Histochemical and biochemical analysis of myosin heavy chain expression during cardiogenesis in the rat.

Little is known about the tissue-specific expression of contractile proteins during cardiogenesis in the mammalian heart. Since the myosin heavy chain (HC) isoform expressed in the adult correlates with myocardial functional capacity, we undertook an analysis of myosin HC expression in atrial and ventricular myocardia during fetal cardiogenesis in the rat heart. Cardiac HCs were separated by electrophoresis under denaturing conditions. The expression of the predominant adult isoform HC alpha was localized within developing fetal cardiac chambers by immunohistochemistry with a specific monoclonal antibody (R 37). Results demonstrated that myosin HC isoform expression followed tissue-specific patterns during cardiogenesis in the rat. Atrial myocytes expressed HC alpha throughout development. The ventricles expressed exclusively HC alpha in the adult, but HC beta expression predominated in fetal ventricles. Fetal ventricles also expressed minor amounts of HC alpha, whose amount and distribution varied with developmental stage. HC alpha was initially confined to tracts of cells in the trabeculae, suggestive of future conduction system cells. A more extensive population of HC alpha-expressing cells appeared several days before birth in a pattern which could represent the prenatal initiation of HC alpha expression in working myocardial cells. These results indicate that there is tissue-specific developmental modulation of myosin isoform expression during fetal development. Results also demonstrated that this modulation may include expression of a third electrophoretically distinct myosin HC in fetal hearts.

Ventricular myosin expression in developing and regenerating muscle, cultured myotubes, and nascent myofibers of overloaded muscle in the chicken.

In embryonic chicken skeletal muscle, the presence of a ventricular myosin heavy chain (MHC) is demonstrated by reactivity with an anti-ventricular MHC antibody. Developmental repression of the ventricular MHC expression occurs earlier in embryonic fast twitch posterior latissimus dorsi (PLD) than in embryonic slow-tonic anterior latissimus dorsi (ALD) muscles. Ventricular MHC expression also occurs in developing myotube cultures and in regenerating ALD muscles. Following the application of a weight overload, a population of nascent myofibers emerges in ALD but not PLD muscles which express a ventricular MHC. Localization of cells reactive with anti-ventricular and anti-fast MHC antibodies suggests that satellite cells participate in nascent myofiber formation. In addition, some mature ALD muscle fibers demonstrate reactivity with these antibodies, suggesting that satellite cell fusion with mature myofibers also occurs in overloaded ALD muscles and results in the reinitiation of the embryonic phenotype in hypertrophying fibers. The presence of cells reacting exclusively with the anti-fast antibody after removal of the overload for 9 wk indicates that nascent myofiber formation results in the establishment of a new myofiber population with an abnormal phenotype in overloaded ALD muscles. These cells form a lattice-like network around normal fascicles.

Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian embryogenesis.

We have utilized a key biochemical determinant of muscle fiber type, myosin isoform expression, to investigate the initial developmental program of future fast and slow skeletal muscle fibers. We examined myosin heavy chain (HC) phenotype from the onset of myogenesis in the limb bud muscle masses of the chick embryo through the differentiation of individual fast and slow muscle masses, as well as in newly formed myotubes generated in adult muscle by weight overload. Myosin HC isoform expression was analyzed by immunofluorescence localization with a battery of anti-myosin antibodies and by electrophoretic separation with SDS-PAGE. Results showed that the initial myosin phenotype in all skeletal muscle cells formed during the embryonic period (until at least 8 days in ovo) consisted of expression of a myosin HC which shares antigenic and electrophoretic migratory properties with ventricular myosin and a distinct myosin HC which shares antigenic and electrophoretic migratory properties with fast skeletal isomyosin. Similar results were observed in newly formed myotubes in adult muscle. Future fast and slow muscle fibers could only be discriminated from each other in developing limb bud muscles by the onset of expression of slow skeletal myosin HC at 6 days in ovo. Slow skeletal myosin HC was expressed only in myotubes which became slow fibers. These findings suggest that the initial commitment of skeletal muscle progenitor cells is to a common skeletal muscle lineage and that commitment to a fiber-specific lineage may not occur until after localization of myogenic cells in appropriate premuscle masses. Thus, the process of localization, or events which occur soon thereafter, may be involved in determining fiber type.

