Ischemic Preconditioning Activates Phosphatidylinositol-3-Kinase Upstream of Protein Kinase C
Abstract—The present study is designed to test whether phosphatidylinositol 3-kinase (PI3-kinase) has a role in the signaling pathway in ischemic preconditioning (PC) and whether it is proximal or distal to protein kinase C (PKC). Before 20 minutes of global ischemia, Langendorff-perfused rat hearts were perfused for 20 minutes (control); preconditioned with 4 cycles of 5-minute ischemia and 5-minute reflow (PC); treated with either wortmannin (WM) or LY 294002 (LY), each of which is a PI3-kinase inhibitor, for 5 minutes before and throughout PC; treated with 1,2-dioctanoyl-sn-glycerol (DOG), an activator of PKC for 10 minutes (DOG); treated identically to the DOG group except with WM added 10 minutes before and during perfusion with DOG; or treated with either WM or LY for 25 minutes. Recovery of left ventricular developed pressure (LVDP; percentage of initial preischemic LVDP), measured after 30 minutes of reflow, was improved by PC (72±2% versus 36±4% in control; P<0.001), and this was blocked by WM and LY (41±4% and 43±5%, respectively; P<0.05 compared with PC). DOG addition improved postischemic LVDP (67±6%; P<0.001 compared with control), but in contrast to its effect on PC, WM did not completely eliminate the protective effect of DOG (52±4%; P>0.05 compared with DOG; P<0.05 compared with control). PC induced phosphorylation of protein kinase B and translocation of PKCε, and it increased NO production, and these effects were blocked by WM, which suggests a role for PI3-kinase in PC upstream of PKC and NO.
Ischemic preconditioning (PC) describes the phenomenon whereby repeated brief episodes of ischemia and reperfusion increase the resistance to myocardial infarction and contractile dysfunction induced by a subsequent sustained episode of ischemia.1 2 The signaling pathways involved in PC have been investigated intensively. There is strong support for the hypothesis that PC results in protein kinase C (PKC) activation and translocation.3 4 5 6 7 8 9 Downstream targets of PKC include activation of the 12-lipoxygenase pathway of arachidonic acid metabolism10 and activation of an ATP-sensitive potassium channel KATP, most likely the mitochondrial KATP channel.11 12 The mechanism by which PC activates PKC has not been elucidated; however, NO,13 reactive oxygen species,14 and diacylglycerol generated by phospholipase D15 have been suggested as mediators of PKC activation in PC.
Phosphatidylinositol 3-kinase (PI3-kinase) is a key signaling enzyme implicated in cell survival and metabolic control. The PI3-kinases are a subfamily of lipid kinases that catalyze the phosphorylation of the inositol ring of phosphoinositides specifically at the 3 position. Downstream targets of PI3-kinase include protein kinase B (PKB)/Akt,16 17 p70S6 kinase,18 and several isoforms of PKC (ie, PKCε, -δ, -η, and -ζ).19 20 21 PKB activation in vivo appears to be dependent on the 3-phosphoinositide–dependent protein kinase (PDK1) that phosphorylates and activates PKB.22 23 PDK1 also phosphorylates several isoforms of PKC.21 24 It is reported that full activation of PKC requires phosphorylation by PDK1 as well as allosteric activation.21 Activated PKB phosphorylates and inactivates glycogen synthase (GS) kinase (GSK), and inactivation of GSK leads to activation of GS.25 Thus, PI3-kinase is a key regulator of glycogen metabolism. PKB also directly activates endothelial NO synthase (eNOS),26 27 and NO generated by eNOS is proposed to initiate PC.13 Wortmannin (WM) binds covalently to the 110-kDa subunit of PI3-kinase and has been shown to inhibit PI3-kinase irreversibly (IC50=5 nmol/L).28 LY 294002 (LY) is another selective PI3-kinase inhibitor (IC50=1.4 μmol/L) that acts on the ATP binding site of the enzyme.29
Because PI3-kinase is reported to activate PKC and eNOS, which are both involved in PC, and because of the suggested role of PI3-kinase in cell survival, we investigated the role of PI3-kinase in PC. The objective of this investigation was to test the hypothesis that PI3-kinase is involved in the signaling pathway of PC. The specific goals of this study were to determine whether administration of WM and LY, potent and specific PI3-kinase inhibitors, block the protective effect of PC, and to determine whether PI3-kinase is upstream or downstream of PKC and NO by testing whether WM blocks the PC-induced phosphorylation of PKB, translocation of PKC, and increase in NO production.
