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.

Angiotensin II receptor blockade prevents microangiopathy and preserves diastolic function in the diabetic rat heart.

BACKGROUND: Cardiac microangiopathy may be involved in the development of heart failure in diabetes mellitus. OBJECTIVE: To evaluate the effect of angiotensin II receptor blockade on cardiac function and fine structures in diabetes. METHODS: Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats (n = 30), a model of spontaneously developing diabetes mellitus, and their diabetes resistant counterparts (n = 20) were used. At 30 weeks of age, when the OLETF rats show hyperglycaemic obesity with hyperinsulinaemia, the animals were divided into two groups and given candesartan, an angiotensin II receptor blocker, 0.2 mg/kg/day, or vehicle for six weeks. Capillary density was evaluated in the left ventricular myocardium by electron microscopy, matrix metalloproteinase (MMP) activity by zymography, and cytokines by reverse transcriptase polymerase chain reaction. RESULTS: Compared with the control rats, the OLETF rats at 36 weeks showed decreased peak negative dP/dt (mean (SD): 2350 (250) v 3492 (286) mm Hg/s) and increased cardiomyocyte diameter (24.3 (0.6) v 18.9 (0.6) microm) (both p < 0.05). Thickening of the capillary basement membranes and decreased capillary density were observed. Angiotensin receptor blockade improved almost all the haemodynamic variables, and the histological findings became similar to those of the controls. Angiotensin receptor blockade also activated MMP-2 and prevented an increase of inflammatory cytokines, especially interleukin (IL)-1beta and IL-6, in the diabetic heart. CONCLUSIONS: Angiotensin II receptor blockade preserved left ventricular diastolic function. It was also potent at improving cardiomyocyte diameter and the thickening of the capillary basement membrane, increasing MMP-2 activity, and decreasing inflammatory cytokines. With all these changes, candesartan could contribute to cardioprotection in diabetes mellitus.

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.

Enterovirus RNA replication in cases of dilated cardiomyopathy: light microscopic in situ hybridization and virological analyses of myocardial specimens obtained at partial left ventriculectomy.

OBJECTIVE: Recently, attention has been focused on enteroviral infection of the heart in the genesis of dilated cardiomyopathy (DCM). To determine the location of enteroviral RNA in the myocardium, we performed light microscopic in situ hybridization (ISH) and virological analyses of myocardial specimens obtained at partial left ventriculectomy (PLV). METHODS: Posterolateral walls of the left ventricle from 26 DCM patients were examined. Myocardial specimens were tested for the presence of enteroviral genomes by polymerase chain reaction (PCR). We selected two age-matched groups (10 patients each) in which enteroviruses were either present (EV-plus group) or not (EV-minus group). For both groups, we examined in situ localization of enteroviral RNA in the myocardium by ISH. RESULTS: In PCR studies, both sense and antisense enteroviral RNA were detected in the myocardium of seven patients in the EV-plus group. The presence of this RNA indicates active viral replication in the myocardium. Five of seven patients who exhibited both sense and antisense enteroviral RNA died early after surgery. On ISH, three patients had evidence of active replication of enteroviral genomes. Viral genomes were present in myocardial lesions, especially in endocardial sites. Viral signals were found in degenerating myocardial cells, interstitial inflammatory cells, and endothelial cells of small vessels. These positive signals were not detected in the myocardium of the EV-negative group. CONCLUSIONS: We detected both sense and antisense enteroviral RNA in various myocardial lesions. This suggests that active enteroviral replication plays a role in the development of myocardial lesions in DCM patients. Active viral replication appears to be a prognostic factor for DCM after PLV. Further study of active viral replication in myocardial lesions will provide information useful for evaluating different therapeutic strategies for DCM.

Myocardial inflammatory cell infiltrates in cases of dilated cardiomyopathy as a determinant of outcome following partial left ventriculectomy.

Partial left ventriculectomy (PLV) can be used to treat refractory congestive heart failure caused by dilated cardiomyopathy (DCM). In order to understand the relationship between the underlying myocardial injury and early clinical outcomes after PLV, histopathologic, immunohistochemical and virologic studies of the resected myocardium were performed. The posterolateral left ventricular walls from 27 patients with idiopathic DCM were examined. Cardiomyocyte diameter, degree of myocardial fibrosis, degree of cardiomyocyte degeneration, and degree of inflammatory cell infiltration were compared with mortality rates. Polymerase chain reaction was performed to detect enterovirus genome in the myocardium. Some patients had inflammatory cell infiltrates with focal accumulations of lymphocytes and macrophages, including both cytotoxic/suppressor T-cells and helper/inducer T-cells. The number of inflammatory cells (activated lymphocytes plus macrophages/mm2) was significantly greater in patients who died of cardiac insufficiency after surgery (27.8 +/- 5.7; n = 7) than in the survivors (11.1 +/- 2.5; n = 15). There was no significant difference in the degree of myocardial fibrosis, cardiomyocyte diameter or degree of cardiomyocyte degeneration between the 2 groups. Enterovirus genome was detected in the myocardium of 9 (38%) of 24 patients examined and 5 of these enterovirus-positive hearts had severe inflammatory cell infiltrates (37.9 +/- 2.5/mm2). Early survival in patients undergoing PLV for DCM is significantly affected by the degree of myocardial inflammation, so patients with more severe or ongoing inflammation may have poor clinical outcomes. Chronic myocarditis may play an important role in the etiology and pathophysiology of idiopathic DCM.

A case of cardiomyopathy showing progression from the hypertrophic to the dilated form: association of Mt8348A-->G mutation in the mitochondrial tRNA(Lys) gene with severe ultrastructural alterations of mitochondria in cardiomyocytes.

