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(Circulation Research. 2004;95:325.)
© 2004 American Heart Association, Inc.

Cellular Biology

Selective Inhibition of Inward Rectifier K⁺ Channels (Kir2.1 or Kir2.2) Abolishes Protection by Ischemic Preconditioning in Rabbit Ventricular Cardiomyocytes

Roberto J. Diaz^*, Carsten Zobel^*, Hee Cheol Cho, Michelle Batthish, Alina Hinek, Peter H. Backx, Gregory J. Wilson

From the Divisions of Cardiovascular Research (R.J.D., M.B., A.H., G.J.W.) and Pathology (G.J.W.), Research Institute, The Hospital for Sick Children; the Departments of Physiology (H.C.C., M.B., P.H.B., G.J.W.) and Medicine (P.H.B.), The University of Toronto; and the Division of Cardiology (C. Z., P.H.B.), University Health Network, Toronto, Canada. Present address of C.Z. is Laboratory of Muscle Research and Molecular Cardiology, Clinic III for Internal Medicine, University of Cologne, Cologne, Germany.

Correspondence to Dr Gregory J. Wilson, Division of Cardiovascular Research, McMaster Bldg, Rm 7019C, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario M5G 1X8, Canada. E-mail Diazport{at}sickkids.ca

Volume regulatory Cl^– channels are key regulators of ischemic preconditioning (IPC). Because Cl^– efflux must be balanced by an efflux of cations to maintain cell membrane electroneutrality during volume regulation, we hypothesize that I_K1 channels may play a role in IPC. We subjected cultured cardiomyocytes to 60-minute simulated ischemia (SI) followed by 60-minute of simulated reperfusion (SR) and assessed percent cell death using trypan blue staining. Ischemic preconditioning (10-minute SI/10-minute SR) significantly (P<0.0001) reduced the percent cell death in nontransfected cardiomyocytes [IPC_CM 18.0±2.1% versus control (C_CM) 48.3±1.0%]. IPC protection was not altered by overexpression of the reporter gene (enhanced green fluorescent protein, EGFP). However, overexpression of dominant-negative Kir2.1 or Kir2.2 genes using adenoviruses (AdEGFPKir2.1DN or AdEGFPKir2.2DN) encoding the reporter gene EGFP prevented IPC protection [both IPC_CM+AdEGFPKir2.1DN 45.8±2.3% (mean±SEM) and IPC_CM+AdEGFPKir2.2DN 47.9±1.4% versus IPC_CM; P<0.0001] in cultured cardiomyocytes (n=8 hearts). Transfection of cardiomyocytes with AdEGFPKir2.1DN or AdEGFPKir2.2DN did not affect cell death in control (nonpreconditioned) cardiomyocytes (both C_CM+ AdEGFPKir2.1DN 45.8±0.7% and C_CM+AdEGFPKir2.2DN 46.2±1.3% versus C_CM; not statistically significant). Similar effects were observed in both cultured (n=5 hearts) and freshly isolated (n=4 hearts) ventricular cardiomyocytes after I_K1 blockade with 20 µmol/L BaCl₂ plus 1 µmol/L nifedipine (to prevent Ba²⁺ uptake). Nifedipine alone neither protected against ischemic injury nor blocked IPC protection. Our findings establish that I_K1 channels play an important role in IPC protection.

Key Words: ischemic preconditioning • cardiomyocytes • ischemia • potassium channels • gene transfer

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