Heart-specific activation of LTK results in cardiac hypertrophy, cardiomyocyte degeneration and gene reprogramming in transgenic mice.

Leukocyte tyrosine kinase (LTK) is a receptor-type protein tyrosine kinase belonging to the insulin receptor superfamily. To elucidate its biological role, we generated transgenic mice expressing LTK under the control of cytomegarovirus enhancer and beta-actin promoter. The transgenic mice exhibited growth retardation and most of the transgenic mice died within several months after birth. Interestingly, although LTK was expressed in several major organs, the activation (tyrosine-phosphorylation, kinase activity, and multimerization) of LTK was observed selectively in the heart, where LTK was localized on intracellular membrane, presumably on endoplasmic reticulum. Echocardiography showed that the transgenic heart underwent severe concentric hypertrophy, which resulted in reduced cardiac output, low blood pressure, and increased heart rate. Histological examination of the heart exhibited focal degeneration of cardiomyocytes. These histological changes were considered to be due to apoptosis, based on the finding that the sarcolemmas of the degenerative cardiomyocytes were well preserved. In addition, expression of fetal genes, such as atrial natriuretic peptide and skeletal alpha-actin, was markedly induced in the transgenic heart. These results indicate that a certain tissue-specific mechanism of activating LTK exists in the heart and that the activated LTK resulted in cardiac hypertrophy, cardiomyocyte degeneration and gene reprogramming. These findings will provide novel insights into the activating mechanism and biological role of LTK in vivo.

Backscattered electron imaging: A new method for the study of cardiomyocyte architecture using scanning electron microscopy.

Scanning electron microscopy (SEM) with secondary electron emissions is useful for the study of cardiomyocyte architecture, however, the information is limited from the cell surface. Whereas backscattered electron (BSE) emission can give a high-resolution image of the specimen's intracellular structure after heavy metal staining. In this study, we applied BSE imaging analysis to the study of the arrangement of cardiomyocytes in the myocardium. The tissue specimens from a normal fresh monkey heart, normal human heart obtained at autopsy, and surgically resected tissue from a patient with old myocardial infarction in the left ventricular aneurysmectomy were used. The tissue specimens were fixed in neutral formalin, treated with NaOH and then stained with Gomori's silver methenamine reagent followed by tannic acid and osmium tetroxide. After dehydration and drying, the specimens were coated with carbon and examined by SEM with a BSE detector. In the tissue preparations, the A bands of sarcomeres were selectively stained with silver so that the arrangements of subsarcolemmal myofibrils and the intercalated discs were clearly seen in the BSE images. In the left ventricular aneurysmal walls of old myocardial infarction, atrophied cardiomyocytes with disarray of subsarcolemmal myofibrils were observed. The results strongly suggest that BSE images are further applicable to the study of the architecture of cardiac myocytes and their branches, and the arrangement of intracellular myofibrils in various diseased myocardium.

Ultrastructural and immunohistochemical studies on myocardial biopsies from a patient with eosinophilic endomyocarditis.

Right ventricular endomyocardial biopsy specimens from a 13-year-old boy with hypereosinophilia were studied by light and electron microscopy using the EG2 monoclonal antibody, which recognizes a common epitope of eosinophil cationic protein and eosinophil protein-X. Although the endocardial layer was of normal thickness, many eosinophils, mononuclear cells, and free eosinophil granules were observed in the endocardium and in the vicinity of degenerated myocardial cells. Under electron microscopy, many of the specific granules in and out of eosinophils had lost their crystalloid internae and displayed reversed density, and there were many degranulated eosinophils with reduced number of granules. Immunohistochemically, large amounts of eosinophil cationic protein and protein-X were observed within cardiocytes when many of them were degenerated. Deposits of the proteins were also found in some small vessels. On electron microscopy, accumulations of gold particles, which bind to eosinophil cationic protein and protein-X, were seen in association with specific granules and on the myofilaments in both degenerated and normal-appearing cardiocytes. The presence of eosinophil cationic proteins within cardiocytes may play an important role in the pathogenesis of eosinophilic endomyocardial disease.

Collagen subtypes and matrix metalloproteinase in idiopathic restrictive cardiomyopathy.

