Free heme is a danger signal inducing expression of proinflammatory proteins in cultured cells derived from normal rat hearts.

Endogenous molecules from damaged tissue act as danger signals to trigger or amplify the immune/inflammatory response. In this study, we examined whether free heme induced pro-inflammatory proteins in cultured cells derived from normal hearts and investigated the cells targeted by heme, together with its mechanism of action in these cells. We cultured collagenase-isolated heart-derived cells from normal rats and examined whether free heme induced pro-inflammatory proteins, reactive oxygen species (ROS) production and NF-κB activation, by quantitative RT-PCR, ELISA and flow cytometry. Free heme increased mRNA of various pro-inflammatory proteins in cultured cardiac resident cells (CCRC) (at least 100-fold) and induced intracellular ROS formation. Approximately 85-90% of CCRC are fibroblast/smooth muscle cells and 10-15% are CD11bc-positive macrophages; therefore to examine individual target cells, macrophage-deleted (CD11bc-negative) CCRC, primary cultured cells (cardiac fibroblasts, arterial smooth muscle cells and cardiac microvascular endothelial cells) and macrophage cells lines (NR8383) were similarly treated. Free heme activated NF-κB and induced expression of some pro-inflammatory proteins, including IL-1 and TNF-α in NR8383. On the other hand, macrophage-deleted CCRC strongly increased expression of these proteins on treatment with IL-1 or TNF-α, but not free heme. Induction of expression of pro-inflammatory proteins by free heme was not inhibited by intracellular ROS reduction, but by protease and proteasome inhibitors capable of regulating NF-κB. These data suggest that free heme strongly induces various pro-inflammatory proteins in injured hearts through NF-κB activation in cardiac resident macrophages and through cross-talk between macrophages and fibroblast/smooth muscle cells mediated inter alia by IL-1, TNF-α.

IL-1 receptor accessory protein-Ig/IL-1 receptor type II-Ig heterodimer inhibits IL-1 response more strongly than other IL-1 blocking biopharmaceutical agents.

INTRODUCTION: Interleukin (IL)-1 is a key orchestrator of inflammation and IL-1 inhibitors are expected to be promising pharmaceutical agents for such pathologies. IL-1 is bound to the complex of two receptor components with much higher affinity than with either receptor component alone. MATERIALS AND METHODS: We examined the effect of a heterodimer of IL-1 receptor accessory protein (Acp)-immunoglobulin (Ig) and IL-1R type II (IL1R2)-Ig named AcP-Ig/IL1R2-Ig heterodimer, and compared its effects with other IL-1 inhibitors reported previously. RESULTS AND DISCUSSION: Our results demonstrated that the rat AcP-Ig/IL1R2-Ig heterodimer (IC50=1.95 pM) inhibited IL-1 response to a greater extent than IL1RA (IC50=1,935 pM), Acp-IL1R type I (IL1R1)-Ig homodimer (IC50=73.7 pM) and Acp-IL1R2-Ig homodimer (IC50=72.8 pM). Moreover, human AcP-Ig/IL1R2-Ig heterodimer (IC50=0.14 pM) inhibited it to a greater extent than Acp-IL1R1-Ig homodimer (IC50=4.48 pM) and strongly inhibited responses of both IL-1α and IL-1ß. CONCLUSIONS: The AcP-Ig/IL1R2-Ig heterodimer, which is similar to the original extracellular structure of the Acp/IL1R1 complex, may inhibit the IL-1 response more vigorously than other IL-1 blocking biopharmaceutical agents.

Lipocalin-2/neutrophil gelatinase-B associated lipocalin is strongly induced in hearts of rats with autoimmune myocarditis and in human myocarditis.

