Koichi Fuse

Myeloid Differentiation Factor-88 Plays a Crucial Role in the Pathogenesis of Coxsackievirus B3–Induced Myocarditis and Influences Type I Interferon Production

Background— Myeloid differentiation factor (MyD)-88 is a key adaptor protein that plays a major role in the innate immune pathway. How MyD88 may regulate host response in inflammatory heart disease is unknown. Methods and Results— We found that the cardiac protein level of MyD88 was significantly increased in the hearts of wild-type mice after exposure to Coxsackievirus B3 (CVB3). MyD88−/− mice showed a dramatic higher survival rate (86%) in contrast to the low survival (35%) in the MyD88+/+ mice after CVB3 infection (P<0.0001). Pathological examination showed a significant decrease of cardiac and pancreatic inflammation in the MyD88−/− mice. Viral concentrations in the hearts were significantly decreased in the MyD88−/− mice. Cardiac mRNA levels for interleukin (IL)-1&bgr;, tumor necrosis factor (TNF)-&agr;, interferon (IFN)-&ggr;, and IL-18 were significantly decreased in the MyD88−/− mice. Similarly, serum levels of T-helper 1 cytokines were significantly decreased in the MyD88−/− mice. In contrast, cardiac protein levels of the activated interferon regulatory factor (IRF)-3 and IFN-&bgr; were significantly increased in the MyD88−/− mice but not other usual upstream signals to IRF-3. The cardiac expression of coxsackie-adenoviral receptor and p56lck were also significantly decreased. Conclusions— MyD88 appears to be a key contributor to cardiac inflammation, mediating cytokine production and T-helper-1/2 cytokine balance, increasing coxsackie-adenoviral receptor and p56lck expression and viral titers after CVB3 exposure. Absence of MyD88 confers host protection possibly through novel direct activation of IRF-3 and IFN-&bgr;.

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.

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.

Polarity of helper T cell subsets represents disease nature and clinical course of experimental autoimmune myocarditis in rats

The mechanisms of progression, remission and relapse of myocarditis remain unclear. To clarify these mechanisms, we focused on T helper‐1 (Th1)/T helper‐2 (Th2) subsets balance of peripheral lymphocytes and serum cytokine levels during disease progression in rats with experimental autoimmune myocarditis (EAM). Lewis rats were immunized with cardiac myosin on day 0. Blood samples were collected on days 0, 7, 15, 18, 21, 28, 35, 42, 49 and 56 following immunization. We examined percentages of interferon (IFN)‐γ and/or interleukin (IL)‐4 producing cells in stimulated peripheral CD4‐positive lymphocytes using flow cytometry analysis. Serum IFN‐γ, IL‐2, IL‐6 and IL‐10 levels were measured by enzyme‐linked immunosorbent assay (ELISA). The percentage of Th1/Th2 subsets in EAM on days 0, 15, 28 and 56 were 2·5 ± 0·5/0·5 ± 0·1%, 19·4 ± 3·2/1·6 ± 0·3%, 2·0 ± 0·5/22·1 ± 5·7% and 3·0 ± 0·4/1·7 ± 0·3%, respectively. Serum levels of Th1 cytokines, IFN‐γ and IL‐2 significantly increased in the acute phase (from day 15–18) and immediately decreased in the early recovery phase. On the other hand, serum levels of Th2 cytokine, IL‐10 significantly increased in the early recovery phase (from day 24–30). These results suggest that induction of acute myocarditis might be associated with systemic Th1 dominance, while recovery is related to systemic Th2 polarity. Thus, analysis of Th1/Th2 balance in peripheral T cells may be useful in disease monitoring in patients with myocarditis and postmyocarditic dilated cardiomyopathy.

Carvedilol enhances atrial and brain natriuretic peptide mRNA expression and release in rat heart.

To clarify the role of the natriuretic peptide (NP) system in the myocardial protective effects of carvedilol, a beta-blocking agent, we investigated the effects of carvedilol on the NP system in the rat heart. After oral administration of carvedilol (low-dose group: 2 mg/kg/day, group C2; high-dose group: 20 mg/kg/day, group C20) for 1 week, plasma rat atrial NP (r-ANP), atrial mRNA levels of ANP, left ventricular mRNA of brain NP (BNP), NP receptor-A and NP receptor-C (NPR-C) (as a clearance receptor) were measured. Values were compared with those in vehicle-treatment rats (group V). The concentration of r-ANP was significantly higher in group C2 (135 +/- 9 pg/ml) and group C20 (161 +/- 11 pg/ml) than group V (75 +/- 6 pg/ml; both p < 0.01). ANP and BNP mRNA levels were significantly increased and NPR-C was significantly down regulated in group C2 (151 +/- 7, 120 +/- 8 and 78 +/- 7%, respectively, vs. group V) and group C20 (164 +/- 8. 133 +/- 7 and 72 +/- 8%, respectively, vs. group V) compared with group V (all p < 0.01). These results suggest that not only a high dose, but a low dose of carvedilol has the effect of increasing plasma ANP and BNP levels. This effect was closely related to the upregulation of ANP and BNP mRNA expression, and the down regulation of NPR-C mRNA expression in the heart. These mechanisms seem to account for a sizable portion of the protective effect of carvedilol for heart diseases.