BACKGROUND: Idiopathic restrictive cardiomyopathy is a rare disease characterized by diastolic dysfunction, and the pathogenesis of the stiff heart remains unclear. The purpose of this study was to analyze the subpopulation of collagen fibers and determine the expression of matrix metalloproteinase in restrictive cardiomyopathy. METHODS AND RESULTS: In endomyocardial biopsy specimens obtained from seven patients with restrictive cardiomyopathy, collagen fiber types I, III, and IV, and matrix metalloproteinase- and two were observed by light and electron microscopy, using monoclonal antibodies. Type I collagen was less prominent in the interstitium, whereas the immunoreactivity for type III collagen was marked. The immunoreactivity against matrix metalloproteinase-1 was observed along with types I and III collagen fibers and in the cytoplasm of some fibrocytes/fibroblasts. The matrix metalloproteinase-1 tended to increase when the reactivity against types I and III collagen was prominent. Both type IV collagen and matrix metalloproteinase-2 were observed along arterial walls and the basement membrane of cardiocytes. CONCLUSIONS: Increased type III collagen may play an important role as the cause of left ventricular stiffness in restrictive cardiomyopathy. The matrix metalloproteinase appeared to be involved in a cascade of collagen synthesis and the remodeling of the heart in patients with restrictive cardiomyopathy.

Increased serum levels and expression of S100A8/A9 complex in infiltrated neutrophils in atherosclerotic plaque of unstable angina.

BACKGROUND: The S100A8/A9 complex is expressed in a subset of activated neutrophils and macrophages in acute inflammatory lesions associated with various diseases. OBJECTIVE: To investigate (a) whether serum S100A8/A9 levels are increased in patients with unstable angina (UA); and (b) whether S100A8/A9 expression is upregulated in coronary atherosclerotic plaques of patients with UA. DESIGN: Serum S100A8/A9 levels in 39 patients with stable angina (SA) and 53 patients with UA were measured. In addition, the presence of the S100A8/A9 complex in directional coronary atherectomy specimens was studied immunohistochemically. Cell types which stain positive for S100A8/A9 were identified by immunodouble staining with neutrophils and macrophages. RESULTS: Mean (SD) serum S100A8/A9 levels were significantly higher in patients with UA than in those with SA (3.25 (3.08) microg/ml vs 0.77 (0.31) microg/ml, p<0.05). In patients with UA, immunodouble staining clearly showed that the S100A8/A9 complex was expressed in infiltrated neutrophils and occasional macrophages. The S100A8/A9-positive area was significantly higher in UA than in SA (mean (SD) 18.3 (14.2)% vs 1.3 (2.4)%, respectively, p<0.001). CONCLUSIONS: The S100A8/A9 complex may be involved in the inflammatory process of coronary atherosclerotic plaques in patients with UA.

Cardiac conduction system in the chicken: gross anatomy plus light and electron microscopy.

Twenty-three chicken hearts were used to study the cardiac conduction system by light and electron microscopy. In addition to a sinus node, atrioventricular node (AVN), His bundle, left and right bundle branches (LBB, RBB), the chicken also has an AV Purkinje ring and a special middle bundle branch (MBB). The sinus node lies near the base of the lower portion of the right sinoatrial valve. The AV node is just above the tricuspid valve and anterior to the coronary sinus. The His bundle descends from the anterior and inferior margin of the AV node into the interventricular septum, then dividing into right, left and middle branches some distance below the septal crest. The middle bundle branch turns posteriorly toward the root of the aorta. The AV Purkinje ring originates from the proximal AV node and then encircles the right AV orifice, joining the MBB to form a figure-of-eight loop. The chicken conduction system contains four types of myocytes: 1) The P cell is small and rounded, with a relatively large nucleus and sparse myofibrils. 2) The transitional cell is slender and full of myofibrils. 3) The Purkinje-like cell resembles the typical Purkinje cell, but is smaller and darker. 4) The Purkinje cell is found in the His bundle, its branches, and the periarterial and subendocardial Purkinje network.

Electron microscopic demonstration of intercellular junctions between subendocardial smooth muscle and myocardium in the sheep heart.

