Abstract-The critical time for opening mitochondrial (mito) K ATP channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29Ϯ3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 mol/L diazoxide, a mito K ATP channel opener, reduced infarction to 3Ϯ1% and 8Ϯ1%, respectively. The mito K ATP channel closer 5-hydroxydecanoate (200 mol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24Ϯ3 and 28Ϯ6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 mol/L), another K ATP channel closer, blocked the protection by PC only when administered early. These data suggest that K ATP channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8Ϯ3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 mol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 mol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N-(2-mercaptopropionyl) glycine (300 mol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 mol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K ATP channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases. (Circ Res. 2000;87:460-466.)

Multiple, short, regional coronary occlusions immediately after prolonged myocardial ischemia are an effective cardioprotective intervention in the rabbit, and the mechanism of protection involves activation of ERK, production of nitric oxide, and opening of mitochondrial K(ATP) channels. These observations suggest that a similar approach could be applied in the cardiac catheterization laboratory to protect reperfused myocardium after primary angioplasty in patients with acute myocardial infarction.

Bradykinin receptor activation has been proposed to be involved in ischemic preconditioning. In the present study, we further investigated the role of this agent in preconditioning in both isolated and in situ rabbit hearts. All hearts were subjected to 30 minutes of regional ischemia followed by reperfusion for 2 hours (in vitro hearts) and 3 hours (in situ hearts). Infarct size was measured by tetrazolium staining and expressed as a percentage of the size of the risk zone. Preconditioning in situ hearts with 5 minutes of ischemia and 10 minutes of reperfusion significantly reduced infarct size to 10.2 +/- 2.2% of the risk region (P < .0005 versus control infarct size of 36.7 +/- 2.6%). Pretreatment with HOE 140 (26 micrograms/kg), a bradykinin B2 receptor blocker, did not alter infarct size in nonpreconditioned hearts (40.6 +/- 5.3% infarction) but abolished protection from ischemic preconditioning (34.1 +/- 1.6% infarction). However, when HOE 140 was administered during the initial reflow period following 5 minutes of ischemia, protection was no longer abolished (15.6 +/- 3.9% infarction versus 13.3 +/- 3.8% without HOE 140, P = NS). Bradykinin infusion in isolated hearts mimicked preconditioning, and protection was not affected by pretreatment with the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester or the prostaglandin synthesis inhibitor indomethacin but could be completely abolished by the protein kinase C (PKC) inhibitors polymyxin B and staurosporine as well as by HOE 140. HOE 140 could not block the protection of ischemic preconditioning in isolated hearts. That failure was apparently due to the absence of blood-borne kininogens rather than autonomic nerves. When the preconditioning stimulus in the in situ model was amplified with four cycles of 5-minute ischemia/10-minute reperfusion, HOE 140 pretreatment could no longer block protection (infarct size was 10.7 +/- 3.5% versus 6.4 +/- 2.0% without HOE 140, P = NS). We propose that bradykinin receptors protect by coupling to PKC as do adenosine receptors, and blockade of either receptor will diminish the total stimulus of PKC below threshold and prevent protection. A more intense preconditioning ischemic stimulus can overcome bradykinin receptor blockade, however, by simply enhancing the amount of adenosine and possibly other agonists released.

The tumor suppressor PTEN is frequently inactivated in human cancers. A major downstream effector of PTEN is Akt, which is hyperactivated via PTEN inactivation. It is not known, however, whether diminished Akt activity is sufficient to inhibit tumorigenesis initiated by Pten deficiency. Here we showed that the deficiency of Akt1 is sufficient to dramatically inhibit tumor development in Pten +/− mice. Akt1 deficiency had a profound effect on endometrium and prostate neoplasia, two types of human cancer, in which PTEN is frequently mutated, and also affected thyroid and adrenal medulla tumors and intestinal polyps. Even haplodeficiency of Akt1 was sufficient to markedly attenuate the development of highgrade prostate intraepithelial neoplasia (PIN) and endometrial carcinoma. These results have significant implications for cancer therapy.Supplemental material is available at http://www.genesdev.org.

Salvage of ischemic myocardium by postconditioning is dependent on activation of A2b receptors, which in turn depends on activation of PKC. It is still unclear why PKC activation is required to make the heart's adenosine become protective.

