Limin Ding

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.

Role of mineralocorticoid receptor on atrial structural remodeling and inducibility of atrial fibrillation in hypertensive rats

Hypertension is well known to increase atrial fibrillation (AF) and the development of AF is associated with atrial chamber remodeling. Although mineralocorticoid receptor (MR) inhibition provides cardiovascular protection, the role of MR on atrial structural remodeling and inducibility of AF in hypertension remains unclear. Here, we investigated roles of the MR on atrial structural remodeling and inducibility of AF in hypertensive rats by using MR antagonist eplerenone (EPL). Dahl salt-sensitive (DS) rats were fed a normal-salt or a high-salt (HS) diet from 7 weeks, and a non-antihypertensive dose of EPL or vehicle was administrated from 13 weeks, at which time myocytes hypertrophy, interstitial fibrosis in the atrium and AF inducibility had increased, until 20 weeks. There was no significant difference in systolic blood pressure between DS+HS (186±4 mm Hg) and DS+HS+EPL (184±5 mm Hg) at 20 weeks. Burst atrial pacing demonstrated decreased AF inducibility in DS+HS+EPL (0 of 10) compared with DS+HS (7 of 10). Fibrosis and myocytes hypertrophy in the atrium were decreased in DS+HS+EPL with the reduction of atrial inflammatory cytokines. These beneficial effects of EPL were associated with less atrial oxidative stress, as assessed by 4-hydroxy-2-nonenal staining, and reduced activation of the Rho GTPase Rac1 in the atrium. Thus, MR has important roles in atrial structural remodeling and AF inducibility in Dahl rats. The effects of MR are associated, at least in part, with activation of Rac1-oxidative stress/inflammatory axis.

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.

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.

Improvement of anemia with decreasing hepcidin levels following valve replacement for severe tricuspid regurgitation

To the Editor: Severe right heart failure is occasionally accompanied by anemia (1, 2), but the underlying mechanisms for this association remain unknown. Hepcidin, a circulating hormone that is synthesized mainly by the liver, has emerged as a key regulator of systemic iron homeostasis (3). Hepcidin suppresses the expression of the iron transporter, ferroportin-1, thereby inhibiting the absorption of iron from the duodenum and the release of iron from the reticuloendothelial system. Therefore, the excessive production of hepcidin decreases serum iron levels, consequently causing and consequently causes anemia (3). Here, we report on a sixty-two-year-old woman of severe tricuspid regurgitation with refractory anemia in a patient with a very high serum hepcidin level who recovered from anemia after valve replacement, which was accompanied by a decrease in the hepcidin level. She had undergone mitral valve replacement for mitral stenosis 24 yrs before this episode. An echocardiogram revealed severe tricuspid regurgitation and dilatation of the right atrium. Right heart catheterization was performed after admission. Her right atrial pressure (RAP) was high (mean RAP, 15 mmHg; prominent V wave, 21 mmHg). Abdominal CT revealed hepatic vein dilatation and massive ascites. She received drug therapy for right heart failure, but this failed to reduce the massive ascites. She also had normocytic, normochromic anemia, but fecal occult blood tests were negative, and no bleeding source was found. She received oral iron preparation and red blood cell transfusions; however, her anemia did not resolve. As the massive ascites and progressive abdominal distension were believed to be life threatening, we decided to perform valve replacement surgery. The patient’s preoperative hemoglobin (Hb) level was 7.9 g ⁄dL, red blood cell (RBC) count was 271 million ⁄L, hematocrit (Ht) was 25.0%, serum iron level was low (26 lg ⁄dL), and serum ferritin level was high (724 ng ⁄mL; Table 1). Moreover, using a hepcidin EIA kit (Bachem Americans Inc., San Carlos, CA, USA), we found that her serum hepcidin levels (828 ng ⁄mL) were very high compared with the normal range (normal range, 1.8– 48.7 ng ⁄mL). Her preoperative serum C-reactive protein (CRP) level (15.91 mg ⁄dL) and interleukin-6 (IL-6) level (695 pg ⁄mL) were very high, but infectious disease was not found in any region of her body. Tricuspid valve replacement for severe tricuspid valve regurgitation and mitral valve replacement for the old ball valve were performed on July 1, 2010. Three months after the operation, the massive ascites completely disappeared. Postoperative right heart catheterization showed a decrease in RAP (mean RAP, 7 mmHg; V wave, 9 mmHg). Serum