mitoKATP Channel Opener Research Articles

Selective Pharmacological Agents Implicate Mitochondrial but Not Sarcolemmal K ATP Channels in Ischemic Cardioprotection

Pharmacological evidence has implicated ATP-sensitive K(+) (K(ATP)) channels as the effectors of cardioprotection, but the relative roles of mitochondrial (mitoK(ATP)) and sarcolemmal (surfaceK(ATP)) channels remain controversial. We examined the effects of the K(ATP) channel blocker HMR1098 and the K(ATP) channel opener P-1075 on surfaceK(ATP) and mitoK(ATP) channels in rabbit ventricular myocytes. HMR1098 (30 micromol/L) inhibited the surfaceK(ATP) current activated by metabolic inhibition, whereas the drug did not blunt diazoxide (100 micromol/L)-induced flavoprotein oxidation, an index of mitoK(ATP) channel activity. P-1075 (30 micromol/L) did not increase flavoprotein oxidation but did elicit a robust surfaceK(ATP) current that was completely inhibited by HMR1098. These results indicate that HMR1098 selectively inhibits surfaceK(ATP) channels, whereas P-1075 selectively activates surface K(ATP) channels. In a cellular model of simulated ischemia, the mitoK(ATP) channel opener diazoxide (100 micromol/L), but not P-1075, blunted cellular injury. The cardioprotection afforded by diazoxide or by preconditioning was prevented by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 micromol/L) but not by the surfaceK(ATP) channel blocker HMR1098 (30 micromol/L). The cellular effects of mitochondria- or surface-selective agents provide further support for the emerging consensus that mitoK(ATP) channels rather than surfaceK(ATP) channels are the likely effectors of cardioprotection.

Roles of mitochondrial ATP-sensitive K channels and PKC in anti-infarct tolerance afforded by adenosine A1receptor activation

OBJECTIVESThis study intended to assess the role of mitochondrial ATP-sensitive potassium (mitoKatp) channels and the sequence of signal transduction with protein kinase C (PKC) and adenosine A1receptors in rabbits.BACKGROUNDTo our knowledge, the link between trigger receptors of preconditioning, PKC and mitoKatpchannels has not been examined in a whole heart model of infarction.METHODSIn the first series of experiments, myocardial infarction was induced in isolated buffer-perfused rabbit hearts by 30-min global ischemia and 2-h reperfusion. Infarct size in the left ventricle was determined by tetrazolium staining and expressed as a percentage of area at risk (i.e., the whole left ventricle) (%IS/AR). In the second series of experiments, mitochondria were isolated from the heart, and their respiratory function was examined using glutamate as a substrate.RESULTSPretreatment with r-phenylisopropyladenosine (r-PIA, 1 μmol/liter), an A1-receptor agonist, reduced %IS/AR from 49.8 ± 6.5% to 13.4 ± 2.9%. This protection was abolished by calphostin C, a PKC inhibitor, and by 5-hydroxydecanoate (5-HD), a selective inhibitor of mitoKatpchannels. A selective mitoKatpchannel opener, diazoxide (100 μmol/liter), mimicked the effect of r-PIA on infarct size (%IS/AR = 11.6 ± 4.0%), and this protective effect was also abolished by 5-HD. However, calphostin C failed to block the infarct size–limiting effect of diazoxide. Neither calphostin C nor 5-HD alone modified %IS/AR. State III respiration (Qo2) and respiratory control index (RCI) were reduced after 30 min of ischemia (Qo2= 147.3 ± 5.3 vs. 108.5 ± 12.3, RCI = 22.3 ± 1.1 vs. 12.1 ± 1.8, p < 0.05). This mitochondrial dysfunction was persistent after 10 min of reperfusion (Qo2= 96.1 ± 15.5, RCI = 9.5 ± 1.9). Diazoxide significantly attenuated the respiratory dysfunction after 30 min of ischemia (Qo2= 142.8 ± 9.7, RCI = 16.2 ± 0.8) and subsequent 10-min reperfusion (Qo2= 135.3 ± 7.2, RCI = 19.1 ± 0.8).CONCLUSIONSThese results suggest that mitoKatpchannels are downstream of PKC in the mechanism of infarct-size limitation by A1-receptor activation and that the anti-infarct tolerance afforded by opening of mitoKatpchannels is associated with preservation of mitochondrial function during ischemia/reperfusion.

Opening of mitochondrial KATP channel induces early and delayed cardioprotective effect: role of nitric oxide.

