In vitro study of flow regulation for pulmonary insufficiency.

Given the tolerance of the right heart circulation to mild regurgitation and gradient, we study the potential of using motionless devices to regulate the pulmonary circulation. In addition, we document the flow performance of two mechanical valves. A motionless diode, a nozzle, a mechanical bileaflet valve, and a tilting disk valve were tested in a pulmonary mock circulatory system over the normal human range of pulmonary vascular resistance (PVR). For the mechanical valves, regurgitant fractions (RFs) and transvalvular pressure gradients were found to be weak functions of PVR. On the low end of normal PVR, the bileaflet and tilting disk valves fluttered and would not fully close. Despite this anomaly, the regurgitant fraction of either valve did not change significantly. The values for RF and transvalvular gradient measured varied from 4 to 7% and 4 to 7 mm Hg, respectively, at 5 lpm for all tests. The diode valve was able to regulate flow with mild regurgitant fraction and trivial gradient but with values higher than either mechanical valve tested. Regurgitant fraction ranged from 2 to 17% in tests extending from PVR values of 1 to 4.5 mm Hg/lpm at 5 lpm and with concomitant increases in gradient up to 17 mm Hg. The regurgitant fraction for the nozzle increased from 2 to 23% over the range of PVR with gradients increasing to 18 mm Hg. The significant findings were: (1) the mechanical valves controlled regurgitation at normal physiological cardiac output and PVR even though they failed to close at some normal values of PVR and showed leaflet flutter; and (2) it may be possible to regulate the pulmonary circulation to tolerable levels using a motionless pulmonary valve device.

Cardiopulmonary malformations in the inv/inv mouse.

The inv/inv mouse carries an insertional mutation in the inversin gene, (inv, for inversion of embryonic turning). Previously it had been reported that almost 100% of the homozygous offspring (inv/inv) were characterized by situs inversus totalis. In this report we identify the spectrum of cardiopulmonary anatomical abnormalities in inv/inv mice surviving to birth to determine whether the abnormalities seen are of the categories classically associated with human situs abnormalities. Stillborn mice, offspring that died unexpectedly (within 48 hr after birth), and neonates with phenotypic characteristics of situs inversus (right-sided stomachs, growth failure or jaundice) were processed for standard histological examination. Of 173 offspring, 34 (20%) neonates (11 stillborn, 9 unexpected deaths, and 14 mice with situs inversus phenotype) were examined, 27 of which were genotyped to be inv/inv. Interestingly, three inv/inv mice (11%) were found to have situs solitus. Twenty-four had situs inversus with normal, mirror-image cardiac anatomy (dextrocardia with atrioventricular concordance, ventriculoarterial concordance and a right aortic arch). The overall incidence of cardiovascular anomalies observed was 10 out of 27 (37%). The most frequent severe malformation, identified in 3 out of 27 animals, was a complex consisting of pulmonary infundibular stenosis/atresia with absence of pulmonary valve tissue and a ventricular septal defect. The pulmonary phenotype in inv/inv mice was situs inversus with occasional minor lobar abnormalities. We conclude that 1) cardiopulmonary malformations in inv/inv mice are not rare (37%), 2) the cardiopulmonary malformations observed in inv/inv specimens are not of the spectrum typically associated with human heterotaxia. In particular, inv/inv mice have a propensity for defects in the development of the right ventricular outflow tract and the interventricular septum, and 3) approximately one out of ten inv/inv mice is born with situs solitus and shows cardiac anomalies that correspond to those observed in inv/inv specimens with situs inversus. Our data therefore suggest that inversin, the product of the inv locus, may have specific roles in cardiac morphogenesis independent of its role in situs determination.

Conotruncal anomalies in the trisomy 16 mouse: an immunohistochemical analysis with emphasis on the involvement of the neural crest.

The trisomy 16 (Ts16) mouse is generally considered a model for human Down's syndrome (trisomy 21). However, many of the cardiac defects in the Ts16 mouse do not reflect the heart malformations seen in patients suffering from this chromosomal disorder. In this study we describe the conotruncal malformations in mice with trisomy 16. The development of the outflow tract was immunohistochemically studied in serially sectioned hearts from 34 normal and 26 Ts16 mouse embryos ranging from 8.5 to 14.5 embryonic days. Conotruncal malformations observed in the Ts 16 embryos included double outlet right ventricle, persistent truncus arteriosus, Tetralogy of Fallot, and right-sided aortic arch. This spectrum of malformations is remarkably similar to that seen in humans suffering from DiGeorge syndrome (DGS). As perturbation of neural crest development has been proposed in the pathogenesis of DGS we specifically focussed on the fate of neural crest derived cells during outflow tract development of the Ts16 mouse using an antibody that enabled us to trace these cells during development. Severe perturbation of the neural crest-derived cell population was observed in each trisomic specimen. The abnormalities pertained to: 1) the size of the columns of neural crest-derived cells (or prongs); 2) the spatial orientation of these prongs within the mesenchymal tissues of the outflow tract; and 3) the location in which the neural crest cells interact with the myocardium. The latter abnormality appeared to be responsible for ectopic myocardialization found in trisomic embryos. Our observations strongly suggest that abnormal neural crest cell behavior is involved in the pathogenesis of the conotruncal malformations in the Ts16 mouse.

Regulation of SERCA 2 expression by thyroid hormone in cultured chick embryo cardiomyocytes.