BACKGROUND: Lipocalin-2/neutrophil gelatinase-B associated lipocalin (Lcn2/NGAL) is involved in the transport of iron and seems to play an important role in inflammation. A recent study has reported that it is also expressed in the failing heart and may be a biomarker not only for renal failure but also for heart failure. Because Lcn2/NGAL is thought to be induced by interleukin-1, it might be strongly induced in the presence of myocarditis. METHODS AND RESULTS: This study investigated the expression of Lcn2/NGAL in rat experimental autoimmune myocarditis (EAM) and in human myocarditis. In EAM hearts, the expression of Lcn2/NGAL was markedly increased (>100-fold at an early stage), and in human myocarditis it was also highly expressed compared with non-inflammatory failing hearts. Lcn2/NGAL expressing cells in hearts with EAM and human myocarditis were identified as cardiomyocytes, vascular wall cells, fibroblasts and neutrophils. Lcn2/NGAL in EAM rats was also expressed in the liver. Plasma Lcn2/NGAL levels abruptly increased at an early stage of EAM, and high levels were initially sustained during the inflammatory stage, then decreased with recovery. In contrast, levels of B-type natriuretic peptide increased only slowly as the disease progressed. CONCLUSIONS: Cardiomyocytes, vascular wall cells and fibroblasts in myocarditis strongly express Lcn2/NGAL via proinflammatory cytokines.

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.

Gene expression profiles of cardiomyocytes in rat autoimmune myocarditis by DNA microarray and increase of regenerating gene family.

Cardiomyocytes with myocarditis compared with the normal state are thought to change the expressions of various genes greatly, some of which may be new biomarkers or new biologic medicinal products. However, until now, little comprehensive analysis has been made of gene-expression changes in cardiomyocytes with myocarditis. In this study, we performed a DNA microarray analysis by using cardiomyocytes from rat experimental autoimmune myocarditis (EAM). On day 0, rats were immunized with porcine cardiac myosin and cardiomyocytes were isolated and purified from EAM hearts and normal hearts by a method that is hardly thought to change gene expressions in cardiomyocytes. RNA from normal cardiomyocytes and cardiomyocytes of EAM on day 18 was analyzed for 7711 gene expressions by DNA microarray. Some gene expressions showed over 10-fold changes. In particular, the regenerated gene (Reg)2/pancreatitis-associated protein (PAP)1 messenger RNA (mRNA) level most markedly increased in the genes, which were clearly expressed in cardiomyocytes rather than in noncardiomyocytes, and it was approximately 2000-fold greater in cardiomyocytes under active myocarditis than normal by real-time reverse transcription polymerase chain reaction analysis. Moreover, we demonstrated that Reg2/PAP1 proteins determined by Western blot analysis and immunohistochemistry and other Reg/PAP family gene expressions were remarkably increased in EAM hearts; in addition, interleukin (IL)-6 expression was significantly related to Reg2/PAP1. It seemed that these data were useful as a reference database of gene-expression changes in cardiomyocytes with myocarditis. The Reg/PAP family, which was found to show dramatically increasing gene expressions by DNA microarray analysis, was suspected to play an important role in myocarditis.

Comparative analysis of systolic and isolated diastolic dysfunction: Sado heart failure study.

Determining the type of cardiac dysfunction is important for implementing therapeutic strategies and for prognostic insights. We characterized systolic dysfunction (SD) and isolated diastolic dysfunction (IDD) in adults referred for echocardiographic evaluation, and compared their clinical and other characteristics. In the present work, we studied 218 patients (137 males) with cardiac dysfunction (mean age, 66.3 +/- 8.3 years). SD was defined as a left ventricular ejection fraction (LVEF) of < 45%, whereas IDD was defined as a LVEF >or= 45% in addition to the standard Doppler-echocardiography diagnostic criteria for IDD. Approximately 68% of subjects had SD (70% males). The proportions of hypertension, diabetes, and dyslipidemia were 44%, 26%, and 22%, respectively, without significant association with the type of dysfunction. Myocardial infarction (MI) was found in 31% of patients, and was significantly (P < 0.001) more prevalent among SD compared with IDD cases. Cerebral stroke (18%) and malignancy (16%) were significantly associated with IDD (29% versus 13% for SD in the case of stroke, and 26% versus 11% for SD in the case of malignancy; P = 0.008 for each). In multivariately-adjusted logistic regression analysis, the following variables were found to be significantly (P < 0.05) and independently associated with IDD: female gender (odds ratio [OR] = 2.207 [95% CI = 1.302-4.608]), stroke (OR = 2.009 [1.119-3.980]), and malignancy (OR = 2.016 [1.230-4.010]). On the other hand, previous MI (OR = 2.075 [1.769-4.808]) was independently associated with SD. In conclusion, some factors/comorbidities were more likely to associate with IDD (female gender, stroke, and malignancy) or SD (previous MI) when IDD and SD were compared with each other.

