Sumoylation of Hypoxia-Inducible Factor-1a Ameliorates Failure of Brain Stem Cardiovascular Regulation in Experimental Brain Death Julie Y. H. Chan1,2, Ching-Yi Tsai2, Carol H. Y. Wu2, Faith C. H. Li2, Kuang-Yu Dai2, Enya Y. H. Sun 2 , Samuel H. H. Chan2*, Alice Y. W. Chang 2 * 1 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, Republic of China, 2 Center for Translational Research in Biomedical Sciences, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan, Republic of China Abstract Background: One aspect of brain death is cardiovascular deregulation because asystole invariably occurs shortly after its diagnosis. A suitable neural substrate for mechanistic delineation of this aspect of brain death resides in the rostral ventrolateral medulla (RVLM). RVLM is the origin of a life-and-death signal that our laboratory detected from blood pressure of comatose patients that disappears before brain death ensues. At the same time, transcriptional upregulation of heme oxygenase-1 in RVLM by hypoxia-inducible factor-1a (HIF-1a) plays a pro-life role in experimental brain death, and HIF-1a is subject to sumoylation activated by transient cerebral ischemia. It follows that sumoylation of HIF-1a in RVLM in response to hypoxia may play a modulatory role on brain stem cardiovascular regulation during experimental brain death. Methodology/Principal Findings: A clinically relevant animal model that employed mevinphos as the experimental insult in Sprague-Dawley rat was used. Biochemical changes in RVLM during distinct phenotypes in systemic arterial pressure spectrum that reflect maintained or defunct brain stem cardiovascular regulation were studied. Western blot analysis, EMSA, ELISA, confocal microscopy and immunoprecipitation demonstrated that drastic tissue hypoxia, elevated levels of proteins conjugated by small ubiquitin-related modifier-1 (SUMO-1), Ubc9 (the only known conjugating enzyme for the sumoylation pathway) or HIF-1a, augmented sumoylation of HIF-1a, nucleus-bound translocation and enhanced transcriptional activity of HIF-1a in RVLM neurons took place preferentially during the pro-life phase of experimental brain death. Furthermore, loss-of-function manipulations by immunoneutralization of SUMO-1, Ubc9 or HIF-1a in RVLM blunted the upregulated nitric oxide synthase I/protein kinase G signaling cascade, which sustains the brain stem cardiovascular regulatory machinery during the pro-life phase. Conclusions/Significance: We conclude that sumoylation of HIF-1a in RVLM ameliorates brain stem cardiovascular regulatory failure during experimental brain death via upregulation of nitric oxide synthase I/protein kinase G signaling. This information should offer new therapeutic initiatives against this fatal eventuality. Citation: Chan JYH, Tsai C-Y, Wu CHY, Li FCH, Dai K-Y, et al. (2011) Sumoylation of Hypoxia-Inducible Factor-1a Ameliorates Failure of Brain Stem Cardiovascular Regulation in Experimental Brain Death. PLoS ONE 6(3): e17375. doi:10.1371/journal.pone.0017375 Editor: Holger Eltzschig, University of Colorado Denver, United States of America Received September 3, 2010; Accepted February 1, 2011; Published March 3, 2011 Copyright: 2011 Chan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Science Council, Taiwan, Republic of China (NSC98-2321-B075-B-001 to J.Y.H.C.; NSC97-2320-B-182A-007-MY3 to S.H.H.C.; and NSC97-2321-B-182A-006, NSC98-2321-B-182A-003 and NSC99-2321-B-182A-006 (A.Y.W.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: shhchan@adm.cgmh.org.tw (SHHC); cgmf.kmc@gmail.com (AYWC) Introduction As much as brain death is currently the legal definition of death in the United States [1], European Union [2] or Taiwan [3] and is of paramount clinical importance, its cellular and molecular underpinning remains relatively unsettled. Despite the complexity of brain death, two clinical observations suggest that mechanistic delineation of permanent impairment of the brain stem cardio- vascular regulatory machinery associated with this fatal phenom- enon may shed light on its underlying mechanisms. First, asystole invariably takes place within hours or days after the diagnosis of brain death [4]. Second, a unique clinical phenotype exists in the low-frequency (LF) component (0.04–0.15 Hz in human) of the systemic arterial pressure (SAP) spectrum, which reflects the status of brain stem cardiovascular regulation. The power density of the LF component undergoes a dramatic reduction or loss before brain death ensues in comatose patients [5–7]. That this life-and- death signal originates from the rostral ventrolateral medulla (RVLM) [8], which is known classically for its role in tonic maintenance of vasomotor tone and SAP [9], allows this brain stem site to be a suitable neural substrate for mechanistic assessment of the brain stem cardiovascular regulatory functions associated with brain death [10]. In an animal model that employs mevinphos (3-[dimethox- yphosphinyl-oxyl]-2-butenoic acid methyl ester; Mev) as the experimental insult [10–12], our laboratory found previously that on systemic administration, this organophosphate pesticide acts directly on RVLM to elicit an augmentation followed by a PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17375