Procoagulant activity of endocardial vegetations and blood monocytes in rabbits with Streptococcus sanguis endocarditis.

To investigate activation of the coagulation system in bacterial endocarditis, we determined the procoagulant activity of blood monocytes isolated from rabbits with Streptococcus sanguis-infected or sterile catheter-induced endocardial vegetations. This activity was determined directly after isolation from the peripheral blood and after stimulation in vitro by either endotoxin or by phagocytosis of S. sanguis. The procoagulant activity of the vegetations of these rabbits was also determined. The procoagulant activity of blood monocytes of rabbits with S. sanguis endocarditis was found to be similar to the activity of monocytes of rabbits with sterile vegetations, both at the time of isolation and after stimulation in vitro by exposure to endotoxin or phagocytosis of bacteria. The procoagulant activity of infected vegetations was significantly higher than that of sterile vegetations. We conclude that in bacterial endocarditis the coagulation system is activated locally at the site of the vegetation. Triggering probably occurs by thromboplastin generated by monocytes activated by phagocytosis of bacteria on the vegetational surface.

Tissue distribution of atrial natriuretic factor in normal and pathologic human hearts.

The tissue distribution and possible neuroendocrine nature of atrial natriuretic factor (ANF) were studied, using the avidin-biotin-peroxidase technique and antibodies to ANF, chromogranin (Ch), and neuron-specific enolase (NSE). Tissues examined included: Group 1, formalin-fixed and fresh frozen atrial tissue from adjacent areas of the hearts from two heart-lung-transplant patients; Group 2, the entire atria and sampling of other areas from formalin-fixed hearts of five gunshot wound or automobile accident victims; and Group 3, formalin-fixed right auricular tissue from 19 open-heart-surgery patients. In each case of Group 3, the ANF score, expressed as the product of the percentage of stained areas by the staining intensity, was correlated with age, weight, height, blood pressure, ejection fraction, and degree of coronary arterial stenosis. It was found that: (a) ANF was limited to atrial myocytes; the staining was significantly stronger in the right atrium, diffuse and most intense in auricles and pectinate muscles, diffuse and strong in subendocardium, focal and weak in other areas; (b) although ANF has been reported to be a peptide hormone stored in dense-core granules, it does not seem to belong to the diffuse neuroendocrine system because Ch and NSE were consistently absent in cardiac myocytes; and (c) although the limited numbers of evaluable clinical parameters do not significantly correlate with ANF scores, a change in the pattern and intensity of ANF staining was noted in some cases of Group 3.

Ultrastructural aspects of atrium development: demonstration of endocardial discontinuities and immunolabeling of atrial natriuretic factor in the Syrian hamster.

The endocardium ultrastructure of 13 embryonic day old hamsters was examined, especially in relationship with the atrial myocytes. The endothelial morphology was described, including the junctional attachments and their relationships with subjacent atrial myocytes. Characteristic atrial myocytes organelles were identified: myofibrils, atrial granules, lipidic inclusions, and polysomes. Immunogold labeling demonstrated that atrial natriuretic factor (ANF)-containing granules were already present in the differentiating cardiomyocytes, even before the myofibrils were completely organized. At this stage of development, while the endothelium was a narrow barrier between the blood and the cardiomyocytes, it displayed fenestrations, but also epithelial discontinuities. In addition it also contains immunoreactive-ANF products. In light of the current knowledge about ANF processing it was proposed that the endocardium lining could be an obligated passageway for transport or activating proANF into ANF before its release into the blood stream. In addition the endocardial gaps could suggest that, until about 13 to 14 days of fetal development, heart atrial tissue could be more susceptible to the effects of pathogenetic compounds than in a later state of development.

Enzyme pattern and lipid peroxides in endomyocardial biopsies from patients with cardiomyopathy and myocarditis.

Methods have been developed for measuring several biochemical parameters (isoenzymes of LDH and ASAT, glycogen phosphorylase, lipid peroxides) in extremely small tissue samples (0.2-1.8 mg) taken using a left ventricular biopsy technique. Endomyocardial biopsies from patients with dilative and hypertrophic cardiomyopathy (CMP) and with myocarditis were investigated and compared with a reference group without actual functional and morphological evidence of chronic heart disease. Patients with myocarditis showed the highest activities of LDH and its isoenzymes, ASAT, ASATm and glycogen phosphorylase and the highest concentration of lipid peroxides. In patients with hypertrophic CMP increased activities of glycogen phosphorylase and decreased activities of ASAT and ASATm have been found. In patients with dilative CMP slightly elevated ASAT and ASATm activities have been observed. The results obtained in this study suggest that the parameters investigated could be useful in differentiating between cardiomyopathies and myocarditis.

