Kiminori Kato

Expression of Coxsackievirus and Adenovirus Receptor in Hearts of Rats With Experimental Autoimmune Myocarditis

The expression of coxsackievirus and adenovirus receptor (CAR) was dominant in the brains and hearts of mice until the newborn phase. There is no detailed information concerning the relation between the expression of CAR and development of hearts. It is also uncertain whether CAR is able to be induced in adult hearts after cardiac injury. We demonstrated that CAR was abundant in the hearts of newborn rats but was barely detectable in the hearts of adult rats. The expression of CAR in rat hearts with experimental autoimmune myocarditis, which was induced by immunization of purified cardiac myosin, was serially investigated. Active myocarditis was observed from day 15 after immunization. By immunohistochemistry, cardiomyocytes were strongly stained for CAR antibody from days 24 to 42. CAR mRNA was also detected from days 18 to 30 by using reverse transcription-polymerase chain reaction. In the next experiment, the induction of CAR on isolated cardiomyocytes was investigated. CAR was barely detectable in cultured cardiomyocytes by Western blot analysis after isolation. This molecule gradually appeared along with the creation of clusters and beating of cardiomyocytes. Furthermore, the induction of CAR in cultured cardiomyocytes increased after supplement with conditioned medium of rat splenocytes activated by concanavalin A. In conclusion, rat CAR is expressed strongly in the hearts of newborn rats and is suppressed in those of adult rats. The expression of CAR is enhanced during the active phase of experimental autoimmune myocarditis and is induced by inflammatory mediators. CAR may play a role in cell-to-cell contact and adhesion of cardiomyocytes.

Hydrodynamic-Based Delivery of an Interleukin-22-Ig Fusion Gene Ameliorates Experimental Autoimmune Myocarditis in Rats

IL-22 is one of several cytokines with limited homology to IL-10. However, the biological activities of IL-22 are mostly unknown. The purpose of this study was to evaluate the effect of IL-22 on rat experimental autoimmune myocarditis (EAM) and elucidate an aspect of the biological activities of IL-22. Rats were immunized on day 0; IL-22-Ig-treated rats were injected with pCAGGS-IL-22-Ig and control rats with pCAGGS-Ig using hydrodynamics-based gene delivery on day 1 or day 6. IL-22-Ig gene therapy administered on day 1 or day 6 after immunization was effective in controlling EAM as monitored by the heart weight to body weight ratio, and the myocarditis area in rats was sacrificed on day 17. Examination of the expression of IL-22-related genes in purified cells from EAM hearts suggested that IL-22-Ig acting target cells were noncardiomyocytic (NC) noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells. Therefore, we examined the effect of rIL-22 or serum containing IL-22-Ig on the expression of immune-relevant genes in IL-1-stimulated NC cells cultured from EAM hearts. Results showed that the expression of immunologic molecules (PGE synthase, cyclooxygenase-2, MIP-2, MCP-1, IL-6, and cytokine-induced neutrophil chemoattractant-2) in IL-1-stimulated NC cells was significantly decreased by rIL-22 or serum containing IL-22-Ig. EAM was suppressed by hydrodynamics-based delivery of plasmid DNA encoding IL-22-Ig, and the reason for this effectiveness may be that IL-22 suppressed gene expression of PG synthases, IL-6, and chemokines in activated NC noninflammatory cells.