The eustachian ridge of 11 sheep hearts was examined by light and electron microscopy to elucidate the morphologic relationship between cardiac smooth muscle cells and myocardial cells. Smooth muscle cells were commonly observed in the subendocardial connective tissue and they were usually arranged in strands. When mixed with myocardial cells at the margin of these strands, smooth muscle cells exhibited numerous direct connections with the myocardial cells. These intercellular junctions were composed primarily of simple appositions (undifferentiated regions), but occasional desmosomes and fasciae adherents (intermediate junctions) were seen. We found no nexus (gap junctions) at these sites. The existence of direct intercellular junctions between smooth muscle and myocardium suggests several possible electrical and mechanical interactions.

Histological organization of the right and left atrioventricular valves of the chicken heart and their relationship to the atrioventricular Purkinje ring and the middle bundle branch.

In the avian heart the right and left atrioventricular (AV) valves not only exhibit their own special anatomical characteristics, but they also are in close proximity to the conduction system. The right AV valve is a single, spiral plane of myocardium, in remarkable contrast to the fibrous structure characteristic of the mammalian tricuspid valve. A ring of Purkinje tissue encircles the avian right AV orifice and connects to the muscular valve. The chicken has no crista supraventricularis, its right AV valve serving that function as well as opening and closing the right AV orifice. The left AV valve consists of three leaflets instead of the two typical of mammalian hearts. Its anterior and posterior leaflets are small; its large aortic (medial) leaflet merges with the bases of both the left and noncoronary cusps of the aortic valve by fibrous tissue, resembling that of the mammalian heart. However, unlike in mammals, there is a slim cylinder of continuous myocardium coursing parallel to this fibrous junction. This unusual arc of myocardium in the chicken serves to complete an entire subaortic ring of myocardium and is thus potentially capable of constricting the outflow tract of the chicken's left ventricle. The middle bundle branch connects with both the muscle arch and the AV Purkinje ring. Thus the myocardium in or near both AV valves (and the left ventricular outflow tract) in the chicken heart is so arranged that it may receive direct early activation from the conduction system.

Remodeling of cardiomyocytes and their branches in juvenile, adult, and senescent spontaneously hypertensive rats and Wistar Kyoto rats: comparative morphometric analyses by scanning electron microscopy.

Scanning electron microscopy was used to compare the shape, size, and connection of left ventricular (LV) myocytes between spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) at 3, 8, 15, 35, and 63 weeks of age. For either strain at each age, five rats were studied, in which LV myocytes consisted of a cylindrical trunk with series (SB) and/ or lateral branch(es) (LB) and step formations; cell junctions had 12 common basic patterns. The length (L), width (W), and L/W ratio of the myocytes, and various indices for SB, LB, and three selected types of cell junctions were measured in 100 cells from each heart and averaged for comparison studies. In the growing period (3-8 weeks of age), the LV myocytes were similar in shape and width in the two age-matched strains and grew similarly with the same L/W ratio. In adolescent (15-week-old) WKY, LV cells grew with the same L/W ratio as in the younger rats, whereas in adolescent SHR, the cells showed a much greater increase in width than in length (disproportionate hypertrophy), the LB proliferated significantly, and the numbers of step-to-step and side-to-side junctions were diminished. In adult (15-35-week-old) WKY, LV cells continued to grow without much change in SB, LB, and the cell junctions, whereas in adult SHR, LV hypertrophy progressed with enhanced cardiomyocyte hypertrophy, increased number of SB, LB, and step-to-end junctions, and reduction in the number of step-to-step and side-to-side junctions per cell. In aged (63-week-old) WKY and SHR, the indices of LV myocytes, SB, LB, and cell junctions did not differ from those in adult WKY and SHR, except for LB thinning in the WKY and significant LB loss in the SHR. Age-related reductions in side-to-side- and step-to-step junctions, and LB loss with myocardial fibrosis in adult and aged SHR may indicate increased loss of gap junctions which couple the cells for transverse conduction, and contribute to anisotropic discontinuous propagation and potential reentrant LV arrhythmias.

