Takeshi Kasajima

Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes.

We examined whether apoptosis occurs in cardiomyocytes by hypoxia in vitro. Neonatal rat cardiomyocytes and nonmyocytes were cultured in 95% N2-5% CO2 atmosphere to produce hypoxic conditions. DNA fragmentation into integer multiples of the internucleosomal DNA length was observed in cardiomyocytes as early as 12 hours, whereas nonmyocytes did not show fragmentation of DNA up to 72 hours. DNA fragmentation of cardiomyocytes induced by hypoxia was also confirmed by nick-end labeling in situ. Messenger RNA for Fas antigen, a mediator of apoptotic cell death, was expressed in both cardiomyocytes and nonmyocytes as revealed by Northern blotting and in situ hybridization. In hypoxic condition, Fas messenger RNA levels in cardiomyocytes were upregulated by twofold over controls, whereas those of nonmyocytes were downregulated. These results indicate that cardiomyocyte death by hypoxia can occur via apoptosis and that Fas antigen may be associated with the mechanism of this apoptotic process.

Nitric Oxide Contributes to the Progression of Myocardial Damage in Experimental Autoimmune Myocarditis in Rats

BACKGROUNDnExcess amounts of NO produced by an inducible NO synthase (iNOS) in response to cytokines may be cytotoxic and can be destructive to tissue. We investigated the role of NO in the development of myocardial damage and the effects of aminoguanidine (AG), an inhibitor of iNOS, on experimental autoimmune myocarditis in rats.nnnMETHODS AND RESULTSnAutoimmune myocarditis was induced in 20 Lewis rats by injection of porcine cardiac myosin. Ten of the 20 rats were administered AG. The severity of myocarditis was evaluated by measuring the size of myocarditic lesion and serum levels of CK-MB. Serum NO levels were determined using the Cd/Cu method. Tissue specimens were immunohistochemically examined for iNOS and nitrotyrosine. Histopathological study revealed extensive myocardial destruction and massive inflammatory cell infiltration in AG-untreated rats but only focal mononuclear cell infiltration in AG-treated rats. The mean percent areas of inflammatory lesions in the untreated and treated rats were 56 +/- 13% and 3 +/- 2%, respectively (P < .001). NO levels were 102 +/- 23 and 25 +/- 9 IU/L, respectively (P < .01). CK-MB levels were 68 +/- 13 and 16 +/- 13 nmol/L, respectively (P < .01). Superoxide production as measured with an ex vivo monitoring system was also significantly decreased in the treated rats. Nitrotyrosine relating to the generation of peroxynitrite was detected through immunostaining in the inflammatory lesions of untreated rats but not in those of treated rats.nnnCONCLUSIONSnExcess amounts of NO produced by iNOS appear to contribute to the progression of myocardial damage in myocarditis. AG may prove to be useful in the treatment of myocarditis.

Expression of Costimulatory Molecules B7–1, B7–2, and CD40 in the Heart of Patients With Acute Myocarditis and Dilated Cardiomyopathy

BACKGROUNDnIn patients with acute myocarditis and dilated cardiomyopathy (DCM), we previously reported that antigen-specific T cells infiltrate the heart and play an important role in the myocardial damage involved. For antigen-specific T-cell activation to occur, it is necessary for T cells to receive a costimulatory signal provided by costimulatory molecules expressed on antigen-presenting cells (APCs) as well as the main signal provided by binding of T-cell receptors to the antigen.nnnMETHODS AND RESULTSnTo investigate the roles of the costimulatory molecules B7-1, B7-2, and CD40 in the development of acute myocarditis and DCM, we analyzed the expression of these antigens in the myocardial tissues of patients with acute myocarditis and DCM. We also examined the expression of a cytolytic factor, perforin, in the infiltrating cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, because both killer lymphocytes are thought to damage B7-1-expressing APCs. We found that B7-1, B7-2, and CD40 were moderately to strongly expressed in the cardiac myocytes of patients with acute myocarditis. Weak to moderate expression of these antigens was also found in the cardiac myocytes of patients with DCM. There was infiltration of perforin-expressing CTLs and NK cells in the myocardial tissues of patients with acute myocarditis and DCM.nnnCONCLUSIONSnOur findings strongly suggest that expression of B7-1, B7-2, and CD40 antigens on cardiac myocytes may make them APCs for CTLs and NK cells and that they may play an important role in the direct myocardial damage by these killer cells in acute myocarditis and DCM.