Materials and Methods
Isolated Rat Heart Preparation
Male Sprague-Dawley rats (170 to 350 g) were used in this study. All animals received humane care in accordance with NIH guidelines. Rats were anesthetized with intraperitoneal pentobarbitone (≈25 mg), and hearts were isolated and perfused in the Langendorff mode as described previously.9 To monitor left ventricular developed pressure (LVDP), a latex balloon connected to a Statham pressure transducer was inserted into the left ventricle. 31P NMR spectra were obtained at 161.9 MHz using a Varian Unity Plus, 400-MHz wide-bore spectrometer.
The protocols are illustrated in Figure 1⇓. Groups differed only in their treatments as shown in Figure 1⇓ and described in the online Materials and Methods (available at http://www.circresaha.org). Recovery of LVDP, expressed as a percentage of the initial LVDP before PC or drug administration, was measured at 30 minutes of reflow after 20 minutes of ischemia.
Subcellular Fractionation for PKCε Translocation
The subcellular fractionation method was modified from Kawamura et al.8 Briefly, the frozen hearts were homogenized with a Polytron and centrifuged at 1000g for 10 minutes, and the supernatants were centrifuged at 100 000g for 60 minutes. The resulting supernatant and pellet were the cytosolic and particulate fractions, respectively.
PKCε and Phospho-PKB Western Blotting Analysis
Frozen hearts were homogenized, electrophoresed, and transferred as described previously.30 The membranes were incubated in 5% BSA in Tris-buffered saline–Tween (150 mmol/L NaCl, 10 mmol/L Tris-HCl [pH 7.4], and 0.1% Tween-20) containing PKB, phospho-PKB antibodies (1:1000 dilution) (New England Biolabs, Inc), or PKCε antibody (1:200 dilution) (Santa Cruz Biotechnology) and then incubated with anti-rabbit IgG (1:2000 dilution). The immunoreactive bands were visualized by a chemiluminescence reagent and analyzed by densitometry.
Tissue Nitrate and Nitrite Assays
Rat hearts were homogenized in PBS (pH 7.4) and centrifuged at 10,000g for 20 minutes, and the supernatant was centrifuged again at 100 000g for 15 minutes. The supernatant was filtered through a Centricon-30 filter. Total NO products (nitrate and nitrite) were measured using a commercial assay kit (Cayman Chemical).
Values are expressed as mean±SE. Significance (P≤0.05) was determined by ANOVA followed by a Fisher post hoc test.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
Hemodynamics Before Ischemia
Treatment with the PI3-kinase inhibitors WM and LY, and the PKC activator 1,2-dioctanoyl-sn-glycerol (DOG) before ischemia resulted in no significant differences in LVDP, heart rate, and coronary flow rate among the groups at the end of the control period (see Table 1⇓ online, available at http://www.circresaha.org). As observed previously,2 PC resulted in a decline in LVDP to 74% of the initial value at the end of the fourth reflow. The decrease in LVDP in hearts preconditioned in the presence of WM and LY were similar to that in hearts with PC alone. The recovery of coronary flow after 20 minutes of sustained ischemia was not significantly different among the groups, which indicates that the differences in functional recovery observed on reflow were not due to differences in tissue perfusion.
WM and LY Block the Protective Effect of PC on Recovery of Function
Consistent with previous data,2 PC resulted in improved recovery of postischemic LVDP (72±2% for PC versus 36±4% for non-PC control). To investigate whether PI3-kinase was involved in the signaling pathway of PC, we examined whether the specific PI3-kinase inhibitors WM and LY blocked the protective effects of PC. As shown in Figure 2A⇓, pretreatment with WM blocked the PC-induced improvement in postischemic LVDP (41±4%; P<0.001 compared with PC). Postischemic function in the group treated with WM alone (41±8%) was not significantly different from that of the control group (P>0.05). Similar to the data obtained with WM, pretreatment with LY also blocked the protective effect of PC (43±5%; P<0.05 compared with PC) (Figure 2A⇓). Postischemic function in the group treated with LY alone (37±2%) was not significantly different than that in the control group (P>0.05).
