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.
Diaz
Roberto J
RJ
Division of Cardiovascular Research, The Hospital for Sick Children, Toronto, Ontario, Canada.
Zobel
Carsten
C
Cho
Hee Cheol
HC
Batthish
Michelle
M
Hinek
Alina
A
Backx
Peter H
PH
Wilson
Gregory J
GJ
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D005822:Genetic Vectors / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D019194:Ischemic Preconditioning; D015425:Myocardial Reperfusion; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D009543:Nifedipine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors* / Q000502:physiology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D002712:Chlorides; C489461:Kir2.1 channel; C489463:Kir2.2 channel; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D001464:Barium; D009543:Nifedipine; D011188:Potassium