Evidence for mitochondrial K ATP channels as effectors of human myocardial preconditioning.
BACKGROUND
Sublethal periods of ischemia preceding a prolonged interval of ischaemia protect the myocardium. This myocardial preconditioning (PC) appears to be effected by KATP channels. These channels occur both in the sarcolemma and the mitochondrial membrane. We investigated whether mitochondrial KATP channels are the end-effector of PC in the human myocardium.


METHODS
Right atrium specimens obtained from patients undergoing cardiac surgery were prepared and incubated in buffer solution at 37 degrees C. After 30-min stabilisation, the muscles were made ischemic for 90 min and then reperfused for 120 min. The preparations were randomised into eight experimental groups (n = 6/group): (1) Aerobic control--incubated in oxygenated buffer for 210 min, (2) ischemia alone--90 min ischemia followed by 120 min reperfusion, (3) PC--preconditioned with 5 min ischemia/5 min reperfusion, (4) Glibenclamide (10 microM) in the incubation media for 10 min before PC, (5) 5-hydroxydecanoate (5-HD, MitoKATP blocker, 1 mM) in the incubation media for 10 min before PC, (6) HMR 1883 (SarcKATP blocker, 10 microM) in the incubation media for 10 min before PC, (7) Pinacidil (0.5 mM) in the incubation media for 10 min before ischemia, and (8) Diazoxide (MitoKATP opener, 0.1 mM) in the incubation media for 10 min before ischemia. Creatinine kinase leakage into the medium (CK, IU/g wet wt) and MTT reduction (OD/mg wet wt.), an index of cell viability, were assessed at the end of the experiment.


RESULTS
Ischemia alone resulted in a significant increase in CK leakage (8.01 +/- 0.35) and decrease in MTT (0.15 +/- 0.01) from the values seen in the aerobic control (2.24 +/- 0.52 and 0.78 +/- 0.10 respectively, P < 0.05 in both instances). PC fully reversed the effect of ischemia (CK = 2.97 +/- 0.31 and MTT = 0.61 +/- 0.05; P < 0.05 vs. ischemia alone group but P = NS vs. aerobic control group). Both Glibenclamide and 5-HD abolished the protection induced by PC (CK = 6.23 +/- 0.5 and 7.84 +/- 0.64; MTT = 0.18 +/- 0.03 and 0.13 +/- 0.02, respectively, P < 0.05 vs. PC), but interestingly, the protective effect of PC was not abolished by HMR 1883 (CK = 2.85 +/- 0.24 and MTT = 0.58 +/- 0.05, P = NS vs. PC). Diazoxide mimicked the protective effect of PC (CK = 3.56 +/- 0.32 and MTT = 0.58 +/- 0.02, P = NS vs. PC), however pinacidil exhibited less protection than PC (CK = 4.02 +/- 0.16 and MTT = 0.30 +/- 0.02, P < 0.05 vs. PC).


CONCLUSIONS
These studies demonstrate that KATP channels are the end-effectors of ischemic preconditioning and that protection is mediated by mitochondrial KATP channels in human right atrial myocardium.
Ghosh
S
S
Department of Surgery, University of Leicester, Glenfield Hospital, UK.
Standen
N B
NB
Galiñanes
M
M
D000704:Analysis of Variance; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003652:Decanoic Acids / Q000494:pharmacology; D003981:Diazoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005905:Glyburide / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D006801:Humans; D006880:Hydroxy Acids / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D019157:Ischemic Preconditioning, Myocardial; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D017202:Myocardial Ischemia / Q000378:metabolism*; D020110:Pinacidil / Q000494:pharmacology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D011897:Random Allocation; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology
D003652:Decanoic Acids; D006880:Hydroxy Acids; D007004:Hypoglycemic Agents; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D013449:Sulfonamides; C052853:5-hydroxydecanoic acid; D020110:Pinacidil; C114623:clamikalant; D013890:Thiourea; D003981:Diazoxide; D005905:Glyburide