A molecular view of cardiogenesis.

Cardiac development involves a complex integration of subcellular processes into multicellular and, finally, whole organ effects. Until recently it has been difficult to investigate the genetic control of this organ level differentiation of the heart. The proliferation of molecular biology methodologies has provided mechanisms to directly investigate the control of these processes. This article focuses on molecular lines of research on two key areas in cardiac development: the regulation of expression of sarcomeric contractile and regulatory proteins, and atrial natriuretic factor. Molecular approaches are described which have allowed investigators to begin to determine the tissue and stage-specific expression of genes, to locate those genes in the genome, determine their sequences, and to directly investigate the mechanisms controlling their expression.

Nascent muscle fiber appearance in overloaded chicken slow-tonic muscle.

The application of a weight overload to the humerus of chickens induces a hypertrophy of anterior latissimus dorsi (ALD) muscle fibers. This growth is accompanied by a rapid and almost complete replacement of one slow-tonic myosin isoform, SM-1, by another slow-tonic isoform, SM-2. In addition, a population of small fibers appears mainly in extrafascicular spaces and, concurrently, three additional myosin bands are detected by gel electrophoresis. Five antibodies against myosin heavy chain (MHC) isoforms were selected as immunocytochemical probes to determine the cellular location and nature of these myosins. The antibodies react with ventricular, fast skeletal muscle and either SM-1 or SM-2, or both the slow-tonic MHCs. The antifast and antiventricular antibodies react with myosin present in the 10-day embryonic ALD muscle but do not react with myosin in posthatch ALD muscle. The small fibers in overloaded muscle contain a myosin isoform characteristically expressed during the embryonic stage of ALD muscle development and therefore are named nascent myofibers. Some of the nascent myofibers do not react with the antibody to both slow-tonic MHCs, indicating the lack of the normal adult slow-tonic myosins which are expressed in 10-day embryos. In order to explore the origin of the nascent fibers, an electron microscopic study was performed. Stereological analysis of the existing fibers shows a stimulation of numbers and sizes of satellite cells. In addition, the volume occupied by nonmuscle and undifferentiated cells increases dramatically. Myotube formation with incipient myofibrils is seen in extrafascicular spaces. These data suggest that new muscle fiber formation accompanies hypertrophy in overloaded chicken ALD muscle and the process may involve satellite cell migration.

Transitions in cardiac isomyosin expression during differentiation of the embryonic chick heart.

The expression of different isoforms of the contractile protein myosin plays a major role in determining contractile characteristics in both cardiac and skeletal muscle in the adult. There is little evidence pertaining to putative changes in myosin phenotype during cardiac embryogenesis or if such changes could play a role in modulating the contractile characteristics of the developing heart. We examined isomyosin expression during cardiogenesis in the chick by indirect immunofluorescence microscopy with monoclonal antibodies to adult ventricular and atrial myosin heavy chains. Antibody specificity was characterized in the adult on the basis of immunofluorescence localization, ELISA, and protein blot immunoassay. Results show that the early embryonic chick heart has a different myosin phenotype than the later embryonic or adult heart. Both the embryonic ventricular and atrial myocardia initially expressed a myosin heavy chain that was recognized by antibody specific (in the adult) for ventricular myosin heavy chain. The ventricles remained reactive throughout life with the ventricular antibody, but reactivity of the atrial myocardium was confined to the initial 6 days of embryonic development. On the other hand, reactivity of the embryonic heart with multiple antibodies specific (in the adult) for atrial myosin was confined to the atrial myocardium throughout development. Thus, the distribution of myosin isoforms became similar to that of the adult myocardium by the time the embryonic heart achieved a 4-chambered configuration at 6 days in ovo.

