced by the myocardium was distributed in an uneven, rather than uniform, pattern. An extreme variability in external morphology could be attributed to the uneven distribution of cardiac jelly. In the absence of cardiac neural crest, the myocardium was characterized by somewhat disorganized myofibrils that may be a result of abnormally elevated proliferation. In contrast, endocardial development appeared normal, as evidenced by normal expression of fibrillin-2 protein (JB3 antigen) and normal formation of cushion mesenchyme and trabeculae. The signs of abnormal myocardial development coincident with normal endocardium suggest that the presence of cardiac neural crest cells is necessary for normal differentiation and function of the myocardium during early heart development. These results indicate a novel role for neural crest cells in myocardial maturation.

FGF-8 in the ventral pharynx alters development of myocardial calcium transients after neural crest ablation

Cardiac neural crest ablation results in depressed myocardial calcium transients and elevated proliferation in myocardium at a stage when cardiac neural crest cells are not in contact with the myocardium. To test the hypothesis that cardiac neural crest-derived cells, which migrate into the caudal, ventral pharynx at stage 14, block a signal from the ventral pharynx, we cultured stage 12 chick heart tube or myocardial strips in the presence or absence of ventral pharynx. We found that myocardium cultured with ventral pharynx that had not yet contacted neural crest cells had significantly reduced calcium transients and an increased rate of proliferation. Ventral pharynx from intact embryos at a stage when neural crest-derived cells had reached the pharynx had no effect on myocardial calcium transients. Ventral pharynx from neural crest-ablated embryos continued to suppress myocardial calcium transients at this later stage. Myocardium cultured with FGF-2 also showed a significant reduction in calcium transients. An FGF-2-neutralizing Ab reversed the deleterious effect of the ventral pharynx on myocardial calcium transients and proliferation. We therefore examined the expression of FGF-2 and similar FGFs in the ventral pharynx. Only FGF-8 was expressed in a temporospatial pattern that made it a viable candidate for altering the myocardial calcium transient during stages 14-18. In explant cultures, neutralizing Ab for FGF-8 rescued development of the myocardial calcium transient in neural crest-ablated chick embryos.

Effects of chronic injections of α-bungarotoxin on embryonic cell death

Abstract Effects of chronic and massive amounts of α-bungarotoxin introduced into the yolk sac of the duck embryo on the magnitude of the spontaneously occurring embryonic cell death in the trochlear nucleus were examined. As expected, the embryos continuously treated with the toxin remained paralyzed. As long as the embryos remained continously paralyzed during the period of normal cell death, a significant increase in the number of trochlear neurons occurred. Axon counts of the trochlear nerve indicated a corresponding increase in the number of nerve fibers. Counts of the myelinated and unmyelinated fibers indicated that the increased fiber count was primarily due to increased numbers of unmyelinated axons. Because the increased cell number is due neither to a stimulatory effect of the toxin on cellular proliferation nor to a transient effect, this increase is interpreted to mean a reduction in the magnitude of the embryonic cell death. As revealed by the retrograde axonal flow of horseradish peroxidase, all trochlear neurons including those which had been prevented from dying during the period of paralysis projected to their appropriate peripheral muscle suggesting that they did not survive by virtue of making improper connections. When the toxin treatment was carried out only during the period of cell death and subsequently discontinued, the embryonic motility increased. Cells which were prevented from dying during the period of paralysis degenerated. Counts of the myelinated and unmyelinated fibers indicated a corresponding loss of nerve fibers and that loss of both classes of fibers occurred.

Reduced L-type calcium current in the embryonic chick heart with persistent truncus arteriosus.

Calcium currents were examined in an experimental model in the embryonic chick heart with a congenital malformation known as persistent truncus arteriosus. This is a severe defect characterized by failure of conotruncal and aorticopulmonary septation of the embryonic heart tube. As a result, no separation into the aortic and pulmonary arteries occurs, and there is a common outflow tract. The hearts with persistent truncus arteriosus had a 26% greater ventricular to whole embryo weight, which indicated that the ventricles were enlarged. Both the low-threshold T-type (ICa.T) and the 1,4-dihydropyridine-sensitive L-type (ICa.L) Ca2+ currents were present in the ventricular myocytes from hearts at day 11 of incubation. However, day 11 hearts with persistent truncus arteriosus showed a twofold reduction in the peak magnitude of ICa.L at a test potential of + 10 mV without a concomitant reduction in the number of L channels detected by 1,4-dihydropyridine antagonist [(+)[3H]PN200-110] and agonist (Bay K 8644) receptor binding. The results indicated that an L channel regulatory mechanism other than protein synthesis was affected. These changes are consistent with responses to conditions of excessive hemodynamic burden that have been characterized in adult hearts.

Effects of chronic paralysis with α-bungarotoxin on development of innervation

Abstract Chronic paralysis of the duck embryo with α-bungarotoxin increases the number of trochlear motor neurons that survive by decreasing the magnitude of embryonic cell death. The present study examined how such an increase in the motor neuron projection is accommodated at the peripheral target. The results indicate that the overall size of the duck superior oblique muscle was drastically reduced following chronic paralysis with α-bungarotoxin. The number of motor end-plates was reduced to about 25% of the control. Although the total number of end-plates was significantly reduced, their size was larger and they became innervated by far more nerve terminals than in the control. Thus, increased motor neuron survival occurred while the size of the periphery and the number of synaptic sites (end-plates) were greatly reduced. These observations suggest that the cause of embryonic cell death may not be related to the availability of adequate numbers of synapses with the periphery. They also suggest that the physiologic activity of the embryo may play a significant role in regulation of cell number in the developing nervous system.