Arrhythmias in Coxsackie B3 virus myocarditis. Continuous electrocardiography in conscious mice and histopathology of the heart with special reference to the conduction system.

In patients with acute myocarditis, arrhythmias constitute one of the crucial risk factors for morbidity and mortality. To clarify the incidences and the chronological features of arrhythmias and their correlation with the histopathology of the heart, we conducted continuous electrocardiography in an animal model of viral myocarditis, and light microscopy of the heart with special reference to the conduction system. Forty weanling C3H/He mice were divided into an inoculated group (IG) and a control group (CG), of 20 mice each. IG was injected intraperitoneally with coxsackie B3 virus and CG was injected with virus-free culture medium. Thin electrodes were implanted in their chest walls and connected with an electric impulse transmitting device so that they could move freely. ECGs were recorded continuously without anesthesia up to the 14th postinoculation day. The atrial and ventricular myocardium and the conduction system were studied by light microscopy. In CG, no arrhythmia was noted. In IG, various kinds of arrhythmias were documented. The incidences of the arrhythmia in the 20 mice were: sinus arrest 80%, second or third degree atrioventricular (AV) block 30%, premature atrial complexes 30%, premature ventricular complexes 20%, and ventricular tachycardia 10%. Arrhythmias were usually transient and recurrent, and the majority of them developed between the 6th and 13th day, when the histologic changes of the heart were greatest. There seemed to be a correlation between the kind of arrhythmias and the myocarditic lesions. Mice with sinus arrest or AV block developed histopathologic changes in the sinus node or AV conducting tissue, respectively. In the early stage of myocarditis, no inflammatory changes were apparent in the sinus node in the mice with sinus arrest.

Ultrastructural alterations of the conduction system in mice exhibiting sinus arrest or heart block during Coxsackievirus B3 acute myocarditis.

By light and electron microscopy we studied the sinus nodes and atrioventricular (AV) conducting tissue of six C3H/He mice having coxsackievirus B3 acute myocarditis. Sinus arrest was documented in all six mice, and second- or third-degree AV block was documented in three of the six mice. Although myocarditic changes in the conduction system, especially in the sinus node, were less than those in atrial and ventricular working myocardium, there were distinct abnormalities within both the sinus node and AV conducting tissue in all six hearts. Important ultrastructural alterations were inflammatory cell infiltrates and significant injury of specialized cells and of neural tissue. Specialized cells showed various features of degeneration and necrosis. Neural tissue damage included degeneration of axons and Schwann cells and disorganization of the neuromuscular junctions. Inflammatory cells, particularly macrophages, were often in intimate contact with injured specialized cells and neural tissue. Interstitial edema and bleeding and lymphatic vessel dilatation were also observed. These pathologic changes are considered to play an important role in the development of the documented disturbances of rhythm and conduction.

Autophagic degeneration as a possible mechanism of myocardial cell death in dilated cardiomyopathy.

In failing hearts, cardiomyocytes degenerate and interstitial fibrosis, which indicates cardiomyocyte loss, becomes more prominent in the myocardium. However, the precise mechanism of cardiomyocyte degeneration that leads to cell death is still unclear, although it is presumed that lysosomal function and autophagy play an important role because lysosomal activity increases under stress such as hypoxia. Myocardium that had been resected during partial left ventriculectomy performed in patients with dilated cardiomyopathy (DCM) was examined. Under light microscopy, some cardiomyocytes had a marked scarcity of myofibrils and had prominent cytoplasmic vacuolization. Atrophic and degenerated cardiomyocytes were often observed adjacent to replacement fibrotic tissue. Immunohistochemistry showed positivity for lysosome-associated membrane protein and a lysosomal catheptic enzyme in vacuoles of various sizes in the cardiomyocytes and these lysosomal markers were markedly increased in atrophic and degenerated cardiomyocytes. Electron microscopy revealed that degenerated cardiomyocytes had many vacuoles containing intracellular organelles, such as mitochondria, and were considered to be autophagic vacuoles. In DCM hearts, autophagy appeared to be associated not only with degradation of damaged intracellular organelles but also with progressive destruction of cardiomyocytes. It is possible that autophagic degeneration is one of the mechanisms of myocardial cell death.

