Cardiac myosin light and heavy chain isotypes in tetralogy of Fallot.

Myosin isotype composition was examined in ventricular infundibular muscle from 40 patients with tetralogy of Fallot, aged from 7 months to 38 years. Results were compared with normal samples of ventricular infundibulum from subjects in the same age range, from 18-20 week old fetuses to 6 month old neonates, and from 43-81 year old adults. Myosin light chain isotypes were examined by one dimensional and two dimensional gel electrophoresis and quantified densitometrically. Heavy chain isotypes were examined by electrophoresis of whole heavy chains and peptide mapping after limited proteolytic digestion with chymotrypsin. At mid-gestation in normal tissues, only ventricular light chain 2 was present but light chain 1 consisted almost equally of atrial and ventricular isotypes. Amounts of atrial light chain 1 declined towards birth and disappeared during the first year after birth, gradually being replaced by ventricular light chain 1. Relative amounts of total light chains 1 and 2 remained equal. In tetralogy of Fallot atrial light chain 1 expression did not cease neonatally with mean values of 11.8% of total light chain 1 present between 7 months and 2 years, decreasing to 1.7% at 6.5-12 years and then increasing again to 3.4% in adults. A value of 34% atrial light chain 1 was present in one subject. As in normal subjects, equimolar amounts of total light chains 1 and 2 were retained. No evidence of new light chain isotypes was found in tetralogy of Fallot. Heavy chain expression was constant in normal infundibulum with only beta-heavy chain (V3 isozyme) present in the fetus, neonate, and adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative structural analysis of myosin after limited tryptic hydrolysis by use of two-dimensional gel electrophoresis.

Polypeptides obtained by limited tryptic digestion of several myosins have been analyzed by a two-dimensional electrophoresis technique. The different maps thus obtained exhibit some common and distinct features specific of the myosins studied. Myosins from rabbit, fast and slow muscle and cardiac tissue, as well as beef cardiac myosin, have been compared. The polypeptides obtained vary in molecular weight from 120 000 60 15 000. The light chains LC1 and LC2, have disappeared. A peptide which comigrates with fast type LC3 is however found. The fast myosin hydrolyzate is very different from that obtained by the hydrolysis of slow and cardiac myosin. Numerous peptides are common to cardiac and slow myosins. However a few peptides are specific of the two myosin types. In the hydrolyzate from fetal calf myosin, some of the typical slow, fast and cardiac peptides can be found. However several apparently unique fetal peptides are also present. The comparison of the fetal calf tissue myosin hydrolyzate and that of [35S] methionine labeled myosin from myotubes in cultures shows qualitatively a very great homology. Thus the protein synthesized by cultured cells seems to be very similar to or the same as that of the embryonic tissue.

Immunological distribution of one form of insulin-like growth factor (IGF)-binding protein and IGF peptides in human fetal tissues.

Insulin-like growth factors (IGFs) are expressed by, and are biologically active on, human fetal cells. The mitogenic actions of IGF-I are modulated by the 21-41 kDa class of IGF-binding proteins (IGF-BPs). Using a rabbit anti-human IGF-BP antibody raised against a highly pure 26 kDa IGF-BP derived from amniotic fluid, we have compared the cellular location of IGF-BP and IGF peptides in tissue sections from prostaglandin-induced human abortuses of 14-16 weeks of gestation. The monoclonal and polyclonal antibodies used were raised against human IGF-I, but did not distinguish between IGF-I and IGF-II. Positive staining for IGF-BP was seen in every tissue except brain, spleen and thyroid. With the exception of skin, the cellular distribution of IGF-BP was similar to that of IGF peptides. Strong immunostaining was found in hepatocytes, hepatic erythropoietic cells, pulmonary epithelium, the tubular epithelium of kidney, intestinal epithelia, the fetal adrenal cortex and cardiac and skeletal muscle fibres. In skin, IGF-BP was located throughout the dermis and in the germinal layer of the epidermis. IGF peptide in skin was restricted to the deeper dermal layers. In the tibial epiphyseal growth plate both IGF-BP and IGF peptide were located in chondrocytes throughout the proliferation and hypertrophic zones. The similarity in distribution of IGF-BP and IGF peptides in fetal tissues suggests that the latter may exist predominantly complexed to IGF-BP in or on the surfaces of cells in vivo. The distribution of IGF-BP may define the sites of biological action of IGF peptides.