An autopsied case of Williams syndrome complicated by moyamoya disease

An 18 year old girl with typical clinical features of Williams syndrome suddenly died of intracerebral hemorrhage due to moyamoya disease. Autopsy revealed vascular abnormalities, such as supravalvular aortic stenosis (SAS) and an abnormal complicated cerebrovascular network in the cerebral arteries. The arterial wall of the SAS lesion consisted of thickened medial tissue showing elastic disorganization with prominence of the smooth muscle cells. The narrowed vessels of the circle of Willis showed intimal thickening with an extremely wavy internal elastic lamina and marked thinning of the media. To our knowledge, this is the first report of moyamoya disease associated with Williams syndrome.

Programmed Cell Death in the Myocardium of Arrhythmogenic Right Ventricular Cardiomyopathy in Children and Adults

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by fibrofatty replacement of the right ventricular myocardium. Recently, the myocardial loss in ARVC has been suggested to be related to apoptosis. However, it is still unknown whether this phenomenon is already established in the myocardium of pediatric cases with this disease. We examined the histopathologic characteristics of the ventricular myocardium in specimens obtained from 10 patients, including 3 children with ARVC, and investigated the occurrence of apoptosis in the myocardium by terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay and agarose-gel electrophoresis of DNA. Endomyocardial biopsy specimens from the 10 cases and a necropsy sample from one adult case with ARVC were examined. Histopathologic examination of biopsy specimens from the pediatric cases revealed extensive fibrosis. Typical fatty infiltration was demonstrated in one of the 3 pediatric cases. These findings were similar to those in adult cases; the histopathologic index based on the severity of myocardial damage, including myocyte degeneration and fibrosis, was not significantly different from that in adult cases. TUNEL assay revealed positive reactivity of the myocardial cells. The apoptotic index was 1.4 +/- 0.4% in children and 1.6 +/- 0.5% in adults (difference not statistically significant). Agarose-gel electrophoresis of a DNA extract of the myocardial tissue of the autopsy case revealed DNA fragmentation. Cases with idiopathic ventricular tachycardia and control cases with a cardiac transplant (with no rejection) had minimal histopathologic findings and negative reactivity in the TUNEL assay. These results indicate that myocardial damage is already established in cases diagnosed as ARVC in childhood, and suggest that the myocardial damage is closely related to apoptosis in children, as well as in adults, in this disease.

Expression of tumor necrosis factor ligand superfamily costimulatory molecules CD27L, CD30L, OX40L and 4-1BBL in the heart of patients with acute myocarditis and dilated cardiomyopathy

BACKGROUNDnT-cell-mediated myocardial damage is known to be involved in acute myocarditis and dilated cardiomyopathy. Recently, we found that tumor necrosis factor (TNF) ligand superfamily costimulatory molecules, especially 4-1BBL, played an important role in the myocardial damage of murine acute viral myocarditis.nnnMETHODS AND RESULTSnTo investigate the roles for CD27L, CD30L, OX40L and 4-1BBL, which belong to TNF ligand superfamily, in the development of acute myocarditis and dilated cardiomyopathy, we analyzed the expression of these antigens in the myocardial tissues of patients with acute myocarditis and dilated cardiomyopathy. We also examined expression of the receptors for these molecules, CD27, CD30, OX40 and 4-1BB, which belong to TNF receptor superfamily, on the infiltrating cells. Strong expression of CD27L, CD30L and 4-1BBL and weak to moderate expression of OX40L was found in the cardiac myocytes of patients with acute myocarditis. Moderate expression of CD27L, CD30L and 4-1BBL and weak expression of OX40L was found on the cardiac myocytes of patients with dilated cardiomyopathy. Most of the infiltrating cells expressed CD27, CD30 and 4-1BB and a part of the infiltrating cells expressed OX40.nnnCONCLUSIONSnOur findings suggest that expression of TNF ligand superfamily costimulatory molecules on cardiac myocytes may play a role in the cell-mediated myocardial damage in patients with acute myocarditis and dilated cardiomyopathy as in murine viral myocarditis.