PI3-Kinase Is Upstream of PKC in PC
To investigate whether PI3-kinase was upstream or downstream of PKC, we examined whether WM could block the protection afforded by the PKC activator DOG. As shown in Figure 2B⇑, the recovery of postischemic contractile function in DOG-treated hearts (67±6%) was significantly better than that of control (36±4%; P<0.05). These data are consistent with the previous studies that demonstrate that PKC participates in the protective effect of PC.3 4 5 6 7 8 9 Figure 2B⇑ shows also that, in contrast to the effect on PC, the improved recovery of postischemic function in DOG-treated hearts was not eliminated by WM (LVDP recovery 52±4%; P>0.05 compared with DOG; P<0.05 compared with control), suggesting that PI3-kinase is upstream of PKC. The lack of inhibition with 100 nmol/L WM was not the result of an inadequate concentration, as a higher concentration of WM (200 nmol/L) resulted in a similar outcome (LVDP recovery, 52±6%; P<0.05 versus control).
PC Increases the Phosphorylation of PKB
If PC activates PI3-kinase, one would expect activation of PKB, a kinase downstream of PI3-kinase.22 23 We therefore examined whether PC increased the phosphorylation of PKB. As shown in Figure 3⇓, PC resulted in a ≈1.5-fold increase in phosphorylation of PKB. Addition of WM in PC and non-PC hearts significantly decreased the phosphorylation of PKB to 50% of basal levels. DOG treatment did not change the phosphorylation of PKB, which suggests that PKB is not downstream of PKC. These data suggest that there is a basal level of PKB activation that is inhibited by WM and that PC further stimulates PKB, which is also prevented by WM.
PC Induces the Translocation of PKCε and Increases NO Production
Western blot analysis (Figure 4⇓) showed that PC increased the content of PKCε in the particulate fraction compared with that of control (P<0.05), and this increase was reduced by WM (P<0.05). This observation is consistent with the data in Figure 2⇑ showing that PI3-kinase is upstream of PKC in PC.
It has been reported that NO donors induce and NOS inhibitors block translocation of PKCε in the rabbit heart, which suggests that NO is upstream of PKC.13 The total level of NO products (nitrate and nitrite) increased significantly in PC hearts compared with control hearts (P<0.05), and WM significantly reduced this increase in NO products in PC hearts (P<0.05) (Figure 5⇓), which suggests that NO generation is downstream of PI3-kinase.
Effect on pHi
PC has been shown consistently to reduce the acidification that occurs during sustained global ischemia,2 although the reduced acidification can be dissociated from the protective effects of PC.9 As shown in Figure 6A⇓, PC significantly reduced acidification during the sustained period of ischemia; pHi fell to 5.8±0.09 in control hearts, compared with 6.4±0.04 in PC hearts (P<0.001). However, WM had no effect on the reduced acidification seen with PC (6.4±0.04; P>0.05). Surprisingly, however, WM reduced acidification (6.1±0.07; P<0.05 compared with control) during 20 minutes of ischemia in non-PC hearts. Similar results were found with LY. As shown in Figure 6A⇓, treatment with LY did not block the reduced acidification observed during 20 minutes of ischemia in PC hearts (pHi=6.4±0.04 for PC and 6.5±0.06 for PC+LY; P>0.05). However, LY reduced the acidification during 20 minutes of ischemia in non-PC hearts.
DOG treatment significantly reduced acidification during the 20-minute period of ischemia (Figure 6B⇑) to a pHi value that was intermediate between control hearts and PC hearts (6.1±0.1; P<0.05 compared with control). WM had no statistically significant effect on the reduced acidification observed with DOG treatment (6.3±0.04; P>0.05 compared with DOG) (Figure 6B⇑).
The decrease in acidification during ischemia, in non-PC hearts in which PI3-kinase was inhibited, might be due to a decrease in glycogen, given that PI3-kinase inhibitors reduced basal PKB phosphorylation, which might be expected to decrease glycogen accumulation via GSK and GS. As shown in the Table⇑, both WM and LY slightly but significantly decreased glycogen levels in the hearts during the treatment period. PC reduced glycogen to ≈60% of its preischemic level; however, PC in the presence of WM or LY did not result in a further significant decrease in glycogen. Addition of DOG did not alter glycogen compared with control, but addition of WM to DOG-treated hearts resulted in a decrease in glycogen similar to that observed in hearts treated with WM alone.
Effects on High-Energy Phosphates
ATP content during the sustained 20-minute ischemia was not significantly different among the groups (data not shown). WM and LY treatment significantly increased ATP depletion during the PC protocol. On reperfusion, ATP recovered to 20% to 50% in all groups. There were no significant differences in ATP levels in DOG, DOG+WM, WM, or untreated hearts during the treatment period, ischemia, or reperfusion.
