Selective inhibition of inward rectifier K⁺ channels (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 18.01±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+AdEGFPKM.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 μ-mol/L nifedipine (to prevent Ba ²⁺ 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.