International Journal of Obesity (2015) 39, 755–761 © 2015 Macmillan Publishers Limited All rights reserved 0307-0565/15 www.nature.com/ijo ORIGINAL ARTICLE Interaction of epoxyeicosatrienoic acids and adipocyte fatty acid-binding protein in the modulation of cardiomyocyte contractility V Lamounier-Zepter1, C Look1, W-H Schunck2, I Schlottmann1, C Woischwill1, SR Bornstein1,AXu3 and I Morano2 BACKGROUND: Adipocyte fatty acid-binding protein (FABP4) is a member of a highly conserved family of cytosolic proteins that bind with high affinity to hydrophobic ligands, such as saturated and unsaturated long-chain fatty acids and eicosanoids. Recent evidence has supported a novel role for FABP4 in linking obesity with metabolic and cardiovascular disorders. In this context, we identified FABP4 as a main bioactive factor released from human adipose tissue that directly suppresses heart contraction in vitro. As FABP4 is known to be a transport protein, it cannot be excluded that lipid ligands are involved in the cardiodepressant effect as well, acting in an additional and/or synergistic way. OBJECTIVE: We investigated a possible involvement of lipid ligands in the negative inotropic effect of adipocyte factors in vitro. RESULTS: We verified that blocking the CYP epoxygenase pathway in adipocytes attenuates the inhibitory effect of adipocyte- conditioned medium (AM) on isolated adult rat cardiomyocytes, thus suggesting the participation of epoxyeicosatrienoic acids (EETs) in the cardiodepressant activity. Analysis of AM for EETs revealed the presence of 5,6-, 8,9-, 11,12- and 14,15-EET, whereas 5,6- EET represented about 45% of the total EET concentration in AM. Incubation of isolated cardiomyocytes with EETs in similar concentrations as found in AM showed that 5,6-EET directly suppresses cardiomyocyte contractility. Furthermore, after addition of 5,6-EET to FABP4, the negative inotropic effect of FABP4 was strongly potentiated in a concentration-dependent manner. CONCLUSIONS: These data suggest that adipocytes release 5,6-EET and FABP4 into the extracellular medium and that the interaction of these factors modulates cardiac function. Therefore elevated levels of FABP4 and 5,6-EET in obese patients may contribute to the development of heart dysfunction in these subjects. International Journal of Obesity (2015) 39, 755–761; doi:10.1038/ijo.2014.193 INTRODUCTION previous studies, we verified a direct effect of FABP4, independent Adipocytes are known to produce a wide variety of bioactive of its function as a transport protein, in the modulation of heart molecules, which are released into the bloodstream.1,2 The function function. We demonstrated that FABP4 was released from mature of these molecules is not completely understood; however, some of adipocytes into the extracellular medium and elicited a direct and 2+ them may partly contribute to the development of obesity-related acute Ca -dependent depression of cardiomyocyte contraction 5 metabolic and cardiovascular diseases. In this context, we have in vitro. Furthermore, we recently showed that FABP4 is recently shown that human mature adipocytes release substances associated with cardiac remodeling in overweight and obese 13 that strongly and acutely suppress the contraction of isolated women and that FABP4 serum levels are linked with left 14 perfused hearts3 andisolatedcardiomyocytesbyattenuating ventricular diastolic dysfunction in morbidly obese subjects. intracellular Ca2+ levels,4 suggesting a new direct role of adipose Thus the elevated levels of circulating FABP4 as observed in obese tissue in the pathogenesis of cardiac dysfunction. Based on this patients may be partially responsible for the development of heart initial work, we have further identified adipocyte fatty acid-binding dysfunction in these subjects. As FABP4 is known to be a transport protein (FABP4) as one of the cardiodepressant factors produced protein, it cannot be excluded that lipid ligands are involved in the and released by human adipocytes.5 cardiodepressant effect as well, acting in an additional and/or Fatty acid-binding proteins (FABPs) are members of a highly synergistic way. Therefore, in the present in vitro study we conserved family of cytosolic proteins, which are found in investigated a possible involvement of lipid ligands in the different cell types and show a high affinity for long-chain fatty negative inotropic effect of adipocyte factors in interaction acids and other hydrophobic ligands. FABPs have important roles with FABP4. in fatty acid solubilization, transfer and storage of fatty acids in eukaryotic cells.6 FABP4 is predominantly present in adipose tissue.7 It is also abundantly present in human serum, probably METHODS due to its release from adipocytes.5,8 Recent evidence has Substances supported a novel role for FABP4 in linking obesity with metabolic FABP4, the epoxyeicosatrienoic acids (EETs) 5,6-, 8,9-, 11,12- and 14,15 and – syndrome, diabetes mellitus and cardiovascular disease.9 12 In the the cytochrome P450 (CYP) epoxygenase inhibitor 2-(2-propynyloxy)- 1Medical Clinic III, University of Technology Dresden, Dresden, Germany; 2Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany and 3State Key Laboratory of Pharmaceutical Biotechnology and Department of Medicine, University of Hong Kong, Hong Kong, China. Correspondence: Dr V Lamounier-Zepter, Medical Clinic III, University of Technology Dresden, Fetscherstr. 