Creatine phosphate content (data not shown) decreased rapidly during each brief cycle of ischemia in the PC protocol and recovered to >100% of control during each 5-minute reflow period, with or without WM. During the 20-minute period of sustained ischemia, creatine phosphate levels fell to undetectable values in all groups and recovered rapidly during the final reperfusion period. Creatine phosphate recovered to significantly higher levels in PC hearts but recovered to nearly 100% in all groups.
Role of PI3-Kinase in PC
The results of this study demonstrate that inhibition of PI3-kinase with either WM or LY blocks the protective effect of PC on functional recovery, whereas inhibition of PI3-kinase has no effect on ischemic injury in non-PC hearts. These data suggest an important role for PI3-kinase in the protective effects of PC. In further support of a role for activation of PI3-kinase in PC, we find that PC stimulates phosphorylation of PKB, a kinase directly downstream of PI3-kinase. Addition of WM blocks the PC-induced increase in PKB phosphorylation. Thus, the data provide evidence that there is activation of PI3-kinase in PC hearts and that inhibition of PI3-kinase blocks downstream effects, such as activation of PKB, and the protective effects of PC.
A previous study by Baines et al31 reported that WM did not block the protective effect of PC using infarct size as a measure of protection. However, the effect of WM was intermediate between PC and non-PC hearts. Infarct size in non-PC hearts was 32.6% of the area at risk, which was significantly higher than that found in PC hearts (12.8%). In hearts preconditioned with WM, infarct size was 22.1%, a value that was not significantly different from that of PC hearts. In the present study, we found that PI3-kinase inhibition blocked the PC-induced improvement in postischemic LVDP, the increase in NO production, and the translocation of PKCε. The difference between this study and the study of Baines et al31 might be due to a difference in the PC protocol.
Relationship of PI3-Kinase, NO, and PKC in PC
Considerable evidence suggests that PKC activation is a critical event in PC.3 4 5 6 7 8 9 Selective isoforms of PKCs, such as PKC-δ, ε, and ζ, have been proposed to be involved in PC. Ping et al5 demonstrated that translocation of PKCε and PKCη increased with the number of cycles of PC in rabbit hearts, which correlates with the protective effect. Gray et al6 showed that a PKCε-selective antagonist inhibits PKCε translocation and abolishes the protective effect of hypoxic PC in cardiac myocytes. Our data also indicated that PC increased PKCε in the particulate fraction. Because activation of both PKC and PI3-kinase are important components in the signaling pathway of PC, we were interested in examining their relationship. Many PKC isoforms (ie, PKCε, -δ, -η, and -ζ) are activated by the lipid products of PI3-kinase.19 However, there are also data suggesting that PKC activation can lead to production of phosphatidylinositol-3,4,5-trisphosphate.32 If PKC is downstream of PI3-kinase, then the PC-induced translocation of PKCε should be blocked by WM, and WM should not block the protection induced by PKC activation by DOG pretreatment. We found that the protective effect of the PKC activator DOG is comparable with the protective effect of PC. Although inhibition of PI3-kinase abolished the protective effect of PC, it did not eliminate the protective effect of the PKC activator DOG. In further support of a role for PKC downstream of PI3-kinase, our data showed that DOG treatment did not affect PKB phosphorylation and WM decreased the PC-induced translocation of PKCε.
Activation of many G protein–coupled receptors (GPCRs) can mimic PC, and addition of pertussis toxin blocks the protective effects of PC. There are data suggesting that PC may activate phospholipase D, which is also known to generate diacylglycerol, an activator of PKC.15 Interestingly, PI3-kinase is activated by many GPCRs,33 and PI3-kinase may be an important mechanism by which GPCRs activate PKC, as PKC has been shown to be activated by PI3-kinase. Furthermore, it has been reported that full activation of PKC by diacylglycerol requires phosphorylation by PDK1.21
It has been demonstrated that NO generated during PC is a trigger for late PC.13 34 The role of NO in early PC is controversial.35 36 It has been reported that inhibition of NOS blocks PC35 ; however, others36 failed to implicate NOS in early PC. Ping et al13 have reported that NO donors stimulate translocation of PKCε and that NOS inhibitors block the PC-induced translocation of PKCε. Interestingly, PKB is reported to activate eNOS,26 27 which has been proposed to mediate the PC-induced activation of PKC.13 We therefore investigated the relationship between PI3-kinase and NO. The data in this study show that PC-induced activation of PI3-kinase occurs upstream of PKC, PKB, and NO. These data are consistent with a linear pathway in which PI3-kinase activates PKB, which stimulates NO production, which in turn activates PKC. However, the data are also consistent with a more complex branched pathway, in which PI3-kinase activates both NOS and PKC, which in turn activate separate pathways. The data show that WM blocks the PC-dependent accumulation of NO products, but they do not show that PKB and NO are required for acute PC. Ping et al.13 suggested that NOS inhibitors blocked the translocation of PKCε, which might suggest a linear pathway. If there is a linear pathway, then NO should be required for the acute protection of PC; however, the role of NO in acute PC is very controversial, perhaps suggesting more complex signaling. The ability of NOS inhibitors to block acute PC may relate to the strength of the signal necessary to activate PKC. PI3-kinase may activate PKC either via direct activation by lipid products or via phosphorylation by PDK1, which enhances activation by allosteric mediators such as diacylglycerol, as well as through activation of eNOS. It may be that the level of PKC activation required for acute PC can be met without activation of eNOS and generation of NO, but with some PC protocols the signals activating PKC are weaker and may require the additional signal from NO. Because eNOS is required for the second window of PC, it is likely that both pathways are necessary for the second window of protection.