We investigated the role of thyroid hormone in the physiological perinatal increase in cardiac sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) expression. We isolated and cultured the cardiomyocytes in 10(-8) M triiodothyronine (T3) for 48 h and then measured SR Ca(2+)-ATPase mRNA and immunodetectable protein contents as well as SR-dependent 45Ca2+ uptake rate. We also examined the effect of T3 on expression of the same gene in monkey kidney CV-1 cells, which do not express thyroid hormone receptors. T3 increased cardiomyocyte SR Ca2+ pump mRNA content by 289 +/- 35%, and immunodetectable SR Ca2+ pump protein content by 255 +/- 44%, and SR-specific 45Ca2+ uptake rate by 189 +/- 22% (P < 0.01 for each). In contrast, T3 had no significant effect on the total cellular RNA or protein contents in the cardiomyocyte, and there was no effect of T3 on Ca(2+)-ATPase mRNA content in the thyroid hormone receptor-negative CV-1 cells. These data demonstrate that T3 increases expression of the cardiac SR Ca2+ pump, that the effect can be localized to the cardiomyocyte, and that the effect is dependent on thyroid hormone receptors. These data are consistent with pretranslational and possibly transcriptional level effect of thyroid hormone on the cardiac SR Ca2+ pump gene (SERCA 2). The gestation-associated increase in thyroid hormone may be at least partially responsible for the previously demonstrated perinatal increase in cardiac SR Ca2+ pump expression.

Activation of the cardiac alpha-actin promoter depends upon serum response factor, Tinman homologue, Nkx-2.5, and intact serum response elements.

A murine cardiac specific homeoboxgene, Nkx-2.5/CSX, a potential Drosophila tinman homologue, may have a fundamental role in cardiac myocyte differentiation. DNA binding targets for Nkx-2.5 were recently shown to represent novel homeodomain binding sequences, some of which resembled serum response elements (SREs); [Chen CY, Schwartz RJ (1995): J Biol Chem 270: 15628-15633]. In this study, Nkx-2.5 facilitated serum response factor (SRF) DNA-binding activity to the multiple SREs found on the cardiac alpha-actin promoter and together stimulated cardiac alpha-actin promoter dependent transcription in 10T1/2 fibroblasts. Analysis of cardiac alpha-actin promoter mutants demonstrated the importance of the multiple upstream SREs and an obligatory requirement for an intact proximal SRE1, for providing high levels of activity in the presence of Nkx-2.5 and SRF coexpression. Transfection assays with mutant SRF species indicated that the C-terminal activation domain and DNA-binding MADS box were necessary for transcriptional activity in the presence of Nkx-2.5. Expression of Nkx-2.5 mutants also demonstrated that the homeodomain alone was insufficient for directing promoter activity in the presence of SRF. The central role of SRF in regulating striated alpha-actin gene activity also was revealed by its embryonic expression restricted primarily to myocardium of the developing heart and the myotomal portion of somites. Thus the function of the cardiac actin promoter SREs appeared to provide binding sites for SRF and Nkx-2.5 to interact and elicit striated muscle specific transcription that was independent of the MyoD family.

The avian cardiac alpha-actin promoter is regulated through a pair of complex elements composed of E boxes and serum response elements that bind both positive- and negative-acting factors.

The chicken alpha-cardiac actin is one of the earliest contractile protein genes selectively expressed during embryonic skeletal and cardiac muscle differentiation. Cardiac actin promoter elements were examined in these two sarcomeric cell types. A portion of the alpha-cardiac actin promoter responsible for striated muscle specificity has been delineated (1, 2) and shown to contain four serum response elements (SRE). Previously, SRE3 was shown to be part of a complex element in conjunction with a functional E box (2), and we now show that SRE4 is also part of an upstream SRE.E box cis-element complex. The SREs function similarly, but the E boxes have dissimilar properties within and between striated muscle types. The SRE3.E1 box binds myogenic basic helix-loop-helix factors and is required for cardiac actin trans-activation in primary muscle cell cultures but functions as a negative regulatory element in cardiac muscle cells. The SRE4.E2 box, on the other hand, fails to bind basic helix-loop-helix (bHLH) factors, is negative acting in skeletal muscle cells, and is positive acting in cardiac myocytes. A DNA binding factor similar to HF1a (3) was identified that interacts specifically with the SRE4.E2 box. This study shows that the avian cardiac actin promoter elements are differentially used between skeletal and cardiac striated muscle cell lineages.

Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation.

To assess the role of cis-acting elements within the smooth muscle alpha-actin gene in smooth muscle cells (SMC), we transfected chicken smooth muscle alpha-actin promoter-chloramphenicol acetyltransferase gene fusion plasmids into SMC derived from rat and chicken aortas. In marked contrast to effects in chicken skeletal myoblasts and fibroblasts, p122CAT (positions -122 to +19), containing two conserved CArG elements, elicited a modest increase in chloramphenicol acetyltransferase reporter activity in chicken SMC. Addition of upstream sequences between -122 and -151 (p151CAT) increased activity in adult chicken SMC. Addition of sequence between positions -151 and -257 (p257CAT) resulted in a 7-fold increase in chloramphenicol acetyltransferase activity over that of p151CAT in rat SMC, but not in chicken SMC. A genomic clone encoding the rat smooth muscle alpha-actin gene was isolated, and the 5'-flanking region was partially characterized. Comparison of primary sequence between rat and chicken promoters showed a conserved E box motif at position -214 in the chicken gene and at position -213 in the rat gene. Results of these studies demonstrate that regions upstream of the conserved CArG elements exert potent regulatory effects on transcription and that SMC require different cis-acting elements than other cell types to transcriptionally regulate this gene.