A Promising Culture Model for Analyzing the Interaction between Adipose Tissue and Cardiomyocytes

RENAL-CARDIAC-VASCULAR A Promising Culture Model for Analyzing the Interaction between Adipose Tissue and Cardiomyocytes Mayumi Anan, Kazuyoshi Uchihashi, Shigehisa Aoki, Aki Matsunobu, Akifumi Ootani, Koichi Node, and Shuji Toda Department of Pathology and Microbiology (M.A., K.U., S.A., A.M., S.T.), and Divisions of Cardiovascular and Renal Medicine (M.A., K.N.) and Gastroenterology (A.O.), Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501, Japan The heart has epicardial adipose tissue that produces adipokines and mesenchymal stem cells. Systemic adipose tissue is involved in the pathophysiology of obesity-related heart diseases. However, the method for analyzing the direct interaction between adipose tissue and cardiomyocytes has not been established. Here we show the novel model, using collagen gel coculture of adipose tissue fragments (ATFs) and HL-1 cardiomyocytes, and electron microscopy, immunohistochemistry, real-time RT-PCR, and ELISA. HL-1 cells formed a stratified layer on ATF-nonembedded gel, whereas they formed almost a monolayer on ATF-embedded gel. ATFs promoted the apoptosis, lipid accumulation, and fatty acid transport protein (FATP) expression of FATP4 and CD36 in HL-1 cells, whereas ATFs inhibited the growth and mRNA expression of myosin, troponin T, and atrial natriuretic peptide. Treatment of leptin (100 ng/ml) and adiponectin (10 ␮g/ml) neither replicated nor abolished the ATF-induced morphology of HL-1 cells, whereas that of FATP4 and CD36 antibodies (25 ␮g/ml) never abolished it. HL-1 cells prohibited the development of CD44⫹/CD105⫹ mesenchymal stem cell-like cells and lipid-laden preadipocytes from ATFs. HL-1 cells increased the production of adiponectin in ATFs, whereas they decreased that of leptin. The data indicate that our model actively creates adipose tissue-HL-1 cardiomyocyte interaction, suggesting first that ATFs may be related to the lipotoxiciy of HL-1 cells via unknown factors plus FATP4 and CD36 and second that HL-1 cells may help to retain the static state of ATFs, affecting adipokine secretion. Our model will serve to study adipose tissue-cardiomyocyte interaction and mechanisms of obesityrelated lipotoxicity and heart diseases. (Endocrinology 152: 1599 –1605, 2011) T he heart has a small amount of epicardial adipose tissue that stores excess energy in the form of lipid droplets. Systemic adipose tissue is suggested to be involved in the pathophysiology of obesity-related heart diseases (1, 2). In general, adipose tissue is an endocrine organ that affects the biological behavior of various cell types through its production of adipokines (3, 4). Furthermore, adipose tissue consists of mature adipocytes and preadipocytes, but it is also shown to contain mesenchymal stem cells (MSCs) that produce various cell types, e.g. adipocytes, osteoblasts, myocytes, and hepatocytes (5). Thus, adipose tissue seems critical for the homeostasis, function, and diseases of the heart. However, any suitable culture methods for analyzing the direct interaction between adipose tissue and cardiomyocytes have not been established. One of the reasons for this is the difficulty in culturing adipose tissue, which has large lipid droplets and thus do not attach to the surface of culture dish due to its buoyancy in culture medium. Recently we established the culture system of adipose tissue fragments (ATFs) that were embedded in three-dimensional collagen gel, which is able to easily entrap buoyant adipose tissue (6). This system retains the unilocular ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/en.2010-1106 Received September 22, 2010. Accepted January 7, 2011. First Published Online February 8, 2011 Abbreviations: ANP, Atrial natriuretic peptide; ATF, adipose tissue fragment; BrdU, bromodeoxyuridine; FATP, fatty acid transport protein; MSC, mesenchymal stem cell; ssDNA, single-stranded DNA. Endocrinology, April 2011, 152(4):1599 –1605 endo.endojournals.org 1599 1600 Anan et al. Adipose Tissue-Cardiomyocyte Interaction structure, proliferative ability, and active functions of mature adipocytes for a long term, generating both preadipocytes and MSC-like cells. By application of this method, here we show for the first time a new model for analyzing adipose tissue-cardiomyocyte interaction. Materials and Methods Cell line and preparation of ATFs All procedures involving animal and human materials were performed in accordance with the regulations laid down by the ethical guidelines of Saga University. Mouse HL-1 cardiomyocytes that have a differentiated adult cardiac phenotype (7) were kindly provided by Dr. W. C. Claycomb (Louisiana State University Health Science Center, New Orleans, LA). Adipose tissue was obtained from visceral and sc adipose tissues of mice and Wistar rats and from epicardial adipose tissue of three human autopsy cases. ATFs were prepared from minced adipose tissues, as described previously (6). As reference, mouse NIH 3T3 fibroblasts (ATCC CCL-92; American Tissue Culture Collection, Manassas, VA) were used. In all interaction cultures, we used the following complete medium: Claycomb medium (JRH Biosciences, Lenexa, KS) supplemented with 10% fetal calf serum (JRH Biosciences), 100 U/ml penicillin/streptomycin, 0.1 mM norepinephrine, and 2 mM L-glutamine. Culture model The coculture model of ATFs and HL-1 cardiomyocytes was organized as follows. First, 0.15 ml ATFs were mixed with 1.0 ml type I collagen gel solution (Nitta Gelatin, Co., Ltd., Osaka, Japan), and then the mixture was poured into a 30-mm-diameter dish (inner dish) of which the bottom was made with nitrocellulose membrane (Millicell-CM; Millipore Corp., Bedford, MA). After the gel was solidified at 37 C for 30 min, 10 ⫻ 105 HL-1 cells were sowed on the gel layer and they grew to be confluent at 2 d in culture. The inner dish was placed in a larger outer dish, and then the complete medium was added to both dishes. As a control, ATFs or HL-1 cells alone were cultured. To estimate the specificity of ATF-induced effects on HL-1 cell behavior, HL-1 cells were also cultured on 10 ⫻ 105 3T3 fibroblast-embedded collagen gel. Figure 1 illustrates the culture model. The cellular behaviors were analyzed at 1 wk in culture, as described below. To estimate the specificity of HL-1 cell-induced effects on ATF behavior, 3T3 cells were cultured on ATF-embedded collagen gel. Histology and morphometric analysis Morphology was analyzed by light microscopy and electron microscopy, as described (6). Oil red O staining for lipid detection was carried out, as described (6). Lipid droplet⫹ and CD44⫹/CD105⫹ spindle cell types were judged as preadipocytes and MSC-like cells, respectively, as described elsewhere (6). The numbers of preadipocytes and MSC-like cells per 10 ATFs were calculated, as described (6). Immunohistochemistry and immunofluorescence S-100 protein antibody (D (...truncated)