Background Blockade of platelet activation during primary percutaneous intervention for acute myocardial infarction is standard care to minimize stent thrombosis. To determine whether antiplatelet agents offer any direct cardioprotective effect, we tested whether they could modify infarction in a rabbit model of ischemia/reperfusion caused by reversible ligation of a coronary artery. Methods and Results The P2Y12 (adenosine diphosphate) receptor blocker cangrelor administered shortly before reperfusion in rabbits undergoing 30-minute regional ischemia/3-hour reperfusion reduced infarction from 38% of ischemic zone in control hearts to only 19%. Protection was dose dependent and correlated with the degree of inhibition of platelet aggregation. Protection was comparable to that seen with ischemic postconditioning (IPOC). Cangrelor protection, but not its inhibition of platelet aggregation, was abolished by the same signaling inhibitors that block protection from IPOC suggesting protection resulted from protective signaling rather than anticoagulation. As with IPOC, protection was lost when cangrelor administration was delayed until 10 minutes after reperfusion and no added protection was seen when cangrelor and IPOC were combined. These findings suggest both IPOC and cangrelor may protect by the same mechanism. No protection was seen when cangrelor was used in crystalloid-perfused isolated hearts indicating some component in whole blood is required for protection. Clopidogrel had a very slow onset of action requiring 2 days of treatment before platelets were inhibited, and only then the hearts were protected. Signaling inhibitors given just prior to reperfusion blocked clopidogrel’s protection. Neither aspirin nor heparin was protective. Conclusions Clopidogrel and cangrelor protected rabbit hearts against infarction. The mechanism appears to involve signal transduction during reperfusion rather than inhibition of intravascular coagulation. We hypothesize that both drugs protect by activating IPOC’s protective signaling to prevent reperfusion injury. If true, patients receiving P2Y12 inhibitors before percutaneous intervention may already be postconditioned thus explaining failure of recent clinical trials of postconditioning drugs

Bradykinin (BK) mimics ischemic preconditioning by generating reactive oxygen species (ROS). To identify intermediate steps that lead to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker Red stain, which becomes fluorescent after exposure to ROS. Fluorescence intensity in treated cells was expressed as a percentage of that in paired, untreated cells. BK (500 nM) caused a 51 +/- 16% increase in ROS generation (P < 0.001). Coincubation with either the BK B2-receptor blocker HOE-140 (5 microM) or the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) prevented this increase, which confirms that the response was receptor mediated and ROS were actually being measured. Closing mitochondrial ATP-sensitive K+ (mitoKATP) channels with 5-hydroxydecanoate (5-HD, 1 mM) prevented increased ROS generation. BK-induced ROS generation was blocked by Nomega-nitro-m-arginine methyl ester (m-NAME, 200 microM), which implicates nitric oxide as an intermediate. Blockade of guanylyl cyclase with 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10 microM) aborted BK-induced ROS generation but not that from diazoxide, a direct opener of mitoKATP channels. The protein kinase G (PKG) blocker 8-bromoguanosine-3',5'-cyclic monophosphorothioate (25 microM) eliminated the effects of BK. Conversely, direct activation of PKG with 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (100 microM) increased ROS generation (39 +/- 15%; P < 0.004) similar to BK. This increase was blocked by 5-HD. Finally, the nitric oxide donor S-nitroso-N-acetylpenicillamine (1 microM) increased ROS by 34 +/- 6%. This increase was also blocked by 5-HD. In intact rabbit hearts, BK (400 nM) decreased infarction from 30.5 +/- 3.0 of the risk zone in control hearts to 11.9 +/- 1.4% (P < 0.01). This protection was aborted by either 200 microM m-NAME or 2 microM ODQ (35.4 +/- 5.7 and 30.4 +/- 3.0% infarction, respectively; P = not significant vs. control). Hence, BK preconditions through receptor-mediated production of nitric oxide, which activates guanylyl cyclase. The resulting cGMP activates PKG, which opens mitoKATP. Subsequent release of ROS triggers cardioprotection.

Protection from postconditioning has been documented in in situ animal models and it has been proposed that it is targeting circulating leukocytes. We therefore tested whether postconditioning can protect leukocyte-free, buffer-perfused rabbit hearts. Infarct size was measured with triphenyltetrazolium staining. In control hearts undergoing 30 min of regional ischemia and 2 h of reperfusion, 33.3 +/- 2.2% of the risk zone infarcted. The protocol previously used in open-chest animals of four postconditioning cycles of 30 s reperfusion/30 s ischemia starting at the beginning of reperfusion decreased infarction to only 24.8 +/- 2.5% of the risk zone in these isolated hearts. Because of the meager protection induced by four 30 s postconditioning cycles, we evaluated the effect of postconditioning with 6 cycles of 10 s reperfusion/10 s ischemia starting at the beginning of reperfusion. Robust salvage was seen with only 10.4 +/- 3.4% of the risk zone infarcting (p < 0.001 vs control and p < 0.003 vs 4 cycles of 30 s ischemia). The 10s protocol was used in all studies of signal transduction. Wortmannin (100 nM), a phosphatidylinositol 3- (PI3-) kinase antagonist, infused for 20 min starting 5 min before reperfusion, blocked postconditioning's, protection (31.2 +/- 4.2% infarction) as did 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) (2 microM) a guanylyl cyclase inhibitor (36.9 +/- 5.3%) and 8-p-(sulfophenyl) theophylline (SPT) (100 microM), a non-specific adenosine receptor blocker (34.2 +/- 2.8%). Thus, postconditioning's protection is not dependent on circulating blood factors or cells, and its anti-infarct effect appears to require PI3-kinase activation, stimulation of guanylyl cyclase and occupancy of adenosine receptors. These signaling steps have also been identified in preconditioning and during pharmacologic cardioprotection and suggest commonality of a protective mechanism.