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Proteomic Profiling Reveals Adaptive Responses to Surgical Myocardial

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Proteomic Profiling Reveals Adaptive Responses to Surgical Myocardial Proteomic Profiling Reveals Adaptive Responses to Surgical Myocardial Ischemia–Reperfusion in Hibernating Arctic Ground Squirrels Compared to Rats PERIOPERATIVE MEDICINE Quintin J. Quinones, M.D., Ph.D., Zhiquan Zhang, Ph.D., Qing Ma, M.D., Michael P. Smith, M.S., Erik Soderblom, Ph.D., M. Arthur Moseley, Ph.D., James Bain, Ph.D., Christopher B. Newgard, Ph.D., Michael J. Muehlbauer, Ph.D., Matthew Hirschey, Ph.D., Kelly L. Drew, Ph.D., Brian M. Barnes, Ph.D., Mihai V. Podgoreanu, M.D. ABSTRACT Background: Hibernation is an adaptation to extreme environments known to provide organ protection against ischemia– Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/124/6/1296/487075/20160600_0-00023.pdf by guest on 02 October 2021 reperfusion (I/R) injury. An unbiased systems approach was utilized to investigate hibernation-induced changes that are char- acteristic of the hibernator cardioprotective phenotype, by comparing the myocardial proteome of winter hibernating arctic ground squirrels (AGS), summer active AGS, and rats subjected to I/R, and further correlating with targeted metabolic changes. Methods: In a well-defined rodent model of I/R by deep hypothermic circulatory arrest followed by 3 or 24 h of reperfusion or sham, myocardial protein abundance in AGS (hibernating summer active) and rats (n = 4 to 5/group) was quantified by label-free proteomics (n = 4 to 5/group) and correlated with metabolic changes. Results: Compared to rats, hibernating AGS displayed markedly reduced plasma levels of troponin I, myocardial apoptosis, and left ventricular contractile dysfunction. Of the 1,320 rat and 1,478 AGS proteins identified, 545 were differentially expressed between hibernating AGS and rat hearts (47% up-regulated and 53% down-regulated). Gene ontology analysis revealed down-regulation in hibernating AGS hearts of most proteins involved in mitochondrial energy transduction, includ- ing electron transport chain complexes, acetyl CoA biosynthesis, Krebs cycle, glycolysis, and ketogenesis. Conversely, fatty acid oxidation enzymes and sirtuin-3 were up-regulated in hibernating AGS, with preserved peroxisome proliferator–activated receptor-α activity and reduced tissue levels of acylcarnitines and ceramides after I/R. Conclusions: Natural cardioprotective adaptations in hibernators involve extensive metabolic remodeling, featuring increased expression of fatty acid metabolic proteins and reduced levels of toxic lipid metabolites. Robust up-regula- tion of sirtuin-3 suggests that posttranslational modifications may underlie organ protection in hibernating mammals. (ANESTHESIOLOGY 2016; 124:1296-310) SCHEMIA–rEPERFUSION (I/R) injury, a con- What We Already Know about This Topic I sequential component of cardiac surgery and organ • Myocardial ischemia–reperfusion injury remains a major transplantation, is a major determinant of morbidity and source of perioperative morbidity and mortality mortality. Cardioprotection after I/R remains an elusive tar- • The study of hibernation, an adaptive response to extreme get despite significant research and numerous compounds environments, has potential to address novel mechanisms with promising preclinical data.1 Mammalian hibernation and potential therapies for perioperative myocardial ischemia– reperfusion injury is a seasonal phenomenon characterized by prolonged bouts (days to weeks) of winter torpor, a state of decreased physi- What This Article Tells Us That Is New ologic activity, reduced body temperature, and decreased • Applying gene ontology analysis to hibernating arctic ground metabolic rate, interspersed with brief (hours to a day) squirrels, the authors have found that the natural cardiac pro- periods of arousal when metabolism and temperature tem- tective adaptations of hibernators involve metabolic shifts in fatty acid metabolism proteins, reductions in toxic lipid me- 2 porarily return to normal. The entry into and exit from tabolites, and up-regulation of sirtuins torpor is akin to I/R, in that blood flow is greatly reduced and then restored. During arousal from torpor, hiberna- consumption, while accelerating their heart rate 100-fold tors rewarm using a combination of shivering and non- with corresponding increases in organ perfusion.3 Hiber- shivering thermogenesis associated with peaks in oxygen nating mammals like arctic ground squirrels (AGS) show This article is featured in “This Month in Anesthesiology,” page 1A. Corresponding article on page 1215. Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are available in both the HTML and PDF versions of this article. Links to the digital files are provided in the HTML text of this article on the Journal’s Web site (www.anesthesiology.org). Submitted for publication May 27, 2015. Accepted for publication February 2, 2016. From the Department of Anesthesiology, Duke Uni- versity, Durham, North Carolina (Q.J.Q., Z.Z., Q.M., M.P.S., M.V.P.); Center for Genomic and Computational Biology – Proteomics Shared Resource, Duke University, Durham, North Carolina (E.S., M.A.M.); Molecular Physiology Institute and Sarah W. Stedman Nutrition and Metabolism Center, Duke University, Durham, North Carolina (J.B., C.B.N., M.J.M., M.H.); and Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska (K.L.D., B.M.B., M.V.P.). Copyright © 2016, the American Society of Anesthesiologists, Inc. Wolters Kluwer Health, Inc. All Rights Reserved. Anesthesiology 2016; 124:1296-310 Anesthesiology, V 124 • No 6 1296 June 2016 Copyright © 2016, the American Society of Anesthesiologists,<zdoi;10.1097/ALN.0000000000001113> Inc. Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. PERIOPERATIVE MEDICINE natural resistance to experimental I/R in multiple organs,4–7 which remains yet uncharacterized for the heart. Moreover, although a suite of adaptations associated with the seasonal entry into torpor have been reported, including metabolic rate depression,8 increased reliance on lipid oxidation for energy production,3,9–11 and immune suppression,12 the mechanisms underlying this naturally evolved organ protec- tion remain unknown. In the current study, we hypothesized that experimen- tal surgical I/R will result in reduced severity of myocardial injury and dysfunction in hibernating AGS compared to a Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/124/6/1296/487075/20160600_0-00023.pdf by guest on 02 October 2021 nonhibernator (rat); furthermore, we postulated that such hibernator cardioprotective phenotype will be attenuated in summer active (SA) AGS. As a first step to delineate the metabolic regulatory events involved, we employed an unbi- ased proteomics approach to identify and quantify differ- ences in myocardial protein abundance between AGS and rats and further correlate with targeted metabolic changes. The potential cardioprotective role of sirtuin-3 up-regulation Fig. 1. Experimental design and perfusion apparatus for identified in hibernating AGS hearts was further assessed in deep hypothermic circulatory arrest (DHCA). B = blood specimen; C = cooling; CPB = cardiopulmonary bypass; vitro using gain-of-function (rat) and loss-of-function (AGS) E = echocardiographic measurements; H = hemodynam- experiments in adult primary ventricular cardiomyocyte ic measurements; M = myocardial specimen collection; (APVC) models of hypoxia–reoxygenation. P = surgical preparation; RR = rewarming and reperfusion; W = weaning from cardiopulmonary bypass. Materials and Methods and n = 7 females. As these animals are wild caught, exact age Animal Ischemia–Reperfusion Protocol—Anesthetic, cannot be determined; however, juvenile and aged animals Surgical, and Perfusion Management were excluded. Adult male Brown Norway and Dahl/Salt Sensitive rat Rats and AGS underwent an identical experimental strains (Charles River, USA) were randomly assigned to protocol, as previously described.14,15 Briefly, animals were three experimental groups: anesthetized and cannulated anesthetized with isoflurane, endotracheally intubated and without undergoing cardiopulmonary bypass (CPB; sham mechanically ventilated; anesthesia was maintained with group) and deep hypothermic circulatory arrest (DHCA; isoflurane, fentanyl, and pancuronium. Surgical prepara- 45 min at 18°C), followed by either 3 h (DHCA R3h group) tion involved cannulation of the ventral tail artery (rats) and or 24 h (DHCA 24h group) of reperfusion (fig. 1). right femoral artery (AGS; 20 gauge, arterial inflow), inser- Arctic ground squirrels (Urocitellus parryii) were trapped tion of a multiorifice dual-stage cannula through the right in July near the Toolik Field Station in northern Alaska and internal jugular vein and advanced into the right atrium (4.5 transported to the University of Alaska Fairbanks (Fairbanks, French, venous outflow), and insertion of a 3.5-mm bal- Alaska). In the fall, animals assigned to the hibernating group loon catheter (Sprinter OTW, Medtronic, USA) via right were housed in temperature- and photoperiod-controlled carotid artery cutdown, positioned above the aortic valve facilities (5°C, photoperiod, 4L:20D), after being implanted under echocardiographic guidance. Electrocardiogram, with intraperitoneal temperature-sensitive radio transmit- pulse oximetry, end-tidal capnography, pericranial and rec- ters to monitor their body temperature and precisely deter- tal temperatures, and invasive arterial blood pressure were mine stages of torpor and arousal, as described
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