Deposits of eosinophil granule proteins in cardiac tissues of patients with eosinophilic endomyocardial disease.

Eosinophilic endomyocardial disease is a complication of the hypereosinophilic syndrome and of several other disorders associated with high blood eosinophil counts. Eosinophil granule proteins may be involved in the development of these lesions. This multi-centre study investigated whether these proteins could be demonstrated within the cardiac tissues of eighteen patients with eosinophilic endomyocardial disease. Serial sections of tissue taken at necropsy or at cardiac biopsy were stained for eosinophil major basic protein by indirect immunofluorescence and for eosinophil cationic protein, eosinophil protein-X, and activated eosinophils by means of alkaline-phosphatase-linked monoclonal antibodies. Activated eosinophils and secreted eosinophil granule proteins were most evident within the necrotic and later stage thrombotic lesions and were found mainly within the areas of acute tissue damage in the endocardium and in the walls of small blood vessels. These findings suggest that eosinophil granule proteins are involved in cardiac injury, producing muscle damage and vascular injury which lead to the development of endomyocardial fibrosis.

Assessment of the beta-adrenergic receptor pathway in the intact failing human heart: progressive receptor down-regulation and subsensitivity to agonist response.

We developed methods for identifying beta-adrenergic receptors in human right ventricular endomyocardial biopsy tissue with the radioligand (-)[125I]iodocyanopindolol (ICYP). Specific ICYP binding in a crude, high-yield membrane preparation derived from endomyocardial biopsy tissue was high (specificity greater than 90%), of high affinity (KD around 20 pM), saturable and stereospecific for the (-) vs the (+) isomer of isoproterenol. Subjects with mild-moderate and severe biventricular dysfunction had respective decreases in beta-adrenergic receptor density of 38.2% and 57.7% when normalization methods were averaged, with no significant differences in ICYP dissociation constant. A subgroup of subjects was subdivided by left ventricular ejection fraction (LVEF) into those with mild cardiac dysfunction (LVEF less than 0.50 greater than 0.40) and severe heart failure (LVEF less than 0.20) and given graded sequential infusions of dobutamine and calcium gluconate. Those with severe cardiac dysfunction had marked impairment of the dobutamine dP/dt and stroke work index response, whereas these responses to calcium did not differ in the two groups. These data indicate that in the intact human heart endomyocardial biopsy may be used for direct analysis of beta-adrenergic receptors, heart failure-associated myocardial beta-adrenergic down-regulation begins with mild-moderate ventricular dysfunction, reduction in myocardial beta-receptor density is related to degree of heart failure, and beta-receptor down-regulation is associated with pharmacologically specific impairment of the beta-agonist-mediated contractile response.

Heterogeneous expression of factor VIII/von Willebrand factor by cardiac myxoma cells.

The expression of factor VIII/von Willebrand factor (FVIII/vWF), a marker for endothelial differentiation, was examined immunohistochemically in normal cardiac tissue and seven cardiac myxomas. In normal tissue, FVIII/vWF was detected in the surface endocardial cells and the vascular endothelial cells. In tumors, the isolated myxoma cells and the small groups and cords of myxoma cells did not express FVIII/vWF. Some of the surface cells contained FVIII/v/WF, but most did not. The predominant sites of FVIII/vWF localization were the multilayered, concentric vascular structures. In most of these, FVIII/vWF was present only in the inner layer of cells immediately surrounding the vascular lumens. In some, however, intense staining was present in both the inner and outer cell layers. In general, the FVIII/vWF-positive cells had the appearance of endothelial cells. These observations correlate with the recognized histologic, histochemical, and ultrastructural heterogeneity of cardiac myxoma cells, and conform to the current view that such cellular heterogeneity arises via the divergent differentiation of multipotential mesenchymal cells.

Reperfusion of the human myocardium by saphenous vein bypass grafts. Biochemical considerations.