Role of PI3-Kinase in Glycogen Metabolism in PC
We find that WM reduced acidification during ischemia in non-PC hearts. As shown in Figure 3⇑, there is measurable basal phosphorylation of PKB, which is significantly reduced by addition of WM. If the basal activity of PI3-kinase is important for setting the balance between glycogen synthesis and degradation, then the addition of WM might be expected to reduce glycogen levels before ischemia, which would be expected to result in a higher pH during ischemia. This hypothesis is supported by the data in the Table⇑, which show that WM and LY both result in slight but significant glycogen depletion before the sustained period of ischemia.
Addition of WM or LY did not significantly alter the pH reached during the sustained period of ischemia in PC hearts. This is consistent with the data in the Table⇑, which show that the glycogen levels at the end of PC, just before the start of the sustained period of ischemia, are not significantly different among the PC, PC+WM, and PC+LY groups.
A prominent feature of the PC myocardium is that the fall in pHi during subsequent ischemia is attenuated relative to non-PC myocardium. However, this attenuated fall in pHi during ischemia in PC hearts can be dissociated from the protective effects of PC.9 Indeed, in this study, WM and LY blocked the protective effects of PC but had no effect on pHi during sustained ischemia. However, because of the reproducibility of the reduced acidification during ischemia in PC hearts, it is of interest to understand the mechanisms responsible. The decrease in pHi during global ischemia has been correlated with a decrease in anaerobic glycolysis. Activation of PI3-kinase is reported to increase glycogen synthesis and inhibit glycogen breakdown, and this would be consistent with decreased glycogen breakdown and decreased glycolysis during ischemia in PC myocardium.1 37 However, the data in the present study suggest that, if PI3-kinase mediates the PC-induced decrease in glycogen breakdown, it is a small effect, at least when measured after 5 minutes of ischemia. The data in this study are most consistent with the concept that reduced glycogen levels are the primary determinant of the magnitude of the fall in pH during sustained ischemia.
DOG also reduced H+ production during ischemia; however, the mechanism responsible is not clear. Inhibition of GS does not appear to be involved in the rat heart, as perfusion with DOG for 10 minutes had no effect on glycogen levels. DOG could alter pH during ischemia by other mechanisms such as alterations of Na+/H+ exchange or alterations in the rate of glycogenolysis or glycolysis. Addition of WM to DOG-treated hearts caused a further reduction in acid production during ischemia, which is largely accounted for by the decrease in glycogen in the DOG+WM hearts. It is interesting that the effects of DOG and WM are additive, consistent with 2 independent mechanisms.
In summary, the present study demonstrates that PC increases phosphorylation of PKB presumably as a result of activation of PI3-kinase, given that WM blocks the PC-induced phosphorylation of PKB. Inhibitors of PI3-kinase during PC block the increase in NO production, the translocation of PKCε, and the cardioprotective effect. PI3-kinase inhibitors do not affect the PC-induced attenuation of the fall in pHi during sustained ischemia, which suggests that PI3-kinase does not regulate anaerobic glycolysis under these conditions. Furthermore, WM does not eliminate the cardioprotective effects due to direct DOG activation of PKC, and DOG does not affect PKB phosphorylation, which suggests that PI3-kinase is upstream of PKC in the signaling pathway of PC.
C.S. and W.C. were supported by National Institutes of Health Grant RO1-HL-39752. H.T. and E.M. were supported by the National Institute of Environmental Health Sciences intramural program.
- Received April 3, 2000.
- Revision received June 20, 2000.
- Accepted June 20, 2000.
- © 2000 American Heart Association, Inc.
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