J-waves in patients with an acute ST-elevation myocardial infarction who underwent successful percutaneous coronary intervention: prevalence, pathogenesis, and clinical implication

AIMS The prevalence, clinical significance, and pathogenesis of J-waves were studied in the patients with an ST-elevation myocardial infarction (MI) after percutaneous coronary intervention (PCI). METHODS AND RESULTS One hundred and fifty-two consecutive patients with an acute ST-elevation MI were included. The mean age was 68.6 ± 13.5 years, and 78.3% of the patients were male. Following successful PCI, 12-lead electrocardiograms (ECGs) were monitored, and J-waves were measured 1 week after the MI and analysed in relation to the location of the MI and arrhythmias. Clinical and ECG parameters were compared between the groups with and without J-waves. The rate dependency of the J-wave amplitude was analysed in the conducted atrial premature beats (APBs). J-waves were present in 60.5% (≥0.1 mV) or 48.9% (≥0.2 mV) of the 152 patients. The J-waves were more often located in the inferior leads and more frequently in an inferior MI. The presence of J-waves was associated with ventricular arrhythmias, including ventricular fibrillation. The J-wave amplitude increased in the conducted APB, mechanistically suggesting a phase 3 block. CONCLUSION Many patients in the early recovery phase after an acute MI had J-waves. This ECG phenomenon was associated with an increased incidence of ventricular arrhythmias. The tachycardia-dependent augmentation of the J-wave amplitude suggested a mechanistic role of conduction delay.

Identification of cryptic splice site, exon skipping, and novel point mutations in type I CD36 deficiency

CD36 is an 88 kDa glycoprotein IV expressed in platelets, monocytes, erythroblasts, capillary endothelial cells, and mammary epithelial cells.1 CD6 was reported to be a receptor for collagen, thrombospondin, P falciparum infected red blood cells,2 apoptotic neutrophils,3 oxidised low density lipoproteins,4 and as a transporter for long chain fatty acids.5 CD36 serves many functions in coagulation, host defence, lipid metabolism and scavenging. CD36 deficiency was first identified in a patient who showed refractoriness to HLA matched platelet transfusion.6 CD36 deficiency can be divided into two subgroups; in type I neither platelets nor monocytes/macrophages express CD36, and in type II monocytes/macrophages express CD36 but platelets do not.7 CD36 deficiency is more often observed in Japanese, Thais, and Africans than in Americans of European descent.8, 9 The incidence of type II CD36 deficiency is approximately 4.0% in Japanese, whereas type I CD36 deficiency is 0.56%.8 However, approximately 17% of Japanese patients with coronary heart disease have CD36 deficiency10 and 40% of Japanese patients with HCM with asymmetrical hypertrophy (ASH) have CD36 deficiency.11 It has been reported that CD36 deficiency might easily result in ischaemic heart disease10 and hypertrophic cardiomyopathy (HCM).11 Recently, CD36 has been reported to play an important role in atherogenicity. It was reported that CD36 deficiency could promote defective insulin action and disordered fatty acid metabolism in spontaneous hypertension12, 13 and antidiabetic thiazolidinedione drug and peroxisome proliferator activated receptor-γ (PPAR-γ) regulated CD36 expression.14, 15 To understand the role of CD36, it is important to clarify the condition of CD36 deficient subjects. Kashiwagi et al 16 and Yanai et al 17 reported three mutations responsible for CD36 deficiency, a substitution of T for C at nt 478 of CD36 cDNA, a dinucleotide deletion at nt 539, …

Abnormal fatty acid metabolism in patients with coronary vasospasm

Although various noninvasive methods have been used to detect vasospasm, none of them are sensitive enough for patients with sporadic attacks. Since abnormal fatty acid metabolism is observed in ischemic myocardium,123I-β-methyl-p-iodophenyl pentadecanoic acid (BMIPP), a radiolabeled fatty acid analog, has recently been proposed as a useful tracer for detecting myocardial damage. The aim of this study was to clarify the clinical implications of decreased myocardial BMIPP uptake in patients with vasospastic angina. We evaluated 53 patients with vasospastic angina (32 with clinically documented vasospasm [Group-A] and 21 with vasospasm induced by ergonovine provocation [Group-B]) and 27 control subjects, 20 in Group-A were re-evaluated 6 months after medical treatment. The territorial regions of vasospasm-induced coronary artery, the wall motion by left ventriculography, and BMIPP uptake were compared. Vasospasm was induced in multiple coronary arteries in 29 (55%) patients. Reduced wall motion and decreased BMIPP uptake were observed in 19 (36%) patients and 47 (89%) patients, respectively. The sensitivity and specificity of determination of vasospasm-induced coronary arteries with BMIPP scintigraphy were 71% (69/97 coronary arteries) and 88% (126/143), respectively. Vasospasm was re-induced by ergonovine provocation in 8 patients (Group-I) and not re-induced in 12 (Group-II) after treatment. In Group-I, improvement of decreased BMIPP uptake was lower than in Group-II (19 ± 11 vs. 59 ± 22%, mean ± SD, p < 0.001). The regions in which vasospasm was re-provoked exhibited decreased BMIPP uptake.Abnormal fatty acid metabolism was more often observed than wall motion abnormality in the vasospastic region in patients with vasospastic angina. BMIPP scintigraphy is a highly accurate and non-invasive technique for determining the presence and location of vasospasm.