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

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 micromol/L BaCl2 plus 1 micromol/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.