Predictors of Disease Course in Patients With Acute Myocarditis

Background—Clinical manifestations of acute myocarditis, with distinct onset, vary from asymptomatic to fatal. The predictors of the course of the disease in patients with acute myocarditis at initial presentation have not yet been established. In this study, we examined the predictive values of various parameters in the disease course of patients with myocarditis. Methods and Results—Twenty-one consecutive patients who had been diagnosed as having acute myocarditis by histological examinations were analyzed. The patients with myocarditis were divided into the survival group (n=13) and the fatal group (n=8). We examined the parameters of the clinical state, hemodynamic variables, required therapies, biochemical laboratory data, and cytokines. The control groups were composed of 23 patients with old myocardial infarction and 20 healthy volunteers. The fatal group had lower blood pressure and higher pulmonary capillary wedge pressure compared with those values in the survival group. Mechanical ventilation support was more frequently required in the fatal group. Serum levels of soluble Fas (sFas) and soluble Fas ligand (sFasL) were significantly higher in the myocarditis group than in the 2 control groups. Furthermore, levels were significantly higher in the fatal group than in the survival group for sFas (13.93±4.77 versus 3.77±0.52 ng/mL, respectively;P <0.001) and sFasL (611.4±127.7 versus 269.5±37.3 pg/mL, respectively;P <0.05). Other clinical states, hemodynamic variables, required therapies, and biochemical laboratory parameters were not different between the 2 groups. Conclusions—Elevation of sFas and sFasL levels at initial presentation appear to be a good serological marker to predict the prognosis of acute myocarditis.

Low dose carvedilol inhibits progression of heart failure in rats with dilated cardiomyopathy

The cardioprotective properties of carvedilol (a vasodilating β‐adrenoceptor blocking agent) were studied in a rat model of dilated cardiomyopathy induced by autoimmune myocarditis. Twenty‐eight days after immunization, surviving Lewis rats (32/43=74%) were divided into three groups to be given 2 mg kg−1 day−1 (Group‐C2, n=10) or 20 mg kg−1 day−1 (Group‐C20, n=10) of carvedilol, or vehicle (0.5% methylcellulose, Group‐V, n=12). After oral administration for 2 months, body weight, heart weight (HW), heart rate (HR), rat α‐atrial natriuretic peptide (r‐ANP) in blood, central venous pressure (CVP), mean blood pressure (mean BP), peak left ventricular pressure (LVP), left ventricular end‐diastolic pressure (LVEDP), ±dP dt−1 and area of myocardial fibrosis were measured. Values were compared with those for normal Lewis rats (Group‐N, n=10). Two out of 12 (17%) rats in Group‐V died from day 28 to day 42 after immunization. No rat died in Groups‐C2, ‐C20 and ‐N. Although the CVP, mean BP, LVP and ±dP dt−1 did not differ among the three groups, the HW, HR and r‐ANP in Group‐C2 (1.14±0.03, 339±16 and 135±31) and Group‐C20 (1.23±0.04, 305±8 and 156±24) were significantly lower than those in Group‐V (1.36±0.04 g, 389±9 beats min−1 and 375±31 pg ml−1, respectively). The LVEDP in Group‐C2 was significantly lower than that in Group‐V (7.4±1.4 and 12.2±1.2 mmHg, respectively, P<0.05). The area of myocardial fibrosis in Group‐C2 was smaller than that in Group‐V (12±1 and 31±2%, P<0.01). These results indicate that a low dose of carvedilol has beneficial effects on dilated cardiomyopathy.

Enhanced expression and production of monocyte chemoattractant protein-1 in myocarditis.

Monocyte chemoattractant protein‐1 (MCP‐1) is a member of the C‐C chemokine family that has been shown to play a major role in the migration of monocytes and T cells to an inflammatory focus. To clarify the role of MCP‐1 in the pathogenesis of myocarditis, we have examined the expression of MCP‐1 in rat hearts with experimental autoimmune myocarditis (EAM), and have also measured serum levels of MCP‐1 in patients with histology‐proven acute myocarditis. Lewis rats were immunized with cardiac myosin and were killed 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 42 and 56 days after immunization. Large mononuclear cells in the myocardial interstitium were stained with an anti‐MCP‐1 antibody. mRNA of MCP‐1 increased in the hearts of EAM rats from days 15–27 as shown by quantitative reverse transcription‐polymerase chain reaction. Serum MCP‐1 levels of the rats with EAM were significantly elevated from days 15–24. In the clinical study, serum levels of MCP‐1 in 24 patients with acute myocarditis at the time of admission (165·2 ± 55·8 pg/ml) were significantly (P = 0·0301) elevated compared with those of 20 healthy volunteers (61·8 ± 10·7 pg/ml). Serum MCP‐1 levels of 8 fatal cases (371·8 ± 145·2 pg/ml) were significantly (P = 0·0058) higher than those of 16 cases who survived (65·5 ± 12·8 pg/ml). In conclusions, MCP‐1 may play an important role in the pathogenesis of human acute myocarditis as well as in the progression of rat EAM.

