J Mol Cell Cardiol 32, 2279–2285 (2000) doi:10.1006/jmcc.2000.1259, available online at http://www.idealibrary.com on No Confirmation for a Causal Role of Volume-regulated Chloride Channels in Ischemic Preconditioning in Rabbits Gerd Heusch 1,2 , Guang S. Liu 1 , Jochen Rose 2 , Michael V. Cohen 1,3 and James M. Downey 1 1 Departments of Physiology and 3 Medicine, University of South Alabama College of Medicine, Mobile, AL, USA and the 2 Department of Pathophysiology, University of Essen Medical School, Essen, Germany (Received 17 July 2000, accepted in revised form 13 September 2000, published electronically 17 October 2000) G. H, G. S. L, J. R, M. V. C J. M. D . No Confirmation for a Causal Role of Volume- regulated Chloride Channels in Ischemic Preconditioning in Rabbits. Journal of Molecular and Cellular Cardiology (2000) 32, 2279–2285. Volume-regulated chloride channels have recently been proposed to be end-effectors in ischemic preconditioning. The present study attempted to confirm this hypothesis by looking both at car- dioprotection and channel activity. In isolated rabbit cardiomyocytes, hypo-osmotic stress (167 mosm/l) induced a current with a magnitude of 2–5 pA/pF at 60mV. That current could be blocked by the selective chloride channel blockers 5-nitro–2–(3-phenylpropylamino) benzoic acid (NPPB) or indanyloxyacetic acid 94 (IAA-94), but only at 100 and 1 m respectively. Lower concentrations were not effective. Because the channel-blocking concentrations were toxic in isolated perfused rabbit hearts, as evidenced by cessation of cardiac contraction and massive infarction, neither agent could be tested against preconditioning’s anti-infarct effect. NPPB and IAA-94 at 1 and 10 , respectively (the doses used in a previous report), did not affect coronary flow, heart rate and developed pressure, and also did not prevent the infarct size reduction of ischemic preconditioning with 5 min global ischemia/10 min reperfusion preceding 30 min of regional ischemia and 120 min of reperfusion [11.4(±3.6) and (11.1(±3.7)% infarction of risk area, respectively]. The volume-regulated chloride and organic osmolyte channel blocker 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) at 100 blocked the hypo- osmotically induced current in myocytes, but again could not be used, since it induced total cessation of cardiac contraction and reduced infarct size in non-preconditioned hearts. Our data do not confirm a prior study on a causal role for volume-regulated chloride channels in the protection of ischemic preconditioning. This hypothesis remains to be adequately tested. 2000 Academic Press K W: Myocardial ischemia; Reperfusion; Infarction; Cardioprotection. recent studies have proposed the activation of mito- Introduction chondrial K ATP channels as the final step in the signal-transduction cascade, 4,5,6 but it remains un- One or several brief episodes of sublethal myocardial ischemia and reperfusion delay infarct development clear how activation of mitochondrial K ATP channels is ultimately cardioprotective. Alternatively, os- during a subsequent prolonged ischemia. 1 The mag- nitude of cardioprotection by such ischemic pre- motic swelling is a key feature of ischemia/re- perfusion injury to cardiomyocytes. 7,8 Indeed, conditioning is impressive, but its underlying signal cascade and mechanisms are still poorly un- preconditioned cardiomyocytes do show increased tolerance to osmotic swelling, 9 and increased sta- derstood. 2,3 In particular, the final end-effector of ischemic preconditioning is still unclear. Several bility of the actin cytoskeleton has been proposed Please address all correspondence to: James M. Downey, Department of Physiology, MSB 3024, University of South Alabama, College of Medicine, Mobile, AL 366 88, USA. Tel: +334-460-6818; Fax: +334-460-6464; E-mail: jdowney@usamail.usouthal.edu 0022–2828/00/122279+07 $35.00/0 2000 Academic Press