The adenine nucleotide content of the human myocardium in the distribution of the left anterior descending coronary artery (LAD) was measured before and after saphenous vein bypass grafting. The purpose of the study were twofold: (1) to relate the level of adenosine triphosphate (ATP) before bypass grafting to the percent stenoses of the LAD and (2) to determine the benefit or lack of benefit of bypass grafting on ATP content. Eighteen patients with angiographically determined LAD lesions of 40% to 100% underwent bypass grafting with standard cardiopulmonary bypass and cardioplegia. Transmural needle biopsy specimens were obtained from the center of the area perfused by the LAD immediately before cross-clamping of the aorta and 30 minutes after reperfusion of the myocardium via the native LAD and the graft. The tissue was divided into thirds: The endocardial and epicardial thirds were analyzed for ATP by high-pressure liquid chromatography and the middle third was viewed by light microscopy. The percent narrowing of the LAD correlated well (r = -0.71) with the ratio of ATP to total adenine nucleotides (TAN) in the endocardium. Epicardial ATP did not correlate with the percent stenoses of the LAD. The endocardial ATP/TAN ratio increased in the group as a whole from 0.51 +/- 0.27 (mean +/- SD) to 0.64 +/- 0.26 (p less than 0.01) after bypass grafting, and this was most impressive in those eight patients with LAD lesions greater than 90% (0.32 +/- 0.20 before grafting to 0.60 +/- 0.29 after grafting, p less than 0.005). However, the epicardial ATP/TAN ratio decreased from 0.75 +/- 0.15 before grafting to 0.64 +/- 0.17 after grafting (p less than 0.05), and this decrease occurred regardless of the percent narrowing of the LAD. There was no difference in vacuolization between the pre-grafting and post-grafting biopsy specimens, and intramyocardial hemorrhage was not observed. This study has demonstrated a close relationship between the degree of LAD stenosis and endocardial ATP content. Also, the endocardium supplied by arteries with greater than 90% lesions had significantly increased ATP while the epicardium had decreased ATP content after bypass grafting.

Regional myocardial lidocaine concentration following continuous intravenous infusion early and later after myocardial infarction.

The regional concentration of lidocaine using a double constant infusion technique (250 micrograms/kg/min x 15 minutes followed by 35 micrograms/kg/mg/min x 120 minutes) was studied immediately (2 hours) in seven dogs and 24 hours (six dogs) after myocardial infarction. Tissue levels were determined by gas chromatography and related to regional myocardial blood flow as determined by the radioactive microsphere technique in multiple samples. At 2 hours after infarction a significantly higher lidocaine concentration (4.1 +/- 0.42 micrograms/g) was found in zones with greatly reduced blood flow (regional myocardial blood flow less than 0.2 ml/min per g) when compared with that (2.6 +/- 0.19 micrograms/g) in zones with normal blood flow (regional myocardial blood flow greater than 0.8 ml/min per g) (p less than 0.01). In contrast, in the 24 hour model the opposite situation was observed. Although the concentration of lidocaine in the infarct zone was substantial, a significant decline in lidocaine tissue concentration was found in the zones of lowest blood flow (regional myocardial blood flow less than 0.2 ml/min per g) when compared with that in normal zones (1.76 +/- 0.21 versus 3.38 +/- 0.21 micrograms/g, p less than 0.001). In addition, no significant differences in lidocaine concentrations were found between endocardium and epicardium in any of the groups other than those related to regional myocardial blood flow. Thus, with the double constant infusion technique, lidocaine reached normal and ischemic myocardium in concentrations equivalent to therapeutic plasma concentrations, even in lower infarct blood flow zones, with no significant differences between endocardium and epicardium. Of perhaps greater significance, the age of the ischemic insult is an important determinant of lidocaine tissue distribution in infarcted myocardium.

Glycogen aggregates in cardiac muscle cell: a cytopathological study on endomyocardial biopsies.