Effect of sulfo-N-succinimidyl palmitate on the rat heart: myocardial long-chain fatty acid uptake and cardiac hypertrophy.

Abnormal long-chain fatty acid metabolism has been suggested as having a role in the genesis of certain cardiac diseases, and depressed myocardial long-chain fatty acid uptake has been clinically demonstrated in some patients with hypertrophic cardiomyopathy. However, the site where long-chain fatty acid metabolism is affected in cardiomyopathy remains unclear. Although cardiac hypertrophy is reported to be induced in rats by a fat-free diet, little is known of the consequences of depressed myocardial long-chain fatty acid uptake. Sulfo-N-succinimidyl derivatives of long-chain fatty acids have been shown to irreversibly inhibit long-chain fatty acid transport. To investigate the possible linkage of abnormal long-chain fatty acid uptake with cardiac hypertrophy, myocardial long-chain fatty acid uptake was blocked in rats using a sulfo-N-succinimidyl derivative of palmitate (SSP). SSP was intraperitoneally administered to rats for 12 weeks, and its effects on physiological parameters, and cardiac morphology were studied, SSP treatment (20 mg/kg) caused a 12% increase in heart weight (663.7 +/- 33.6 mg in controls v 741.2 +/- 26.5 mg after SSP treatment) and an 11% increase in the heart weight to body weight ratio (2.46 +/- 0.10 in controls v 2.72 +/- 0.17 after SSP) without any significant change of body weight. No significant differences were observed in blood pressure, heart rate, and serum hormones (insulin and triiodothyronine) between the control and SSP-treated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Anatomy of the sinus node, AV node, and His bundle of the heart of the sperm whale (Physeter macrocephalus), with a note on the absence of an os cordis.

BACKGROUND: Atrioventricular (AV) conduction time in large whales is only slightly greater than in smaller mammals even though their hearts are enormously larger. Little is known of the detailed histology or cytology of the conduction system of large whales. Such knowledge could be useful in defining the nature of cardiac rhythm and conduction of the whale as well as smaller mammals including humans. METHODS: We studied hearts from seven sperm whales. After fixation in formaldehyde and later dissection, specimens were prepared for histological examination. RESULTS: Cell size, histological organization, and innervation of the sperm whale's sinus node, AV node, and His bundle are similar to most mammalian hearts, except the sinus node is substantially larger. There is no central fibrous body between the atrial and ventricular septa, and the whale has no os cordis. Only the upper quarter of the interventricular septum is fully formed; below that there is only a thin layer of fatty connective tissue between the two ventricles. CONCLUSIONS: Given our morphological findings, we believe that the whale's comparatively short AV conduction time may be best explained by the sinus node and AV node functioning as coupled relaxation oscillators. Absence of an os cordis or central fibrous body or strong attachment between the two ventricles may pose both electrophysiological and hemodynamic hazards when the whale is no longer in its normally buoyant aquatic environment.

Myocardial integrated ultrasonic backscatter in patients with dilated cardiomyopathy: prediction of response to beta-blocker therapy.