Health-related quality of life of outpatients with systolic and isolated diastolic dysfunction: Sado Heart Failure Study.

BACKGROUND: The impact of isolated diastolic dysfunction (IDD) and systolic dysfunction (SD) on health-related quality of life (HRQOL) is unknown. METHODS AND RESULTS: To evaluate HRQOL in patients with IDD and SD under treatment, information on outpatients aged 60-84 years was extracted from the records of 4,500 consecutive individuals who underwent echocardiographic examination at Sado General Hospital. The medical records of these patients were reviewed and a questionnaire, including the Medical Outcome Study Short Form 36, was mailed to 71 IDD and 99 SD patients; answers were obtained from 66 and 91 patients, respectively. The HRQOL of patients with cardiac dysfunction was impaired even when echocardiographic parameters improved with treatment. Patients with IDD showed an impairment of HRQOL similar to those with SD. Compared with males, female patients had a larger and more significant reduction in the physical and mental components of the HRQOL score. These scores correlated positively with exercise capacity in patients with IDD or SD. CONCLUSIONS: Impaired HRQOL, in both its mental and physical components, is a serious problem for IDD and SD patients under treatment. Because exercise intolerance may underlie the reduced HRQOL, improving exercise capacity could be an important target for managing outpatients with heart failure.

Alteration of IL-17 related protein expressions in experimental autoimmune myocarditis and inhibition of IL-17 by IL-10-Ig fusion gene transfer.

BACKGROUND: T-helper (Th)1/Th2 cytokine balance plays an important role in the pathogenesis of myocarditis. Recently, some studies indicate that interleukin (IL)-17, known as a T cell (Th17)-derived proinflammatory cytokine, is the major mediator of tissue inflammation in inflammatory and autoimmune diseases. Experimental autoimmune myocarditis (EAM) is a T cell-mediated autoimmune disease; however, the pathogenic role of IL-17 in the development of rat EAM remains largely unknown. METHODS AND RESULTS: In the present study, alterations of IL-17-related protein expressions were investigated and then the effect of hydrodynamic-based delivery of plasmid DNA encoding the IL-10-Ig gene on rat EAM and the effect of IL-10-Ig on IL-17 was evaluated. The results showed that IL-17 was expressed more highly than IFN-gamma expressed by Th1 cells in alphabetaT cells and the peaks of IL-17 related protein expression in the heart were the early phase of EAM. Moreover, we observed that IL-10-Ig gene therapy was effective in controlling EAM and that IL-10-Ig significantly suppressed the expression of IL-17 as well as other proinflammatory cytokines, IL-1beta and TNF-alpha, in IL-1-stimulated splenocytes cultured from EAM rats. CONCLUSIONS: IL-17 is highly produced by alphabetaT cells in the early phase of EAM hearts and IL-17 inhibition might be a possible mechanism of the amelioration of EAM by IL-10-Ig treatment. These data suggest that IL-17 produced by Th17 plays an important role in the pathogenesis of rat EAM.

Postprandial variations in ST-segment in a patient with Brugada syndrome and partial gastrectomy.

A 74-year-old man with a history of partial gastrectomy presented with an electrocardiogram consistent with Brugada syndrome and marked meal related fluctuations in the ST segment. ST-segment elevation was prominently attenuated at 30 minutes and increased at 120 minutes after meals. Analysis of heart rate variability revealed a relationship between postprandial heightened parasympathetic activity and increase in Brugada-type ECG abnormality. A rapid postprandial increase in blood glucose may initially stimulate sympathetic nervous activity and secondarily increase parasympathetic tone. Food intake can be associated with fluctuations in ST-segment elevation in patients with the Brugada syndrome.