Diazoxide acts more as a PKC-epsilon activator, and indirectly activates the mitochondrial K(ATP) channel conferring cardioprotection against hypoxic injury.
OBJECTIVE
Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoK(ATP) channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoK(ATP) channel in diazoxide-induced cardioprotection.


METHODS
In H9c2 cells and neonatal rat cardiomyocytes, PKC-epsilon activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca(2+) and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay.


RESULTS
Diazoxide (100 microM) induced translocation of PKC-epsilon from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-epsilon by either epsilonV1-2 or dominant negative mutant PKC-epsilon (PKC-epsilon KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either epsilonV1-2 or PKC-epsilon KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-epsilon induced by diazoxide. Transfection with wild type PKC-epsilon mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by epsilonV1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca(2+), mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by epsilonV1-2 or 5-HD.


CONCLUSIONS
Diazoxide induced isoform-specific translocation of PKC-epsilon as an upstream signaling molecule for the mitoK(ATP) channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway.
Kim
M-Y
MY
Department of Physiology, School of Medicine, Ajou University, Suwon, South Korea.
Kim
M J
MJ
Yoon
I S
IS
Ahn
J H
JH
Lee
S H
SH
Baik
E J
EJ
Moon
C-H
CH
Jung
Y-S
YS
D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002316:Cardiotonic Agents; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D003981:Diazoxide / Q000494:pharmacology*; D020536:Enzyme Activators / Q000494:pharmacology*; D005420:Flavoproteins / Q000378:metabolism; D000860:Hypoxia / Q000473:pathology* / Q000517:prevention & control*; D020287:In Situ Nick-End Labeling; D054086:KATP Channels; D008564:Membrane Potentials / Q000187:drug effects; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D024661:Potassium Channels, Inwardly Rectifying / Q000819:agonists* / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D000959:Antihypertensive Agents; D002316:Cardiotonic Agents; D020536:Enzyme Activators; D005420:Flavoproteins; D054086:KATP Channels; D024661:Potassium Channels, Inwardly Rectifying; C092520:uK-ATP-1 potassium channel; D051744:Protein Kinase C-epsilon; D003981:Diazoxide; D002118:Calcium