Elsevier

Translational Research

Volume 152, Issue 3, September 2008, Pages 119-127
Translational Research

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

https://doi.org/10.1016/j.trsl.2008.07.006Get rights and content

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.

Section snippets

Animals

Lewis rats were obtained from Charles River, Japan (Atsugi, Kanagawa, Japan) and were maintained in our animal facilities until they reached 8 weeks of age. Throughout the studies, all animals were treated in accordance with the guidelines for animal experiments of our institute and the guide for the care and use of laboratory animals published by the U.S. National Institutes of Health.

Induction of experimental autoimmune myocarditis (EAM)

Whole cardiac myosin was prepared from the ventricular muscle of porcine hearts as previously described. It

DNA microarray

Scatter plots of 7711 gene expressions in cardiomyocytes show that the expressions of various genes in cardiomyocytes of EAM markedly increased or decreased compared with normal cardiomyocytes (Fig 1). All data for each spot, which include the Cy3 and Cy5 intensities of which were over 100, are shown in an online data supplement (see online data supplement). Areas surrounded by the triangle in Fig 1 indicate that both Cy3 and Cy5 intensities were over 100, and these intensities showed over

Discussion

In this study, we examined the gene expressions in cardiomyocytes with myocarditis comprehensively. Various comprehensive studies for gene expressions with myocarditis by DNA microarray were reported7, 8, 9; however, other cells except cardiomyocytes (inflammatory cells, fibroblast and smooth muscle cells, and so on) increase in hearts with myocarditis; therefore, gene expressions in cardiomyocytes can be analyzed more accurately using purified cardiomyocytes than unpurified cells. Our

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    PAP was among a group of genes whose expression was restored to normal by eplerenone treatment; this response to eplerenone was associated with improvements in left ventricular remodeling. Watanabe et al5 found that PAP was the most significantly upregulated gene in the hearts of rats with experimental autoimmune myocarditis. Finally, induction of PAP expression can be prevented by chemical inhibitors of stress kinase family proteins in vitro.6

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

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    However, the identity of “danger signals” in myocardium remains largely unknown; nor is it known which resident cells react to these signals and produce pro-inflammatory proteins such as cytokines and chemokines. On the other hand, our previous gene expression analysis in hearts of rats with myocarditis by DNA microarray and real time RT-PCR has demonstrated that gene expression of several iron metabolism-related proteins, such as lipocalin-2/NGAL/alpha-2μ globulin-related protein (Lcn2/NGAL), hepcidin and heme oxygenase-1 (HO-1), is markedly increased in such cardiomyocytes (Ding et al., 2010; Isoda et al., 2010; Watanabe et al., 2008). The role of these proteins in injured hearts still remains largely unknown; however, this finding led us to hypothesize that resident cells in injured hearts are rapidly and strongly influenced by a molecule containing iron, which is critically involved in inflammation.

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Supported in part by a Ministry of Health, Labor, and Welfare in Japan Grant “Research on Regulatory Science of Pharmaceutical and Medical Devices.”

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