Original article Paradoxical resistance to myocardial ischemia and age-related cardiomyopathy in NHE1 transgenic mice: A role for ER stress? Alexandra R. Cook a , Sonya C. Bardswell a , Subashini Pretheshan a , Kushal Dighe a , Gajen S. Kanaganayagam a , Rita I. Jabr a , Sabine Merkle b , Michael S. Marber a , Stefan Engelhardt b,c , Metin Avkiran a, ⁎ a Cardiovascular Division, King's College London, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK b Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany c Institute of Pharmacology and Toxicology, Technische Universitaet Muenchen, Munich, Germany abstract article info Article history: Received 1 September 2008 Received in revised form 14 October 2008 Accepted 16 October 2008 Available online 5 November 2008 Keywords: Na+/H+ exchanger Ischemia Infarction Heart failure Cardiac remodelling Endoplasmic reticulum stress Sarcolemmal Na + /H + exchanger (NHE) activity, which is provided by the NHE isoform 1 (NHE1), has been implicated in ischemia/reperfusion-induced myocardial injury in animal models and humans, on the basis of studies with pharmacological NHE1 inhibitors. We generated a transgenic (TG) mouse model with cardiac- specific over-expression of NHE1 to determine whether this would be sufficient to increase myocardial susceptibility to ischemia/reperfusion-induced injury. TG mouse hearts exhibited increased sarcolemmal NHE activity and normal morphology and function. Surprisingly, they also showed reduced susceptibility to ischemia/ reperfusion-induced injury, as reflected by improved functional recovery and smaller infarcts. Such protection was sustained in the presence of NHE1 inhibition with zoniporide, indicating a mechanism that is independent of sarcolemmal NHE activity. Immunoblot analysis revealed accumulation of immature NHE1 protein as well as marked upregulation of both cytoprotective (78/94 kDa glucose-regulated proteins, calreticulin, protein disulfide isomerase) and pro-apoptotic (C/EBP homologous protein) components of the endoplasmic reticulum (ER) stress response in TG myocardium. With increasing age, NHE1 TG mice exhibited increased myocyte apoptosis, developed left ventricular contractile dysfunction, underwent cardiac remodelling and died prematurely. Our findings indicate that: (1) Cardiac-specific NHE1 over-expression induces the ER stress response in mouse myocardium, which may afford protection against ischemia/reperfusion-induced injury despite increased NHE activity; (2) Ageing NHE1 TG mice exhibit myocyte apoptosis, cardiac remodelling and failure, likely as a result of sustained ER stress; (3) The pluripotent effects of the ER stress response may confound studies that are based on the chronic over-expression of complex proteins in myocardium. © 2008 Elsevier Inc. All rights reserved. 1. Introduction The Na + /H + exchanger (NHE) isoform 1 (NHE1) is an ubiquitously expressed membrane glycoprotein that is responsible for sarcolemmal NHE activity in cardiac myocytes [1], in which such activity contributes to the integrated control of intracellular pH (pH i ) [2]. The mature form of NHE1 is heavily glycosylated at both N-linked and O-linked sites and migrates as a diffuse band of 100–110 kDa on SDS-PAGE [3,4]. Confocal immunofluorescence microscopy has revealed endogenous NHE1 protein in rat ventricular myocardium [5] and isolated ventricular myocytes [6] to be concentrated predominantly at intercalated disk regions, although the functional significance of such localization with respect to the spatial control of pH i remains unclear [7]. In addition to its physiological role in pH i regulation, sarcolemmal NHE activity has also been causally implicated in ischemia/reperfu- sion-induced myocardial injury [8]. Consistent with this, NHE1- selective pharmacological inhibitors (which include cariporide [9], eniporide [10] and zoniporide [11]) have been reported to provide marked protection against ischemia/reperfusion-induced myocardial injury in numerous pre-clinical studies, with an unusual degree of conformity between different investigators, species and end-points (see reviews by Avkiran [12] and Karmazyn et al. [13]). Indeed, the encouraging evidence from the pertinent experimental studies has led to the initiation of several clinical trials with NHE1 inhibitors, whose outcomes and implications have been reviewed recently [14,15]. Complementary genetic evidence for a key role for NHE1 in determining myocardial susceptibility to ischemia/reperfusion- induced injury has been provided by a study in mice with global deletion of the NHE1 gene [16], although interpretation of the pertinent data is complicated by the fact that such mice exhibit severe neurological and other deficits, such as ataxia, epileptic Journal of Molecular and Cellular Cardiology 46 (2009) 225–233 ⁎ Corresponding author. Tel.: +44 20 7188 3899; fax: +44 20 7928 0658. E-mail address: metin.avkiran@kcl.ac.uk (M. Avkiran). 0022-2828/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2008.10.013 Contents lists available at ScienceDirect Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc