Role of TGF Beta and PPAR Alpha Signaling Pathways in Radiation Response of Locally Exposed Heart: Integrated Global Transcriptomics and Proteomics Analysis Vikram Subramanian, † Ingar Seemann, ‡ Juliane Merl-Pham, § Stefanie M. Hauck, § Fiona A. Stewart, ‡ Michael J. Atkinson, †,∥ Soile Tapio, † and Omid Azimzadeh* ,† † Helmholtz Zentrum Mü nchen - German Research Center for Environmental Health GmbH, Institute of Radiation Biology, 85764 Neuherberg, Germany ‡ Division of Biological Stress Response, Netherlands Cancer Institute, 1006 BE Amsterdam, The Netherlands § Helmholtz Zentrum Muenchen - German Research Centre for Environmental Health GmbH, Research Unit Protein Science, 80939 Munich, Germany ∥ Chair of Radiation Biology, Technical University of Munich, 81675 Munich, Germany * S Supporting Information ABSTRACT: Epidemiological data from patients undergoing radiotherapy for thoracic tumors clearly show the damaging effect of ionizing radiation on cardiovascular system. The long-term impairment of heart function and structure after local high-dose irradiation is associated with systemic inflammatory response, contraction impairment, microvascular damage, and cardiac fibrosis. The goal of the present study was to investigate molecular mechanisms involved in this process. C57BL/6J mice received a single X-ray dose of 16 Gy given locally to the heart at the age of 8 weeks. Radiation-induced changes in the heart transcriptome and proteome were investigated 40 weeks after the exposure. The omics data were analyzed by bioinformatics tools and validated by immunoblotting. Integrated network analysis of transcriptomics and proteomics data elucidated the signaling pathways that were similarly affected at gene and protein level. Analysis showed induction of transforming growth factor (TGF) beta signaling but inactivation of peroxisome proliferator-activated receptor (PPAR) alpha signaling in irradiated heart. The putative mediator role of mitogen-activated protein kinase cascade linking PPAR alpha and TGF beta signaling was supported by data from immunoblotting and ELISA. This study indicates that both signaling pathways are involved in radiation-induced heart fibrosis, metabolic disordering, and impaired contractility, a pathophysiological condition that is often observed in patients that received high radiation doses in thorax. KEYWORDS: heart, ionizing radiation, proteomics, transcriptomics, label-free quantification, PPAR alpha, TGF beta, cardiovascular disease ■ INTRODUCTION Epidemiological studies show an increased risk of cardiovascular disease (CVD) associated with high local doses of ionizing radiation to the heart. This is observed in patients after thoracic radiotherapy treatment for breast cancer, Hodgkin’s disease, or childhood cancers. 1−4 A significant increase in the mortality from CVD has been reported for patients treated by radiotherapy for left-sided breast cancer compared with those with right-sided cancer. 1,5−7 The recent development of radiation therapy practice and equipment has decreased the heart dose from left- tangential radiotherapy considerably over the past 40 years; however, certain parts of the heart still receive >20 Gy in approximately half of left-sided breast cancer patients. 8 High- dose radiation leads to late adverse cardiac side effects including damage to pericardium, myocardium, valves, and coronary vessels as well as cardiomyopathy and myocardial fibrosis. 9,10 Because breast cancer is by far the most common cancer in women worldwide and most patients are treated by radiotherapy, an estimated increased risk for CVD induced by radiation (2.5%/ Gy) concerns a large number of people 11 and thus remains one of the important health concerns. 12 Therefore, a deep investigation of the molecular mechanisms of CVD following irradiation of the heart is urgently needed. We have previously shown using a mouse model (C57Bl/6J) that local cardiac high-dose radiation caused cardiac metabolic impairment that was coupled to mitochondrial dysfunction and reduction in the activity of peroxisome proliferator-activated receptor (PPAR) alpha complex. 13 It also induced a systemic inflammatory response and increased the level of free fatty acids in blood. 13,14 A similar study showed morphological disorders of the heart such as increased left ventricle (LV) wall thickening and increased interstitial collagen accumulation in LV myocardium Special Issue: The Immune System and the Proteome 2016 Received: August 31, 2016 Published: November 2, 2016 Article pubs.acs.org/jpr © XXXX American Chemical Society A DOI: 10.1021/acs.jproteome.6b00795 J. Proteome Res. XXXX, XXX, XXX−XXX