This report describes a case of cardiomyopathy with a novel point mutation of mitochondrial DNA coding lysine tRNA in association with severe ultrastructural alterations of the mitochondria in the cardiomyocytes. Abnormalities of energy production and/or abnormal protein synthesis because of the mutation of mitochondrial DNA may have played an important role in the pathogenesis of this case, which showed severe cardiomyocyte degeneration and deterioration from hypertrophic cardiomyopathy to severe dilated cardiomyopathy.

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.

Up-regulation of natriuretic peptides in the ventricle of Csx/Nkx2-5 transgenic mice.

A cardiac homeobox-containing gene Csx/Nkx2-5, which is essential for cardiac development, is abundantly expressed in the adult heart as well as in the heart primordia. Targeted disruption of this gene results in embryonic lethality due to abnormal heart morphogenesis. To elucidate the role of Csx/Nkx2-5 in the adult heart, we generated transgenic mice which overexpress human Csx/Nkx2-5. The transgene was expressed abundantly in the heart and the skeletal muscle. mRNA levels of several cardiac genes including natriuretic peptides, CARP, MLC2v, and endogenous Csx/Nkx2-5 were increased in the ventricle of the transgenic mice. Electron microscopic analysis revealed that the ventricular myocardium of the transgenic mice had many secretory granules, which disappeared after administration of vasopressin. These results suggest that Csx/Nkx2-5 regulates many cardiac genes and induces formation of secretory granules in the adult ventricle.

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.

Increased digitalis-like immunoreactive substances in patients with hypertrophic cardiomyopathy.

AIMS: Although increased digitalis-like immunoreactive substances have been found in cases of hypertension and heart failure, no information is available about digitalis-like immunoreactive substances in patients with hypertrophic cardiomyopathy. We investigated digitalis-like immunoreactive substances in the plasma and biopsied specimens of patients with hypertrophic cardiomyopathy. METHODS AND RESULTS: In 40 patients with hypertrophic cardiomyopathy (27 with the non-obstructive type and 13 with the obstructive type), the plasma concentration of digitalis-like immunoreactive substances was studied by fluorescence polarization immunoassay. Right ventricular endomyocardial biopsy specimens were analysed immunohistochemically, using a monoclonal antibody against digoxin. An increase in digitalis-like immunoreactive substances of more than 0.2 ng. ml(-1)in plasma was found in six of 27 patients with non-obstructive hypertrophic cardiomyopathy (22.2%) and five of 13 with obstructive hypertrophic cardiomyopathy (38.4%). Under light microscopy, positive staining against the antibody was observed heterogeneously on some cardiocytes. In non-obstructive hypertrophic cardiomyopathy, digitalis-like immunoreactive substances in the plasma correlated with the left atrial dimension and inversely with the cardiac index. In obstructive hypertrophic cardiomyopathy, plasma and myocardial digitalis-like immunoreactive substances were positively correlated; they also correlated with left ventricular end-diastolic pressures. Under electron microscopy, digitalis-like immunoreactive substances were detected at the sarcolemma in the free wall, T-tubules, intercalated discs and Z-bands of cardiocytes. CONCLUSIONS: Increased digitalis-like immunoreactive substances in plasma and cardiocytes, which may have been caused by pressure and/or volume overload, were found in patients with hypertrophic cardiomyopathy. Digitalis-like immunoreactive substances may act on the sarcolemma of cardiocytes and be transported into the cytoplasm.

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.

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.

Myocardial inflammatory cell infiltrates in cases of dilated cardiomyopathy: light microscopic, immunohistochemical, and virological analyses of myocardium specimens obtained by partial left ventriculectomy.

OBJECTIVE: Partial left ventriculectomy was introduced for the treatment of refractory dilated cardiomyopathy (DCM). To determine the presence and degree of inflammatory cell infiltrates in DCM and the correlation between the underlying myocardial injury and early clinical outcomes after the operation, we performed histopathological, immunohistochemical, and virological studies of the resected myocardium. METHODS: Posterolateral walls of the left ventricle from 13 idiopathic DCM patients (9 males and 4 females; mean age = 53+/-14 years) were examined. Qualitative and quantitative analyses of the interstitial fibrosis and of the infiltrating inflammatory cells were conducted. For the immunohistochemistry, leukocyte surface markers and antibodies to adhesion molecules and cytokines were used. The histopathological findings were compared with the clinical results, including outcome within 1 year, and pre- and postoperative hemodynamic data. Genomic analysis of the myocardium with polymerase chain reaction was performed for enterovirus, mumps, influenza A, cytomegalovirus, and hepatitis C virus. RESULTS: (1) The three patients who died of cardiac insufficiency after surgery had a higher count of infiltrating inflammatory cells than the eight survivors (32.1+/-10.4 vs 16.3+/-11.9 cells/mm2, p = 0.07). The severity of interstitial fibrosis (percent fibrosis) did not differ significantly between these two groups (28.3+/-15.0 vs 24.0+/-11.7%). (2) In patients who died of myocardial dysfunction, focal accumulations of lymphocytes were common, in which cytotoxic/suppressor T cells and helper/inducer T cells were observed. (3) Enterovirus genome was detected in the myocardium of two patients, both of them died after surgery. CONCLUSIONS: Inflammatory cell infiltrates or active myocarditis appear in some cases to play an important role in the etiology and pathophysiology of clinically diagnosed DCM. There is a possibility that those patients with a more severe or ongoing inflammatory process might have poor outcomes after partial left ventriculectomy.

A case of malignant lymphoma with diastolic heart failure.

In patients with malignant lymphoma, the first signs and symptoms are frequently noncardiac and clinical manifestations of cardiac involvement are often nonspecific. This case report presents a patient with malignant lymphoma whose first manifestation was characteristic of heart failure, mainly due to diastolic dysfunction, and whose postmortem examination revealed massive myocardial invasion.

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.