Decreased concentration of myocardial alpha-adrenoceptors with increasing age in foetal lambs.

Using [3H]-dihydroergocryptine, we identified myocardial alpha-adrenoceptor binding sites in foetal lambs and demonstrated that the concentration of receptors decreased with increasing foetal age. The presence of the receptor in the foetus correlated with the presence of myocardial alpha-adrenergic responsiveness. However, we found neither the alpha-receptor binding site nor responsiveness to alpha-adrenoceptor stimulation in the myocardium of adult sheep.

Expression of genes encoding two chains of the collagen type VI molecule during human fetal heart development.

Northern blot analysis was used to demonstrate the expression of genes encoding two chains of the extracellular matrix molecule, collagen type VI, in human fetal heart tissue. Both genes have been mapped to the Down's obligate region of chromosome 21. As congenital heart malformations are a common feature of Down's syndrome, developmentally regulated structural protein genes encoded by chromosome 21 are being investigated.

Development of immunoreactive atrial natriuretic peptide in fetal hearts of spontaneously hypertensive and Wistar-Kyoto rats.

The development of immunoreactive atrial natriuretic peptide (ANP) was studied in fetal hearts of spontaneously hypertensive (SHR) and compared to normotensive Wistar-Kyoto (WKY) rats. While SHR fetal hearts were noticeably less developed than those of WKY at 10 and 11 days gestation, both strains showed ANP immunoreactive cells in some but not all primitive heart tubes. At 12 days additional ANP immunoreactive cells appeared in formative trabeculae of the ventricle and atrium. ANP cells were also observed in the myogenic layer of the truncus and bulbus arteriosus and their derivatives from 11 through 16 days, but not at 18 days. In both strains, there were more ANP cells in the left ventricle than in right beginning at day 13. There were no obvious strain differences in the developmental pattern and timing of ANP producing cells. However, on the day of birth, staining was reduced in hearts from some WKY newborn pups compared with hearts from SHR newborns and ventricular staining was reduced in both strains when compared to fetal hearts. These observations indicate that ANP is one of the earliest peptide hormones produced and that the predisposition to genetic hypertension does not appear to influence the development of ANP.

Glycogen content in human embryonic heart.

Glycogen concentration in the human embryonic heart is about 10 times higher than in that of human adults. Glycogen content of embryonic right atrium is significantly higher than that in ventricles.

Histological studies of normal and mutant mouse embryo hearts (a glycogen content study).

Mutant tail-short (Ts/+) embryos are developmentally retarded compared with normal +/+ litter mates. The development of the heart of Ts/+ embryos is severely affected if the tail-short gene is transferred to a new genetic (50% A/Gr) background. The aim of the present study was to investigate the glycogen content of the sinus muscle, the cushion and the atrial and ventricular walls of the heart. In normal embryos the sinus muscle is well developed by the 15th day post coitum (d.p.c.) and is crowded with glycogen granules. In Ts/+ mutant embryos, on the other hand, the development of this muscle is retarded and it contains only a little, diffusely distributed glycogen. The atrial and ventricular walls of embryos with a normal heart are well trabeculated and contain a large quantity of glycogen granules, while in mutant embryos they are less well trabeculated and contain only a little glycogen in a diffuse of finely granular form.

[Changes in the composition of heart proteins during early ontogenesis in normal subjects and in the presence of reduced uteroplacental blood flow]. / Izmeneniia sostava belkov serdtsa v rannem ontogeneze v norme i v usloviiakh umen'shennogo matochno-platsentarnogo krovotoka.

The heart was studied on the 11th, 12th, 14th, 16th and 18th days of the rabbit embryo development under normal conditions and in decreased uteroplacental circulation. Under normal conditions the heart weight/embryo weight ratio was particularly high during the intensive development of hemodynamic functional "embryo -- placenta -- uterus" system (on the 12th and the 14th day), and then this ratio decreased. An increase of the total heart nitrogen and alterations in fractional composition on account of increase of contractile proteins and stroma proteins was particularly high by the 18th day. In pathology a decrease of the embryo and heart weights was noted in the course of all the days of the experiment. By the 18th day the heart/embryo weight ratio suggested the beginning of spontaneous rehabilitation due to gradual increase of the uteroplacental circulation. However, the total nitrogen in the heart in all the test groups remained the same as in control or increased, this pointing to the heart dehydration. Changes in the fractional composition of the heart proteins indicated deep biochemical disorders in it, and this could serve as one of the causes of the heart functional derangement.