BACKGROUND: Myocardial integrated backscatter (IB) imaging has been reported to be useful for ultrasonic tissue characterization and delineation of myocardial viability or fibrosis. beta-Blocker therapy has beneficial effects for patients with dilated cardiomyopathy (DCM), but there are no clear findings that indicate which patients with DCM will respond to this therapy. This study was performed to evaluate whether myocardial IB analysis can predict the response to beta-blocker therapy. METHODS AND RESULTS: We prospectively performed echocardiographic examination with IB analysis in 29 patients with DCM (20 men, 9 women) before starting bisoprolol therapy and in 15 normal subjects. Standard echocardiographic examination and IB analysis in the left ventricular wall in the 2-dimensional short-axis view were performed and the magnitude of cyclic variation (CV) of IB and calibrated myocardial IB intensity (subtracted pericardial) were obtained from the interventricular septum and the left ventricular posterior wall. Sixteen patients responded to bisoprolol therapy and 13 did not respond after 12 months of full-dose therapy. Calibrated myocardial IB intensity was lower in responders relative to nonresponders in both the interventricular septum (responders, -20.1 +/- 3.6 dB vs nonresponders, -9.8 +/- 5.1 dB, P <.0001; controls, -20.1 +/- 4.4 dB) and posterior wall (responders, -20.6 +/- 3.6 dB vs nonresponders, -14.6 +/- 4.2 dB, P =.0002; controls, -22.7 +/- 3.3 dB). Also, the lower the myocardial intensity in the interventricular septum or posterior wall, the better left ventricular systolic function improved after beta-blocker therapy. However, CV was lower in both DCM groups than in the controls, and CV in the interventricular septum was lower in nonresponders than in responders (responders, 4.0 +/- 4.1 dB vs nonresponders, -0.8 +/- 6. 1 dB, P <.02; controls, 8.3 +/- 2.4 dB). In addition, CV in the posterior wall showed no difference between the 2 DCM groups (responders, 5.6 +/- 1.3 dB vs nonresponders, 5.1 +/- 3.5 dB, P = not significant; controls, 9.6 +/- 2.5 dB). Also, the percent fibrosis on right ventricular endomyocardial biopsy specimens showed no distinctions between these 2 groups (responders, 25.1% +/- 16.1% vs nonresponders, 24.9% +/- 15.0%, P = not significant). CONCLUSIONS: These findings suggest that left ventricular myocardial IB data, especially IB intensity, provide useful information for predicting the response to beta-blocker therapy in patients with DCM. However, right ventricular endomyocardial biopsy findings do not appear to contribute to discriminating between the 2 groups.

Comparative ultrastructure of the tip of ventricular papillary muscle.

Electron microscopic studies of the tips of left ventricular papillary muscles from seven human, two monkey, three sheep, and two chicken hearts were done to elucidate the fine structure of myotendinous junctions. The human specimens were from normal hearts obtained 3 to 9 hours postmortem from persons aged 7 months to 30 years (mean, 13.3 years). We found no significant ultrastructural differences between the human hearts and those of monkey, sheep, and chicken. Myocardial fibers were elongated and thinner (tapered) in the tips of papillary muscles. In addition to usual working myocardial cells, the distal end of narrowing muscle fibers also contained small pale cardiocytes containing fewer myofibrils and smaller mitochondria. These cells were similar to P cells or transitional cells in the conduction system. Nerve axons and Schwann cells were commonly seen in the interstitium, usually in association with capillaries. Fibroblasts and axon varicosities were occasionally seen extremely close to the cardiocytes. These specialized myocardial cells associated with rich neural tissue in the papillary muscle tip possibly function as foci of local automaticity. This histologic organization may also represent neurosensory function responding to and monitoring local pressure changes, efferent adrenergic or cholinergic neural activity, or both.

Apoptosis and pleomorphic micromitochondriosis in the sinus nodes surgically excised from five patients with the long QT syndrome.

Sinus nodes of five symptomatic patients with the long QT syndrome were surgically excised and followed by permanent electronic pacing as part of a new surgical treatment. We examined those sinus nodes by light and electron microscopy with tissue that was promptly fixed at the time of surgery. All five sinus nodes were similarly abnormal. By light microscopy we found distinctive focal fibrosis, some degenerating myocytes and neural elements, and numerous narrowed small vessels. Except in the nerves there was no evidence of inflammation. In electron micrographs the mitochondria within nodal myocytes were abnormally abundant, remarkably pleomorphic, and smaller than those in normal human sinus nodal cells. The ultrastructural features of the degenerated nodal cells were typical of apoptosis, characterized by the absence of inflammation, well-preserved mitochondria, the presence of apoptotic bodies, phagocytosis of these cells by neighboring myocytes, and especially in smooth muscle cells of arterioles, nuclear chromatin margination and nucleolar disintegration. Apoptotic degeneration of nodal myocytes was stochastic, with adjacent cells appearing unaffected. Focal ischemia caused by narrowed vessels may be a contributory factor, and the nerves may harbor some viral infection, but for the nodal myocytes the abnormality appears to be primarily apoptosis, sometimes called programmed cell death. Both the typically episodic clinical features and the terminal event in fatal cases of the long QT syndrome may be due to apoptosis.