Glycogen aggregates in the cardiac muscle cell, which have been frequently demonstrated in endomyocardial biopsies under the transmission electron microscope, were studied using the optical microscope in order to clarify their histopathological significance in various heart diseases. The right ventricular muscle biopsies were obtained from the following 120 patients: 24 cases of congestive cardiomyopathy, 26 of hypertrophic cardiomyopathy, 28 of atrial septal defect, 6 of primary pulmonary hypertension, 31 of bradyarrhythmic hearts and 5 controls. The tissue specimens were fixed with glutaraldehyde and osmium tetraoxide, and embedded in epoxy resin. Semi-thin sections from these specimens were dyed with tribasic staining originated by KUROTAKI (1972). Under the optical microscope, glycogen is clearly identified by its forming red stained areas which are seen not only in the subsarcolemmal layers and the perinuclear region, but also in the intermyofibrillar zones. The glycogen aggregates are more frequently observed in the specimens from the atrial septal defects and the bradyarrhythmic hearts than in the other cases. Thus, observation of glycogen aggregates reveals notable differences in appearance depending on the kinds of diseases. In the bradyarrhythmic hearts, the glycogen aggregates can be more readily observed in specimens from younger patients than in elderly ones. Furthermore, the glycogen aggregates appear regardless of both the grades of hypertrophy and degeneration in myocardial cells. These results do not agree with previous reports that the occurrence of glycogen is proportional with the grade of cardiac cell hypertrophy and/or damage.

Origin and migration of cushion tissue in the developing heart.

The origin of cushion tissue mesenchyme in the developing chick heart was investigated by three basic methods: use of an inert metabolic marker; time lapse recording of organ-cultured heart regions; and scanning stereo-microscopy. All three approaches support the hypothesis that the endocardium is the progenitor of cushion tissue mesenchyme. Additional observations show a cell:matrix interaction by the endocardium prior to the formation of mesenchyme. It is postulated that this activity in some manner alters the underlying matrix and helps to initiate and maintain migratory activity of the mesenchyme.

Biochemical measurements of endomyocardial biopsies.

The purpose of this study is to investigate the validity of performing biochemical analyses on human endomyocardial biopsies. One problem of such analyses is the decay of metabolites between the time of biopsy and the analyses. This was addressed by varying the time delay from biopsy to submersion in liquid nitrogen (-200 degree C). Neither cyclic AMP or cyclic GMP changed significantly up to 90 seconds after biopsy. The reproducibility of the assays in human heart biopsies was determined by submitting paired samples from each procedure. The ATP content was determined by a fluorescent technique and was related to the protein and creatine content. The average ATP of 25 paired samples was 12.3 nm per mg protein; the average difference between the paired samples was -0.6, and the 95% confidence interval was 5.6 nm/mg protein. The average ATP-to-creatine ratio was 0.25; the 95% confidence interval was 0.12. The cyclic AMP and cyclic GMP were found to average 3.3 and 0.25 femtomoles/mcg protein, respectively. The 95% confidence intervals were 1.8 and 0.12 fmoles/mcg protein. The problems of sampling error and blood and connective tissue contamination decrease the reliability of the biochemical analyses. Reproducibility is adequate, however, to acquire meaningful biochemical information on the human heart.

Structural analysis of extracellular matrix prior to the migration of cephalic neural crest cells.

Cephalic neural crest cells enter cell free areas containing abundant extracellular matrix (ECM). Previous histochemical studies have identified both sulfated and non-sulfated glycosaminoglycans within this matrix. In the present study, ultrastructural examination of the ECM demonstrated an anastomosing network of pleomorphic, cetyl pyridinium chloride-dependent strands within cell free spaces and in association with the basement membrane of the surface ectoderm. Thin section analysis revealed that the strands consisted of three components: (1) 3-5 nm filament meshwork; (2) electron dense amorphous material and (3) 30 nm granules. In contrast, the ECM associated with the basement membrane consisted principally of a continuum of electron dense, amorphous material. The molecular ordering of ECM within crest cell pathways was compared to the well-characterized, hyaluronate-rich, premigratory matrix of cardiac jelly.

Cardiac involvement in secondary haemochromatosis: a catheter biopsy study and analysis of myocardium.