Developmental aspects of cardiac contractile proteins.

The change in the isomyosin complement of avian and mammalian hearts was examined during the embryonic period in primary cultures of embryonic myocytes and in the cross-section of the adult ventricular wall. The type of myosin was determined by immunofluorescence using Abs specific for heavy chains of V1 and V3 isomyosins and by cytochemical staining for Ca2+ activated myosin ATPase. Our analysis indicates that the first isomyosin to appear in both chambers of avian heart is of the V3 type (HC beta). With advancing development, however, the atria initiate the expression of HC alpha and repress that of HC beta while the ventricle retains HC beta. In cultured myocytes derived from rat embryos cellular heterogeneity was detected in response to thyroid hormone. The cells are not synchronized in their response. Two populations are discernible with the minor one being thyroid hormone insensitive. Heterogeneity of the cellular populations was also seen in the left ventricle of adult rabbits. Myocytes with a similar isomyosin complement appear clustered with a predominance of V1 in the epicardium. Heterogeneous myocytes are, however, also frequently seen connected by an intercalated disc.

Immunofluorescence analysis of the primordial myosin detectable in embryonic striated muscle.

Immunofluorescence analysis showed that the earliest myosin detectable in both the embryonic chicken heart and somitic myotome, the precursor to skeletal muscle, was strongly reactive with two different monoclonal antibodies specific for the heavy chain of cardiac ventricular myosin, but it showed no reactivity with affinity-purified polyclonal antibodies specific for the heavy chains of either fast-twitch or slow-tonic skeletal myosins. The heart remained reactive exclusively with the antibodies to cardiac myosin throughout development, while late embryonic (day 20) skeletal muscles were strongly reactive only with their homologous skeletal myosin antibodies. Our findings suggest that the primordial myosin heavy chain detectable in both forms of embryonic chicken striated muscle, the myotome and the heart, is immunologically distinct from myosins expressed in later embryonic as well as adult skeletal muscles, but it contains antigenic determinants similar to those present in cardiac ventricular myosin.

Atrial septal thickness and area in normal heart specimens and in those with ostium secundum atrial septal defects.

This anatomic study suggests that an understanding of the thickness of the atrial septum should allow better visualization of most portions of the blade-shaped atrial septum during cross-sectional echocardiography, both in normal subjects and in patients with ostium secundum atrial septal defects. The thickness measurements also demonstrate that difficulties in imaging the atrial septum are a function of resolution limits of existing echocardiographic equipment.

The normal anatomy of the atrial septum in the human heart.

The atrial septum is a blade-shaped structure with a concave anterior margin that reflects the curve of the ascending aorta, a convex posterior margin, and an inferior margin along the mitral annulus. The fossa ovalis comprises an average of 28 per cent of the total septal area, or 43 mm.2 in infants and 240 mm.2 in adults. The channel that persists between the fossa ovalis and the muscular atrial septum is patent except at the limbus, providing a useful explanation for the success of blunt transseptal atrial catheterization and right-to-left shunts in individuals with elevated right atrial pressure.

Psychiatric patients' perceptions of their milieu therapy program.

This study was designed to provide information concerning psychiatric patients' perceptions of their individual treatment program in an inpatient treatment facility with an established therapeutic milieu. The responses of 24 patients aged 16 to 69 years to an 8-item questionnaire, developed and administered by the investigator, constituted the data collected. The 24 patients identified 57 components as part of their treatment programs. Patients with more education mentioned more treatment components than those with less education. Talking with staff members was considered the most helpful treatment component by the most patients, and medication was the most frequently mentioned least helpful treatment component. Six categories were used to organize the data: recreation-physical, recreation-mental, verbal interaction, creature comforts, staff supervision, and diagnostic and medical services. More of the identified treatment components were categorized under verbal interaction than any other category. The second most mentioned category was recreation-physical. As patients listed the most helpful persons during their hospitalization, 36 students and staff members and four other patients were identified. Two psychiatric technicians, a registered nurse, and an occupational therapist were mentioned most often. Patients tended to see persons of their same sex and close to their age as particularly helpful.