Opening of mitochondrial ATP-sensitive (mitoKATP) channel with diazoxide induces an early phase (EP) of cardioprotection. It is unknown whether diazoxide also induces a delayed phase (DP) of cardioprotection. Because nitric oxide (NO) modulates ATP sensitivity of the KATP channel, we hypothesized that NO may play a role in diazoxide-induced cardioprotection. Diazoxide (1 mg/kg) was administered either 30 min (for EP) or 24 h (DP) before 30 min of lethal ischemia. Blockers of mitoK(ATP) channel [5-hydroxydecanoate (5-HD)] or NO synthase [N(G)-nitro-L-arginine methyl ester (L-NAME)] were given 10 min before ischemia-reperfusion performed by 30 min of left anterior descending coronary artery occlusion and 3 h of reperfusion. A risk area (RA) was demarcated by Evans blue dye, and infarct size (IS) was measured by tetrazolium staining. Diazoxide caused a decrease in IS (%RA) from 27.8 +/- 4.2% in the vehicle group to 12.9 +/- 1.2% during EP and from 30.4 +/- 4. 2% in vehicle-treated rabbits to 19.6 +/- 2.4% during DP (P < 0.05). IS increased to 31.3 +/- 1.1% and 27.9 +/- 1.0% (EP) and 29.9 +/- 2. 3% and 35.1 +/- 1.8% (DP) with 5-HD and L-NAME, respectively (P < 0. 05). 5-HD and L-NAME caused no proischemic effect in controls. Diazoxide induced both early and delayed anti-ischemic effects via opening of mitoK(ATP) channels, which was NO dependent.

Mitochondrial potassium channel opener diazoxide preserves neuronal-vascular function after cerebral ischemia in newborn pigs.

N-Methyl-D-aspartate (NMDA) elicits neuronally mediated cerebral arteriolar vasodilation that is reduced by ischemia/reperfusion (I/R). This sequence has been preserved by pretreatment with the ATP-sensitive potassium (K(ATP)) channel opener aprikalim, although the mechanism was unclear. In the heart, mitochondrial K(ATP) channels (mitoK(ATP)) are involved in the ischemic preconditioning-like effect of K(+) channel openers. We determined whether the selective mitoK(ATP) channel opener diazoxide preserves the vascular dilation to NMDA after I/R. Pial arteriolar diameters were determined with the use of closed cranial window/intravital microscopy in anesthetized piglets. Vascular responses to NMDA were assessed before and 1 hour after 10 minutes of global cerebral ischemia induced by raising intracranial pressure. Subgroups received 1 of the following pretreatments before I/R: vehicle; 1 to 10 micromol/L diazoxide; and coapplication of 100 micromol/L 5-hydroxydecanoic acid (5-HD), a K(ATP) antagonist with diazoxide. NMDA-induced dose-dependent pial arteriolar dilation was not affected by diazoxide treatment only but was severely attenuated by I/R. In contrast, diazoxide dose-dependently preserved the NMDA vascular response after I/R; at 10 micromol/L, diazoxide arteriolar responses were unaltered by I/R. The effect of diazoxide was antagonized by coapplication of 5-HD with diazoxide. Percent preservation of 100 micromol/L NMDA-induced vasodilation after I/R was 53+/-19% (mean+/-SEM, n=8) in vehicle-treated controls versus 55+/-10%, 85+/-5%, and 99+/-15% in animals pretreated with 1, 5, and 10 micromol/L diazoxide (n=8, n=8, and n=12, respectively) and 60+/-15% in the group treated with 5-HD+diazoxide (n=5). The mitoK(ATP) channel opener diazoxide in vivo preserves neuronal function after I/R, shown by pial arteriolar responses to NMDA, in a dose-dependent manner. Thus, activation of mitoK(ATP) channels may play a role in mediating the protective effect of other K(+) channel openers.

Modulation of Mitochondrial ATP-Dependent K + Channels by Protein Kinase C

Pharmacological openers of mitochondrial ATP-dependent K+ (mitoKATP) channels mimic ischemic preconditioning, and such cardioprotection can be prevented by mitoKATP channel blockers. It is also known that protein kinase C (PKC) plays a key role in the induction and maintenance of preconditioning. To look for possible mechanistic links between these 2 sets of observations, we measured mitochondrial matrix redox potential as an index of mitoKATP channel activity in rabbit ventricular myocytes. The mitoKATP channel opener diazoxide (100 micromol/L) partially oxidized the matrix redox potential. Exposure to phorbol 12-myristate 13-acetate (PMA, 100 nmol/L) potentiated and accelerated the effect of diazoxide. These effects of PMA were blocked by the mitoKATP channel blocker 5-hydroxydecanoate, which we verified to be a selective blocker of the mitoKATP channel in simultaneous recordings of membrane current and flavoprotein fluorescence. The inactive control compound 4alpha-phorbol (100 nmol/L) did not alter the effects of diazoxide. We conclude that the activity of mitoKATP channels can be regulated by PKC in intact heart cells. Potentiation of mitoKATP channel opening by PKC provides a direct mechanistic link between the signal transduction of ischemic preconditioning and pharmacological cardioprotection targeted at ATP-dependent K+ channels.