Satoru Hirono

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.

Expression of Inducible Nitric Oxide Synthase in Rat Experimental Autoimmune Myocarditis With Special Reference to Changes in Cardiac Hemodynamics

Excessive NO produced by an inducible NO synthase (iNOS) has been implicated in many types of immune-associated disorders of the cardiovascular system, but it remains to be determined whether NO plays a role in myocarditis. Thus, the significance of iNOS expression in the development of experimental autoimmune myocarditis (EAM), an animal model of human giant cell myocarditis, was investigated. Lewis rats were immunized with cardiac myosin and were killed 7, 14, 21, 28, and 49 days after immunization. The development of severe myocarditis was observed on days 14, 21, and 28 in association with significant deterioration of hemodynamics determined by cardiac catheterization, which peaked on day 21. In parallel with histological severity of myocarditis and deterioration of cardiac performance, iNOS activity in the heart measured by [14C]L-citrulline formation was markedly increased on days 14, 21, and 28. The expression of iNOS was confirmed by immunoblotting and was localized to the infiltrating inflammatory cells found in the vicinity of necrotic myocytes by immunohistochemical analysis. Aminoguanidine, a selective inhibitor of iNOS, significantly decreased the iNOS activity (1.04 +/- 0.37 compared with 29.1 +/- 8.62 pmol.min-1.mg protein-1 in untreated myosin-immunized rats, P < .01) and effectively attenuated histopathological changes of EAM on day 21. Hemodynamic parameters were also improved from 64 +/- 3 to 89 +/- 3 mm Hg for mean blood pressure, from 80 +/- 2 to 113 +/- 4 mm Hg for left ventricular systolic pressure, from 7.8 +/- 0.3 to 3.2 +/- 0.3 mm Hg for left ventricular end-diastolic pressure, from 2867 +/- 137 to 4180 +/- 102 mm Hg/s for +dP/dt, and from 2717 +/- 132 to 4180 +/- 184 mm Hg/s for -dP/dt (P < .01). The values after aminoguanidine treatment were not significantly different from the control values. These results suggest an important role for NO in mediating pathophysiological changes in myocarditis of autoimmune origin.

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.

Thromboembolism in Takotsubo cardiomyopathy

BACKGROUND Most patients with Takotsubo cardiomyopathy show a favorable outcome. Although several complications have been reported, the frequency of thromboembolism has not been clarified. METHODS Clinical characteristics and complications of 21 consecutive patients (18 female, aged 72 years) with Takotsubo cardiomyopathy during the past 9 years were investigated. RESULTS The most major complication was heart failure (52%). Thromboembolism was found in 3 patients (14%) and this was the second most frequent cardiovascular complication. One of the 3 patients showed left ventricular thrombus and the other 2 experienced cardioembolic stroke. All 3 patients visited the emergency department more than 48 h after initial chest pain occurred. CONCLUSIONS This study indicates that thromboembolism is a common complication in the acute phase of Takotsubo cardiomyopathy, and anticoagulation therapy should be performed in all patients until wall motion abnormalities improve. Takotsubo cardiomyopathy should be considered one of the important causes of cardioembolic stroke.

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.

Amiodarone Improves Cardiac Sympathetic Nerve Function to Hold Norepinephrine in the Heart, Prevents Left Ventricular Remodeling, and Improves Cardiac Function in Rat Dilated Cardiomyopathy

Background—It is unclear how amiodarone therapy exerts its effects on left ventricular remodeling and cardiac sympathetic nerve function in chronic heart failure. We investigated long-term effects of amiodarone on rat dilated cardiomyopathy after healing of cardiac myosin–induced autoimmune myocarditis. Methods and Results—Rats were treated with oral amiodarone or vehicle for 6 weeks. We determined cardiac function, left ventricular remodeling, and cardiac sympathetic nerve function with iodine-125–labeled metaiodobenzylguanidine ([I125]MIBG). Amiodarone treatment improved left ventricular pressure, central venous pressure, and rate of isovolumetric contraction and decreased ventricular weight (P<0.005). Expression of cytokine mRNA was unchanged; expression of atrial natriuretic peptide, collagen III, and transforming growth factor-&bgr;1 mRNA was decreased in amiodarone-treated rats (P<0.05). Phenotype of myosin heavy chain was moved toward that of normal rats by amiodarone. Initial myocardial uptake of MIBG decreased by 67% (P<0.001) and washout rate accelerated by 221% in rats with chronic heart failure compared with normal rats. Whereas amiodarone decreased the initial uptake by 71% in normal rats, amiodarone decelerated the early washout and the late washout and improved the late myocardial distribution of MIBG in rats with chronic heart failure (257% compared with vehicle-treated rats with chronic heart failure; P<0.01). In proportion to MIBG distributions, cardiac tissue catecholamines were increased by amiodarone treatment. Conclusions—Long-term amiodarone treatment prevented left ventricular remodeling and improved cardiac function in rat dilated cardiomyopathy. Long-term amiodarone treatment also restored cardiac sympathetic tone to hold norepinephrine in the heart.

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.