Myocardial CD36 expression and fatty acid accumulation in patients with type I and II CD36 deficiency

Long-chain fatty acids (LCFA) are one of the major cardiac energy substrates, so understanding LCFA metabolism may help in elucidating the mechanisms of various heart diseases. CD36 is a multifunctional membrane glycoprotein that acts not only as a receptor for thrombospondin, collagen and oxidized low density lipoprotein but also as a receptor for LCFA. We investigated the relationship between CD36 expression in myocardial capillary endothelial cells and myocardial LCFA uptake in patients with CD36 deficiency. We analyzed CD36 expression in blood cells from 250 patients with heart diseases by means of a flow cytometer. In 218 patients, myocardial LCFA scintigraphy was performed with123I-β-methyl-p-iodophenyl pentadecanoic acid (BMIPP). In 5 patients, myocardial capillary endothelial cells were examined immunohistochemically for CD36 expression. Eleven patients (4%) showed signs of type I CD36 deficiency (neither platelets nor monocytes expressed CD36). Twenty patients (8%) had type II CD36 deficiency (monocytes expressed CD36 but platelets did not). In all 11 patients with type I CD36 deficiency, no BMIPP accumulation was observed in the heart, but in 13 patients with type II CD36 deficiency, BMIPP accumulation in the heart was focally reduced, but there were no patients without BMIPP accumulation in the heart. Although the myocardial capillary endothelial cells from two CD36-positive patients expressed CD36, those from two patients with type I CD36 deficiency did not. In a patient with type II CD36 deficiency, some capillary endothelial cells displayed patchy CD36 expression.CD36 deficiency was documented in 31 (12%) patients with heart diseases. Because CD36 was not expressed in the myocardial capillary endothelial cells in patients with type I CD36 deficiency, type I CD36 deficiency is closely related to lack of myocardial LCFA accumulation and metabolism in the myocardium.

Relationship between myoglobin contents and increases in cyclic GMP produced by glyceryl trinitrate and nitric oxide in rabbit aorta, right atrium and papillary muscle

SummaryEffects of glyceryl trinitrate (GTN) and nitric oxide (NO) on the cardiac functions and myocardial cyclic GMP (cGMP) contents were examined in comparison with those in the aorta and correlated with myoglobin (an inhibitor of soluble guanylate cyclase) contents using the preparations isolated from the reserpinized rabbit.GTN (10−10-10−4mol/l) produced a dose-dependent relaxation in the aorta. However, this compound exerted no effect on the rate of the spontaneous beat of the right atrium and the contraction of the papillary muscle. A transient and significant increase in cGMP was observed in the aorta with GTN (3 × 10−6 mol/l). Although the increase was also observed in the right atrium, it was much smaller. No definite change was observed in papillary muscle. Increases in cGMP produced by NO (3 × 10−6 mol/l) were larger and significant in all tissues; (AUCcGMP(GTN)/AUCcGMP(NO)) ratio was 30.1 for the aorta, 65.0 for the right atrium and 16.3% for the papillary muscle. Although higher concentrations of NO were necessary in the right atrium and papillary muscle to induce increases in cGMP, no differences were noted in the three tissues as regards the maximum accumulation of this substance. Furthermore, kinetic analysis of NO-induced increases in tissue cGMP indicated no marked difference in the production rate among the three tissues, while the rate of elimination of cGMP was lower in the aorta than in the atrium or the papillary muscle. The increases in cGMP observed in these three tissues were inversely related to the contents of myoglobin in respective tissues. No effect on myocardial function was observed with NO up to the concentration of 3 × 10−5 mol/l.These results suggest that myoglobin, an endogenous inhibitor of activation of soluble guanylate cyclase by NO, was responsible for the lower production of cGMP by NO and GTN in the myocardial tissue.

Expression of the peptide hormone hepcidin increases in cardiomyocytes under myocarditis and myocardial infarction

The micronutrient iron is an essential component that plays a role in many crucial metabolic reactions. The peptide hormone hepcidin is thought to play a central role in iron homeostasis and its expression is induced by iron overloading and inflammation. Recently, hepcidin has been reported to be expressed also in the heart; however, the kinetics of altered hepcidin expression in diseases of the heart remain unknown. In this study, we examined cardiac expression of hepcidin in rat experimental autoimmune myocarditis (EAM), human myocarditis and rat acute myocardial infarction (AMI). In rat EAM and AMI hearts, hepcidin was expressed in cardiomyocytes; ferroportin, which is a cellular iron exporter bound by hepcidin, was also expressed in various cells. Analysis of the time course of the hepcidin to cytochrome oxidase subunit 6a (Cox6a)2 expression ratio showed that it abruptly increased more than 100-fold in hearts in the very early phase of EAM and in infarcted areas 1 day after MI. The hepcidin/Cox6a2 expression ratio correlated significantly with that of interleukin-6/gamma-actin in both EAM and AMI hearts (r=0.781, P<.0001 and r=0.563, P=.0003). In human hearts with histological myocarditis, the ratio was significantly higher than in those without myocarditis (0.0400+/-0.0195 versus 0.0032+/-0.0017, P=.0045). Hepcidin is strongly induced in cardiomyocytes under myocarditis and MI, conditions in which inflammatory cytokine levels increase and may play an important role in iron homeostasis and free radical generation.

Spatiotemporal changes of coxsackievirus and adenovirus receptor in rat hearts during postnatal development and in cultured cardiomyocytes of neonatal rat

Coxsackievirus B is the most common cause of viral myocarditis and is particularly virulent in neonates and children. Adenovirus is also a leading cause of the disease. The determinant of tropism for both viruses is considered to be the expression of coxsackievirus and adenovirus receptor (CAR) in target organs. However, developmental change and physiological localization of CAR in the heart are unknown. We examined expression levels of CAR in rat hearts by quantitative real-time polymerase chain reaction and Western blot analysis and found that CAR decreased gradually during postnatal development, although CAR was detectable, even in adults. Immunohistochemistry revealed CAR on the whole surface of cardiomyocytes in immature rat hearts. In contrast, CAR was detected predominantly on intercalated disks in the adult heart and was accumulated especially at the contact point between the cultured cardiomyocytes, even though they were prepared from the neonatal rat heart. In conclusion, CAR was expressed abundantly on the whole surface of cardiomyocytes in immature rat hearts. Both the expression level and the localization of CAR are possible determinants of the susceptibility to viral myocarditis of neonates and children.

Appropriate Waist Circumference Cutoff Values for Persons with Multiple Cardiovascular Risk Factors in Japan: a Large Cross-sectional Study

Background In Japan, the current standard waist circumference cutoff value for persons with multiple cardiovascular risk factors remains controversial. In this study we aimed to analyze the health-check examination data from a large Japanese population and propose a revised waist circumference cutoff value. Methods Subjects of this study were 12,725 adults who underwent a health-check by thorough medical examination between April 2006 and March 2007. Medical examinations included measurement of waist circumference, fasting blood triglycerides, HDL cholesterol, glucose concentrations, blood pressure and collection of demographic characteristics. Receiver operating characteristic (ROC) curve analysis was utilized to find appropriate waist circumference cutoff values in relation to multiple cardiovascular risk factors with two or more of the following: dyslipidemia (hypertriglyceridemia or low HDL cholesterol), hypertension, and hyperglycemia defined by the Japanese criteria of metabolic syndrome. Results The average age of the subjects was 50.7 years (standard deviation [SD]: 8.8) for men and 49.7 years (SD: 8.6) for women. ROC curve analysis showed maximum sensitivity plus specificity at a waist circumference of 87 cm in men (0.66 and 0.62, respectively) and 83 cm in women (0.73 and 0.70). When analyzed by ten-year age groups, the ROC curves for younger age groups were shifted up and to the left compared to older age groups, but associations between cutoff values and age were not clear. Conclusion In Japan, the appropriate cutoff value of waist circumference for persons with multiple cardiovascular risk factors is 87cm for men and 83 cm for women.