Inhibitory effects of vesnarinone in the progression of myocardial damage in experimental autoimmune myocarditis in rats

OBJECTIVESnVesnarinone, a positive inotropic and immunomodulatory agent, diminishes nitric oxide (NO) levels by suppressing the induction of inducible NO synthase (iNOS) expressed in cytokine-stimulated macrophages and cardiomyocytes. We examined whether vesnarinone exerts inhibitory effects on the progression of myocardial damage in experimental autoimmune myocarditis in rats through suppression of iNOS.nnnMETHODSnMyocarditis was induced in 30 Lewis rats by injection of porcine cardiac myosin and vesnarinone was orally administered to 20 of the 30 rats. On day 21 after immunization (the climax of inflammation), the hemodynamics were examined and the severity of myocarditis was evaluated by determining the area ratio (%) [affected/entire area] of myocardial lesions in histological sections. Levels of serum CK-MB, NOx (NO2(-)+NO3-), TNF-alpha and IL-1 beta, and cyclic GMP, iNOS mRNA, TNF-alpha and IL-1 beta in heart tissues were determined. Expression of iNOS and TNF-alpha protein were examined by immunohistochemical methods.nnnRESULTSnHistopathological examination revealed extensive myocardial destruction and massive infiltration of inflammatory cells in the vesnarinone-untreated rats. The area ratio of the lesions in the treated rats was significantly lower than that in the untreated ones. Levels of CK-MB, NOx, cyclic GMP, cytokines and iNOS mRNA were significantly lower in the vesnarinone-treated rats. Infiltrating macrophages and cardiomyocytes in the untreated rats showed much higher levels of expression of iNOS and TNF-alpha than those in the vesnarinone-treated rats.nnnCONCLUSIONSnVesnarinone may prove to be useful in the treatment of myocarditis by attenuating NO production through suppression of iNOS induced by cytokines.

Histopathologic aspects of endomyocardial biopsy in pediatric patients with idiopathic ventricular tachycardia

Abstract Purpose: The present study aimed to investigate the clinicopathologic findings and histopathologic characteristics of endomyocardial biopsy in pediatric patients with idiopathic ventricular tachycardia.

The effect of forskolin on the teratogenicity of methylxanthines in the chick embryo heart

Interactions between forskolin and methylxanthines, including caffeine and isobutylmethylxanthine (IBMX), in the developing chick embryo heart were investigated. Forskolin, a potent activator of adenylate cyclase, was administered to young chick embryos (Hamburger-Hamilton stage 24) together with caffeine or IBMX at doses where each agent alone caused minimal embryotoxicity. The incidence of malformation in the embryonic chick heart or aorta induced by caffeine (5 x 10(-7) or 5 x 10(-6) mol) and IBMX (1 or 2.5 x 10(-6) mol) significantly increased with coadministration of forskolin (1 x 10(-9) mol). Cardiovascular malformations included ventricular septal defect, double outlet right ventricle, and aortic arch anomalies. These results indicate that forskolin potentiates the teratogenicity of caffeine or IBMX on the cardiovascular system in the chick embryo and suggest that this potentiation may be related to increase intracellular cAMP due to stimulation of adenylate cyclase (forskolin) and inhibition of phosphodiesterase (methylxanthines).

726-1 Expression of Inducible Nitric Oxide Synthase in the Myocardium of Acute Myocarditis – A Serial Cardiac Biopsy Study

In endotoxin shock, hypotension and impaired myocardial contractility may be caused by increased nitric oxide (NO) formed by inducible NO synthase (iNOS) which is induced by cytokines and bacterial lipopolysaccarides. We have investigated whether iNOS is expressed in serially biopsied samples from the right ventricle of 20 cases of acute myocarditis by immunohistochemistry and in situ hybridization. Distinct iNOS immunoreactivity was observed in cardiomyocytes, endothelial cells, vascular smooth muscle cells and macrephages in all cases during acute active stage, in which cardiac function was severely impaired. Immunoelectron microscopic distribution of iNOS was evident in the cytosol of these cells. However, little staining was disclosed in the myocardium during the convalescent stage, showing normal cardiac function. The distribution of myocytes with elevated iNOS mRNA concentration was identical to that of these immunoreactive cells. Conclusions The present study shows the expression of human myocardial iNOS in acute myocarditis, suggesting that the enhanced production of NO by iNOS is cytotoxic and accounts, in part, for myocardial injury and reduced myocardial contractility during acute illness.