Published online before print July 1, 2004, doi: 10.1161/01.RES.0000137727.34938.35
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Submitted on December 11, 2003
Revised on June 16, 2004
Accepted on June 17, 2004
Selective Inhibition of Inward Rectifier K+ Channels (Kir2.1 or Kir2.2) Abolishes Protection by Ischemic Preconditioning in Rabbit Ventricular Cardiomyocytes
Roberto J. Diaz ;
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.
* To whom correspondence should be addressed. 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 IK1 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 [IPCCM 18.0±2.1% versus control (CCM) 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 IPCCM+AdEGFPKir2.1DN 45.8±2.3% (mean±SEM) and IPCCM+AdEGFPKir2.2DN 47.9±1.4% versus IPCCM; 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 CCM+ AdEGFPKir2.1DN 45.8±0.7% and CCM+AdEGFPKir2.2DN 46.2±1.3% versus CCM; not statistically significant). Similar effects were observed in both cultured (n=5 hearts) and freshly isolated (n=4 hearts) ventricular cardiomyocytes after IK1 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 IK1 channels play an important role in IPC protection.
Key words: ischemic preconditioning • cardiomyocytes • ischemia • potassium channels • gene transfer
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