Although high blood transfusion regimens have improved the life expectancy of the patient with Thalassemia Major, cardiac failure and arrhythmias remain a cause of early death. It is not certain whether the massive myocardial iron deposition found in such patients is preventable by intensive chelation therapy. This study evaluates endomyocardial biopsy as a method of assessing myocardial iron deposition. Of four patients with clinical and biochemical evidence of severe haemochromatosis, only one had a myocardial iron content comparable to that found in severe haemochromatotic myocardium. The one patient with cardiac failure had an endomyocardial iron content within the normal range. Studies of the iron distribution in haemochromatotic myocardium demonstrate that the subendocardial myocardium contains only half the iron content of the subepicardial layer, and there is a large sampling variation. It is concluded that catheter endomyocardial biopsy is an insensitive method of determining early myocardial deposition because of the location of iron and the variability of the sampling. Studies of the nature of the myocardial iron protein with CM32 cation exchange resin chromatography show that there is a large increase in the haemosiderin: ferritin ratio (5:1) in iron overload myocardium as compared with the normal heart (2:1). Similar results have been observed in the liver with iron overload, where the increase in hepatic haemosiderin was associated with greater lysosomal fragility. It is possible that myocardial cell damage may also occur by the rupture of iron engorged lysosomes.

Effects of aging on hydroxyproline in human heart muscle.

Hydroxyproline concentration in the NaCl-soluble, TCA-soluble, and residual fractions of heart muscle was chemically determined in 40 autopsy subjects (25 men and 15 women), to study the effects of aging. In the left ventricle, the total hydroxyproline content of the endocardium and papillary muscle increased significantly in relation to age; the increase was greatest in the TCA-soluble fraction, followed by the residual fraction. Statistical analysis revealed that hydroxyproline in the residual fraction increased from the epicardium to the endocardium and then to the papillary muscle. Significant negative correlations were found between heart weight and total hydroxyproline in the epicardium and endocardium. In the right ventricle, a significant negative correlation was noted between heart weight and total hydroxyproline content. Though no correlations were evident between age and hydroxyproline content in females, significant correlations were observed between age and the total hydroxyproline content of the endocardium and papillary muscle in males. Furthermore, soluble collagen increased in most parts of the heart in females, but insoluble collagen increased in all parts of the heart in males. It is concluded that the hydroxyproline content of heart muscle increases in proportion to age and is in inverse proportion to heart weight. The increase occurs in the TCA-soluble and residual fractions, and extends from the endocardium to the papillary muscle. More profound changes in collagen metabolism are observed in males.

Determination of hydroxyproline content in human heart muscle.

Hydroxyproline content was chemically determined on 45 human hearts obtained at autopsy in order to study whether hydroxyproline contents vary in proportion to heart weight or not. There were no significant differences in the mean hydroxyproline content between the epicardium and the endocardium, between the left ventricle and the right ventricle, or among cardiovascular and renal disease group, malignant disease group and miscellaneous disease group. Total hydroxyproline content per mg dry weight of the left ventricle increased proportionally with heart weight. Hydroxyproline content in neutral salt soluble fraction increased more proportionally than that in acid soluble fraction. This tendency was more evident in the endocardium than in the epicardium of the left ventricle. These results seem to be well consistent with those of histometrical estimation of scar tissue in human heart muscle. Hydroxyproline content in acid soluble fraction of the right ventricle was increased proportionally with heart weight.

Amyloid in the aged heart: frequency and clinical significance.

Cardiac amyloid accumulation is a common autopsy finding in elderly persons, the frequency increasing with each advancing age decade. In most cases the deposits are microscopic, confined to the atrial subendocardium, and of dubious significance. In a few cases, however, there is much more extensive cardiac involvement, with infiltration of the ventricular musculature and intracardiac conduction system. In the authors' recent autopsy study of 100 patients over 60 years of age, 10 had diffuse cardiac amyloidosis with biventricular involvement. Microscopically, the amyloid deposits often resembled foci of interstitial fibrosis. Significant cardiac enlargement (weight greater than 380 gm) was present in only 2 of these patients, and in only one of them were the clinical signs and symptoms clearly attributable attributable to the amyloid infiltration. It was difficult to assess the possible contribution of ventricular amyloid to congestive failure since most of the patients also had coronary arteriosclerosis. In the aged, clinically significant cardiac amyloidosis associated with cardiac enlargement appears to be part of a disease spectrum more commonly characterized by focal, clinically insignificant cardiac amyloid deposits. Yet several features suggest that senile cardiac amyloidosis may differ from the clinically significant cardiac amyloidosis occassionally seen in younger patients.