Localisation of atrial natriuretic peptide immunoreactivity in the ventricular myocardium and conduction system of the human fetal and adult heart.

Atrial natriuretic peptide immunoreactivity was found in ventricular and atrial tissues with specific antisera raised to the amino and carboxy terminal regions of the precursor molecule. In 13 developing human hearts (7-24 weeks' gestation) the immunoreactivity was concentrated in the atrial myocardium and ventricular conduction system but it was also detected in the early fetal ventricular myocardium. Immunoreactivity in five normal adults was largely confined to the atrial myocardium although it was also found in the ventricular conduction tissues of hearts removed from 10 patients who were undergoing cardiac transplantation. The ventricular conduction system is an extra-atrial site for the synthesis of atrial natriuretic peptide. In the failing heart this synthesis may be further supplemented by expression of the gene in the ventricular myocardium. It is possible that ventricular production of the peptide contributes to the raised circulating concentrations of atrial natriuretic peptide immunoreactivity found in severe congestive heart disease, particularly in patients with dilated cardiomyopathy.

Atrial natriuretic peptide in the ventricles of patients with dilated cardiomyopathy and human foetuses.

Immunohistochemical examination of atrial natriuretic peptide (ANP) was performed on endomyocardial biopsy specimens from 8 patients with dilated cardiomyopathy (DCM), 10 human foetal hearts obtained from legal abortions, and 8 adult hearts from autopsy control subjects without cardiovascular diseases. The indirect immunoperoxidase method, using specific monoclonal antibody to alpha-human ANP was employed. Immunoreactivity was observed at the light microscope level in the working ventricular cardiocytes of all patients with DCM as dark-brown, granular deposits. Peripheral plasma levels of ANP in these patients were also increased. In control adult hearts without cardiovascular diseases, immunoreactivity was detected both in the atria and in the ventricular impulse-conducting system, although the working ventricular cardiocytes were not immunoreactive. In foetal hearts, immunoreactivity was detected not only in the atria and ventricular impulse-conducting system, but also in the working ventricular cardiocytes. We conclude that ANP is present in the ventricular impulse-conducting system of the human hearts, and that ANP is also present in the working ventricular cardiocytes in patients with DCM as well as in human foetuses.

[DNA in growing hearts of children. Biochemical and cytophotometric investigations (author's transl)]. / DNS in Kinderherzen. Biochemische und zytophotometrische Untersuchungen

In order to clarify the question of an age-dependent polyploidisation of the myocardium during normal cardiac growth, the DNA-content of 39 hearts of infants and children was investigated. After autopsy the total cardiac weights were estimated and thereafter, following the preparation of the hearts, the pure weight of the myocardium (preparation weight) was determined. Using the diphenylamine reaction according to Dische and Burton, the DNA-concentrations and total amounts of DNA in the heart muscle were estimated. The DNA-content of the heart muscle cell nuclei (ploidy classes) was measured cytophotometrically on Feulgen-stained smears of heart muscle cells. DNA measurements were performed on tissue samples from 6-8 different sites of both heart chambers. Growing human hearts of all ages were investigated including 3 fetal hearts and 3 hearts with malformations.

Body composition studies in the human fetus after intra-amniotic injection of hypertonic saline.

PIP: A systematic investigation of the chemical composition of the total fetus and its individual organs after intraamniotic injection of hypertonic sodium chloride, was performed in 23 fetuses, of 14 to 24 weeks gestation. Fetal death occurs prior to delivery in the great majority of hypertonic saline-induced mid-trimester abortions. This study on the chemical composition of fetal skin, gut, liver, muscle, fat, brain, and heart in 23 cases indicates a high sodium concentration in skin and in intestinal contents and a high and constant sodium concentration in cardiac muscle. Based on these observations, fetal swallowing of hypertonic saline, followed by paralysis of cardiac muscle depolarization when cardiac sodium concentration reaches a threshold level, is suggested as the mechanism of fetal death.^ieng

Differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in the bovine heart: an immuno- and enzyme histochemical study.

The differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in bovine hearts was studied with specific antibodies against M-line proteins (MM-creatine kinase and myomesin) and with enzyme histochemistry (succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase). MM-creatine kinase was detected at an earlier stage in Purkinje fibres and atrial myocytes than in ordinary ventricular myocytes. The findings are in agreement with previous ultrastructural observations that an earlier appearance of a dense M-band occurs in Purkinje fibres than in ordinary ventricular myocytes. Myomesin was detected in all three cell types even at early foetal stages, in accordance with suggestions that it is an integral component of the myofibrillar structure. The activity of succinate dehydrogenase gradually increased in both ordinary ventricular and atrial myocytes, while the activity of mitochondrial glycerol-3-phosphate dehydrogenase was high at different stages of early foetal development in the two tissues, finally becoming low in the adult stage. The activity of succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase seemed to remain unchanged in the Purkinje fibres from early to late foetal stages. The present study shows that the Purkinje fibres are already different from ordinary ventricular myocytes at early foetal stages and that the two cell types differentiate in different ways. It is concluded that there are also developmental differences between ordinary ventricular and atrial myocytes.

Antiserum to a new neuronal growth factor: effects on neurite outgrowth.

A new neuronal growth factor (CMF) isolated from mouse heart-cell-conditioned medium stimulates morphologic and biochemical development of mouse embryo sympathetic neurons [Coughlin et al, 1981]. Further analysis of CMF by chromatographic and electrophoretic procedures has shown that under nondissociating conditions, CMF activity is associated with very high molecular weight material. All biological activity eluted within the included volume of a Sepharose CL-2B column in a molecular weight range corresponding to approximately 5 X 10(6) daltons. Similarly, electrophoresis showed no activity and very little protein entering 3% polyacrylamide gels, whereas both protein and activity migrated through 0.6% agarose-1.2% acrylamide composite gels. To further characterize the biological effects of CMF on normal neuronal development, antibodies to CMF were employed. Rabbits immunized against CMF developed high titres of antibodies with activity specifically directed against CMF (anti-CMF). Although anti-CMF inhibited nerve growth factor (NGF)-stimulated neurite outgrowth from the neonatal superior cervical ganglion, it did not inhibit the NGF-stimulated increase in tyrosine hydroxylase activity. Moreover, ganglia incubated for 3 days in the presence of anti-CMF were subsequently capable of producing neurites when washed and cultured in medium free of antiserum. Thus, anti-CMF specifically blocked neurite extension without causing cell death or irreversible damage in ganglionic explants. Our observations suggest, therefore, that CMF or antigenically similar material is a requirement for neurite extension.

Regulation of glycolytic flux in the heart of the fetal guinea pig.

Functional glycolytic capacity and its regulation have been studied in the fetal guinea-pig heart during O2 deprivation in situ and in the Langendorff perfused heart. Anaerobic glycolytic flux, at 2 mumol/min per g wet wt. was similar in the 48-50 and 60-65 days fetal and adult guinea-pig heart, despite lower fetal phosphofructokinase activity. During O2 deprivation in situ and in the perfused heart glucose was the major substrate, with glycogen making a smaller contribution. Glycolytic capacity became more tightly regulated during fetal heart development. Thus at 48-50 days glycolysis was increased during O2 deprivation by substrate supply, but at 60-65 days activation of phosphofructokinase was required also. Low malate/aspartate cycle activity in the fetal heart was suggested by the absence of an increase in malate and alanine at the expense of aspartate. The large proportion of aerobic glycolytic flux converted to lactate concurred with this. Because of the low O2 consumption and relatively high aerobic glycolytic flux, the proportion of glycolytically-derived ATP was 3-4 fold higher in the fetal than adult heart, and may explain its functional resistance to O2 deprivation.

Cellular development of some embryonic organs and the chorion during the first trimester of human pregnancy.

The DNA, RNA and protein content of chorionic tissue, heart, liver, kidney, lung and brain was estimated in 27 fetuses obtained at termination of pregnancy between 50 and 105 days of menstrual age. For brain, heart and liver, growth appeared to be most rapid at the earliest period studied (50 to 60 days) whereas kidney and lung development was most rapid later in the first trimester. It is suggested that these periods of particularly rapid growth may be the times at which those tissues are most vulnerable to injury.