74, Dresden 01307, Germany. E-mail: [email protected] Received 29 May 2014; revised 15 October 2014; accepted 27 October 2014; accepted article preview online 5 November 2014; advance online publication, 25 November 2014 Eicosanoids and FABP4 in heart function V Lamounier-Zepter et al 756 benzenehexanoic acid (PPOH) were purchased from Cayman Chemicals 27 min. After washing, increasing Ca2+-concentrations were applied to cells (Ann Arbor, MI, USA). 8,9-, 11,12- and 14,15-EETs were dissolved in ethanol, to obtain Ca2+-tolerant cardiomyocytes. Isolated cells were resuspended in and 5,6-EET was dissolved in acetonitrile. medium 199 complemented with 0.2% bovine serum albumin, 5% fetal The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), the bovine serum, 5 mmol l − 1 creatine and taurine, 2 mmol l − 1 carnitine, − 1 cyclooxygenase (COX) inhibitor indomethacin and the CYP epoxygenase 10 μmol l cytosine-D-arabinofuranoside and antibiotics. Cardiomyocytes inhibitor 17-octadecynoic acid (17-ODYA) (all from Sigma, Munich, were cultured in laminin-coated four-well chamber slides (Nunc, Wiesbaden- Germany) and PPOH were dissolved in dimethyl sulfoxide (DMSO). All Schierstein, Germany) for at least 2 h. substances were stored at − 20 °C, FABP4 at − 80 °C. Acetonitrile, ethanol (both from Roth, Karlsruhe, Germany) and DMSO were used as controls at appropriate concentrations. Incubation of adult rat cardiomyocytes Adult cardiomyocytes were electrically stimulated at 1 Hz until both shortening and fura-2 signals reached a steady level. Electric pacing was Human adipose tissue then switched off, and AM (1:6 dilution), EETs (from 0.01–30 nmol l − 1), Human white adipose tissue was obtained from healthy overweight and FABP4 (60 nmol l − 1) or the corresponding vehicle buffers were added obese women (aged 39–70 years, body mass index ranging from 24.1 to directly to the cardiomyocytes and incubated for 5 min. Subsequently, − 33.5 kg m 2, n = 7) undergoing elective surgical mammary and abdominal electrical pacing was restarted, and mechanical and fluorescence signals reduction. All women were otherwise free of metabolic and endocrine were collected again. diseases. Informed consent was obtained from all donors before the surgical procedure. The study was approved by the ethical committee of 2+ the Technical University of Dresden. Measurement of cell shortening and Ca transients Attached cardiomyocytes were loaded with fura-2-AM dissolved in Hank’s balanced salts solution buffered with 10 mmol l − 1 4-(2-hydroxyethyl)-1- Isolation of human adipocytes and preparation of adipocyte- piperazineethanesulfonic acid (HEPES) at pH 7.4 in the dark at room conditioned medium (AM) temperature for 15 min. Subsequently, cardiomyocytes were washed for − Adipocytes were isolated from subcutaneous human adipose tissue as 30 min with Hank’s balanced salts solution buffered with 10 mmol l 1 described before.4 Briefly, after surgical removal, adipose tissue samples of HEPES. Only cardiomyocytes of optically intact rod-shaped morphology 20–60 g wet weight were immediately transported to the laboratory in with clear cross striation were analyzed. We used an IonOptix Contractility Dulbecco’s modified Eagle’s medium/Nutrient Mix F12 (Life Technologies, and Fluorescence System (IonOptix, Milton, MA, USA) to measure cell Karlsruhe, Germany) with 100 U ml − 1 penicillin and 100 μgml− 1 streptomycin. shortening and Ca2+ transients. Cell shortening was measured using the − After removal of fibrous material and blood vessels, adipose tissue video-edge technique at a sampling rate of 240 s 1. Calcium transients was digested in Krebs–Ringer bicarbonate buffer supplemented with were monitored as a ratio of fluorescence emission at 510 nm obtained by 120 U ml − 1 collagenase type IV from Clostridium histolyticum (Serva, alternate excitation at 340 and 380 nm (340/380 ratio). Heidelberg, Germany). Isolated mature adipocytes were kept at 37 °C in fi a humidi ed atmosphere of 5% CO2 and cultured for 24 h in serum-free Statistics Dulbecco’s modified Eagle’s medium/Nutrient Mix F12 culture medium.