Atrial defect size after Blalock-Hanlon atrioseptectomy.

In transposition of the great arteries, a Blalock-Hanlon closed atrial septectomy is performed to improve intracardiac mixing at the atrial level. Although the Blalock-Hanlon septectomy is a common surgical procedure in cyanotic congenital heart disease, it has not been adequately assessed pathologically. In 14 heart specimens from patients (aged 3 days to 19 years) with transposition of the great arteries and Blalock-Hanlon septectomy, the margins of the septectomy, fossa ovalis and atrial septum were identified. The total area of the septum and its defects was calculated using planimetry. The ratio of defect size to atrial septal area was expressed as percent communication, which ranged from 5 to 39 (mean 18) percent in eight specimens with intact limbus of the foramen ovale and 26 to 57 (mean 42) percent in six specimens in which the limbus had been excised. The finding that specimens in which the Blalock-Hanlon defect extended into the fossa ovalis had the largest total communication emphasizes that to obtain optimal bidirectional atrial mixing the surgeon should extend the Blalock-Hanlon procedure across the limbus into the foramen ovale.

Closed foramen ovale: a potential entrance to left atrium.

The anatomy of the limbus and its relationship to the fossa ovalis were studied in 90 normal human hearts in an attempt to explain the clinical success of blunt trans-septal catheterization. Previously reported rates of patent foramen ovale (25-35% of adult specimens) were confirmed, well below the reported rate of success for blunt trans-septal catheterization (67-87%). A possible reason for this success is the fragile attachment of the fossa ovalis to its limbus in nearly all hearts without persistent patency.

The normal spectrum of mitral and aortic valve discontinuity.

The relationship of the base of the left and noncoronary sinuses of the aortic valve and the adjacent aortic leaflet of the mitral valve was studied in 106 normal heart specimens and 184 specimens with isolated VSD. The results show a spectrum of persistence of the tissue along the inner curvature of the heart. This may help settle the recent controversy in the interpretation of echocardiograms of this area because the recorded mitral-aortic discontinuity may be due to this spectrum rather than to variations in technique.

Horseshoe lung in an infant with recurrent pulmonary infections.

A case of horseshoe lung occurred in a 7-month-old infant, the first, we believe, to be associated with horseshoe kidney. This lesion should be considered in any infant with dextrocardia, recurrent cardio-respiratory distress, and pulmonary infections.

Relationships of the tricuspid valve to the membranous ventricular septum in Down's syndrome without endocardial cushion defect: study of 28 specimens, 14 with a ventricular septal defect.

The commissure between the anterior and medial leaflets of the tricuspid valve is commonly absent in Down's syndrome without endocardial cushion defect (19 of 28 specimens). As a result, aneurysm of the membranous ventricular septum may develop (eight of 14 specimens with ventricular septal defect limited to the membranous ventricular septum) and the potential for left ventricle-to-right atrial communication is increased.

Normal variations in tricuspid valve attachments to the membranous ventricular septum: a clue to the etiology of left ventricle-to-right atrial communication.

In 39 out of 95 normal specimens, there was either no commissure between the anterior and medial leaflets of the tricuspid valve, which resulted in an interrupted valve margin at the center of the membranous ventricular septum, or an incompletely formed commissure. It is suggested that one end of this spectrum of normal anatomic variations could have a direct relationship to VSD associated with left ventricle-to-right atrial communication.

Enlargement of the membranous ventricular septum: an internal stigma of Down's syndrome.

A study of heart specimens without ventricular septal defect from patients with down's syndrome showed a significant enlargement of the membranous ventricular septum as compared to the normal heart. This increase in area of the ventricular septum occupied by membranous tissue from 2 to 9% may not only predispose patients with Down's syndrome to congenital heart disease, but may be one end of a spectrum of cardiac anomalies related to malalignment or maldevelopment of the ventricular septum in Down's syndrome.