RESEARCH ARTICLE Protein modulation in mouse heart under acute and chronic hypoxia Agnese Vigano` 1 , Michele Vasso 1,2 , Anna Caretti 3 , Valentina Bravata` 2,4 , Laura Terraneo 3 , Chiara Fania 1 , Daniele Capitanio 1 , Michele Samaja 3 and Cecilia Gelfi 1,2 1 Dipartimento di Scienze e Tecnologie Biomediche, Universita` degli Studi di Milano, Segrate (MI), Italy 2 Istituto di Bioimmagini e Fisiologia Molecolare, CNR, Segrate (MI), Italy 3 Dipartimento di Medicina, Chirurgia e Odontoiatria, Ospedale San Paolo, Universita` degli Studi di Milano, Milano, Italy 4 LATO HSR Giglio, Cefalu` (PA), Italy Received: December 20, 2010 Revised: July 8, 2011 Accepted: August 8, 2011 Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1a), energy balance (AMPK), mitochondrial biogenesis (PGC-1a), autophagic/ apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2-D DIGE, mass spectrometry and antigen–antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF-1a stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up- regulation of adenosylhomocysteinase and acetyl-CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC-1a, a master regulator of mitochondrial biogenesis, was down-regulated. CH was characterized by the up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a general down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic molecules increased accompained by down- regulation of specific target proteins. Keywords: AMPK / Animal proteomics / Apoptosis / Autophagy / Heart / Hypoxia 1 Introduction The result of an imbalance between oxygen supply and demand, hypoxia, is an important component of ischemia. Hypoxia is related to a number of physiological conditions (i.e. fetal development, exercise and high altitude exposure) and to a wide range of clinically relevant disorders where it may initiate, be maintained or be a result of a disease state. It is known to occur in lung diseases, stroke and cancer. Whereas conditions leading to myocardial injury have been well investigated, endogenous protective responses able to preserve cellular and organelle functions in cardiac tissue during oxygen deprivation are not completely understood and are not yet targeted in the current therapeutic strategies. Several studies demonstrate the central role of HIF-1a in hypoxia response indicating it as a regulator of this process [1, 2]. Under hypoxic conditions, dimerization of the stabi- lized HIF-1a with the HIF-1b subunit enables the tran- scriptional activity of a wide range of genes including those involved in cell metabolism, hypoxia tolerance [3], angio- genesis [4] and others. Although it has been recognized that Abbreviations: AH, acute hypoxia; AMPK, AMP-activated protein kinase; BVA, biological variation analysis; CH, chronic hypoxia COX, cytochrome c oxidase; DIA, differential in-gel analysis; FDR, false discovery rate; HIF, hypoxia inducible factor; OXPHOS, oxidative phosphorylation; PDK, pyruvate dehydro- genase kinase 1; p-AMPK, phospho-AMP-activated protein kinase; TCA, tricarboxylic acid cycle Correspondence: Professor Cecilia Gelfi, Dipartimento di Scienze e Tecnologie Biomediche, Universita` degli Studi di Milano, Via Fratelli Cervi 93, 20090 Segrate, MI, Italy E-mail: cecilia.gelfi@unimi.it Fax: 139-0221717558 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 4202 Proteomics 2011, 11, 4202–4217 DOI 10.1002/pmic.201000804