Excitation-Contraction Coupling in the Day 15 Embryonic Chick Heart with Persistent Truncus Arteriosus

Ca2+ transients were examined in embryonic chick hearts with an experimentally induced cardiac neural crest-related outflow tract defect known as persistent truncus arteriosus (PTA). In all of the animal models of neural crest-related heart defects, prenatal mortality is too high to be attributed to structural defects of the heart alone, suggesting that there is altered development of the myocardium. Earlier reports indicating reduced L-type Ca2+ current in hearts with PTA suggest that poor viability may be related to impairment of cardiac excitation-contraction coupling. To test this hypothesis, direct measurements of the systolic Ca2+ transient in fura-2-loaded myocytes from normal hearts and hearts with PTA were carried out. We found that Ca2- transients were severely depressed in hearts with PTA and difficult to measure above background noise unless signal averaged or treated with isoproterenol (ISO). We confirmed that the reduced Ca2+ transients were due, at least partly, to a reduction in L-type Ca2+ current. In addition we found that although ISO could raise the L-type current in hearts with PTA to the level found in normal hearts in the absence of ISO, it could not fully restore the Ca2+ transient. Furthermore, caffeine-stimulated Ca2+ transients were diminished in size and the time-to-peak and the decaying phase were significantly slowed. Interestingly, these observations were not accompanied by a reduction in the number of Ca2+ release channels. These results indicated an impairment of SR function in addition to the reduction in L-type Ca2+ current. These results strongly support our hypothesis that the poor viability of embryos with PTA is due to impaired cardiac excitation-contraction coupling.

The effect of 2,3-butanedione 2-monoxime (BDM) on ventricular trabeculae from the avian heart.

Summary2,3-butanedione 2-monoxime (BDM, 3–30 mm) decreased twitch force of intact ventricular trabeculae isolated from 19-day embryonic chick hearts in a dose-dependent manner. The responses to BDM were rapid and reversible. In an attempt to determine the cellular basis for the inhibitory effect of BDM, experiments were carried out on skinned muscle fibres and isolated myocytes. In trabeculae skinned with Triton X-100, BDM depressed maximum calcium activated force (Fmax) with an IC50 of 14 mm. At 3 mm BDM, the proportional decrease in twitch force in intact tissue was similar to that of Fmax in skinned tissue. At higher BDM concentrations (10 and 30 mm), however, the proportional decrease in twitch force was greater than that of Fmax. BDM (up to 10 mm) had no effect on the normalized force-pCa relationship. In saponin-skinned preparations, BDM (3 and 30 mm) released calcium from the fully loaded sarcoplasmic reticulum to a slightly greater extent in the absence of calcium (pCa 8.5) than in the presence of a fixed level of free calcium (pCa 5.5). Whole cell patch clamping of freshly isolated chick myocytes demonstrated that BDM caused a dose-dependent decrease in the T-and L-type calcium current. Therefore, at low BDM concentrations (3 mm), the decrease in twitch force can be ascribed predominantly to depression of the contractile apparatus while, at higher concentrations of BDM, there is an additional inhibitory effect of BDM on excitation-contraction coupling.

Cardiovascular Development: Neural Crest and New Perspectives

The neural crest provides an important population of cells to the developing great arteries and cardiac outflow tract that are essential for normal development of these structures. Neural crest cells migrate from the entire length of the embryonic neural tube, but only the subdivision originating from the posterior hindbrain is involved in structural development of the cardiovascular system. Ablation of the premigratory cardiac neural crest cells results in outflow tract anomalies similar to those seen in children. Functional anomalies of the heart occur long before abnormal structural development is apparent after ablation of the premigratory cardiac neural crest. The functional deficits that preceed and later accompany neural crest-related cardiovascular dysmorphogenesis persist and are probably responsible for the high mortality associated with neural crest-type malformations. The potential underlying causes of neural crest-related cardiovascular malformations is discussed.

ELEVATION OF INTRACELLULAR CA2+ CONCENTRATION IN RABBIT NONPIGMENTED CILIARY EPITHELIAL CELLS BY ALLICIN

A previous study has shown that allicin produces changes in aqueous humor dynamics, and this study was conducted to examine possible cellular mechanisms. In rabbit nonpigmented ciliary epithelial cells, basal levels of [Ca2+]i were determined to be 164 +/- 34 nM. Allicin, a sulfhydryl-reactive agent, induced Ca2+ transients at 0.01 mM and at 0.2 mM, the Ca2+ transient peaked at 732 +/- 35 nM. Allicin-induced Ca2+ transients were prevented by pretreatment with dithiothreitol which did not affect the basal Ca2+ levels. Allicin had only a slight, insignificant, effect on L-type Ca2+ currents, and allicin-induced Ca2+ transients were also present under extracellular Ca(2+)-free conditions. These data suggest that intracellular Ca2+ stores are the most probable source of allicins effect. Pretreatment of cells with ryanodine, an inhibitor of Ca(2+)-induced-Ca(2+)-release, inhibited allicin-induced Ca2+ transients, but the basal Ca2+ levels were unaffected by ryanodine. Thus, allicin-induced Ca2+ transients are most likely mediated through ryanodine-sensitive intracellular Ca2+ stores.