Cross-talk between the survival kinases during early reperfusion: its contribution to ischemic preconditioning

OBJECTIVES: Recruitment of the survival kinase cascades, PI3K-Akt and Raf-MEK1/2-Erk1/2, at the time of reperfusion, following a lethal ischemic insult, may mediate the protection associated with ischemic preconditioning (IPC). The exact interplay between these two kinase cascades in mediating this effect is not clear. We examine the 'cross-talk' between these kinase cascades in their contribution to IPC-induced protection. METHODS AND RESULTS: In isolated perfused rat hearts subjected to 35 min of lethal ischemia +/- ischemic preconditioning, the phosphorylation states of Akt, Erk1/2, p70S6K were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a threefold increase in Akt, Erk1/2, and p70S6K phosphorylation, at reperfusion. We found that inhibiting the PI3K-Akt (using LY294008) at reperfusion induced the phosphorylation of Erk1/2-p70S6K, and conversely, that inhibiting the MEK1/2-Erk1/2 pathway (using PD 98059) at reperfusion, induced the phosphorylation of Akt, suggesting 'cross-talk' between the two kinase pathways. However, this effect was not accompanied by a reduction in infarct size (43.1 +/- 7.2% with LY 294008 and 57.7 +/- 7.0% with PD 98059 vs. 46.3 +/- 5.8% in control; P = NS), suggesting that both the kinase cascades may need to be activated to mediate IPC-induced protection. IPC reduced the infarct-risk volume ratio to 17.8 +/- 2.3% from 46.3 +/- 5.8% in control (P < 0.01). Inhibiting p70S6K, a kinase situated downstream of both PI3K and Erk1/2, using rapamycin, abolished IPC-induced protection (46.0 +/- 7.7% with IPC+RAPA vs. 17.8 +/- 2.3% with IPC; P < 0.01). CONCLUSIONS: We report that, the survival kinase cascades PI3K-Akt and MEK1/2-Erk1/2, which are recruited at the time of reperfusion in response to ischemic preconditioning, exhibit 'cross-talk' such that inhibiting one cascade activates the other and vice versa. Furthermore, at the time of reperfusion, these kinase cascades mediate IPC-induced protection, by acting in concert via p70S6K.     

丝裂原激活蛋白激酶信号通路在肝脏缺血预处理细胞保护效应中的作用

目的 研究p44/42丝裂原激活蛋白激酶(MAPK)信号通路在肝脏缺血预处理细胞保护效应中的作用。方法 建立体内和体外肝脏缺血预处理模型,应用蛋白激酶C(PKC)抑制剂和MAPK抑制剂,通过检测p44/42MAPK磷酸化水平,细胞活力,血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)的活性变化,同时观察光镜细胞形态学损害,对相关数据进行统计学处理。 结果 体内和体外的缺血预处理两部分实验都观察到相似的结果。和缺血再灌注组比较,预处理组的p44/42 MAPK磷酸化水平显著增高,肝细胞结构损伤改变较小,血清ALT、AST水平显著降低,缺血再灌注组的ALT、AST水平分别为(762.8±130.5)U/L和(820.9±111.3)U/L,预处理组的ALT、AST水平分别为(281.0±35.6)U/L和(407.7±73.7)U/L;和缺血预处理组相比,PKC抑制剂组和丝裂原蛋白激酶(MEK)抑制剂组相应的观察指标呈相反的变化,p44/42 MAPK磷酸化激活显著减少,肝组织细胞结构出现较明显的损伤改变,血清ALT、AST水平显著升高,PKC抑制剂组和MEK抑制剂组的ALT、AST水平分别为(645.61±90.4)U/L、(678.6±136.5)U/L和(466.2±82.8)U/L、(732.9±91.1)U/L。 结论 肝脏缺血预处理细胞保护作用中,p44/42 MAPK通路起到至关重要的作用。     

Intermittent hypoxia-induced delayed cardioprotection is mediated by PKC and triggered by p38 MAP kinase and Erk1/2

We previously reported that acute intermittent hypoxia (IH) confers delayed cardioprotection against a prolonged ischemic insult in the rat, via the involvement of nitric oxide synthase and K(ATP) channels. In the present study, we investigated the role of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K), stress activated p38 MAP kinase (MAPK) and extracellular signal-regulated kinase (ERK1/2) using selective inhibitors of these pathways. Adult male rats were exposed to 1-min cycles of IH (10% O(2), 40 s)/normoxia (21% O(2), 20 s) during 4 h or to normoxic cycles. 24 h later, isolated hearts were perfused in Langendorff mode and subjected to a 30-min global ischemia followed by 120 min of reperfusion. Compared to normoxic conditions, IH significantly reduced infarct size (22.2+/-2.4% vs. 33.8+/-2.6%, p<0.05), improved coronary flow and decreased the contracture at reperfusion. When administered before sustained ischemia, chelerythrine (a PKC inhibitor) abolished both the IH-induced reduction in infarct size (36.1+/-4.9%) and improvement in hemodynamic parameters. In contrast, chelerythrine administration 10 min before IH, did not modify the delayed cardioprotective response. Similarly, wortmannin (a PI3K inhibitor) administration 10 min before IH was unable to block the cardioprotective effects. However, administration of SB203580 (a p38 MAPK inhibitor) and PD98059 (an Erk1/2 inhibitor), 30 min before IH abolished its delayed infarct-sparing effect (32.2+/-3.4% and 33.9+/-2.9%, respectively). In addition, 24 h after IH, a significant increase in p38 MAPK and Erk1/2 phosphorylation was observed by Western blot. These results suggest that the delayed preconditioning induced by intermittent hypoxia does not involve the PI3K signalling pathway and that is mediated by PKC and triggered by p38 MAPK and Erk1/2.     

Role of phasic dynamism of p38 mitogen-activated protein kinase activation in ischemic preconditioning of the canine heart

Although ischemic stress, including ischemic preconditioning (IP), activates p38 mitogen-activated protein kinase (MAPK), the relationship between p38 MAPK activation and the underlying cellular mechanisms of cardioprotection by IP is not verified in vivo. We examined the effects of the selective p38 MAPK inhibition on the cardioprotective effect of IP in the open-chest dogs. The coronary artery was occluded 4 times for 5 minutes, separated by 5 minutes of reperfusion (IP) followed by 90 minutes of occlusion and 6 hours of reperfusion. We infused SB203580 into the coronary artery during IP and 1 hour of reperfusion, during IP alone, and during sustained ischemia in the IP group. p38 MAPK activity markedly increased during IP but did not additionally increase at the onset of ischemia and was even attenuated at 15 minutes of sustained ischemia, and heat-shock protein (HSP) 27 was phosphorylated and translocated from cytosol to myofibril or nucleus without affecting total protein level at the onset of ischemia compared with the control group. SB203580 treatment (1 micromol/L) only during IP blunted the infarct size limitation by IP (37.3+/-6.3% versus 7.4+/-2.1% in the IP group, P:<0.01) and attenuated either phosphorylation or translocation of HSP27 during IP. Although the SB203580 treatment throughout the preischemic and postischemic periods had no significant effect on infarct size (33.3+/-9.4%) in this model, treatment with SB203580 only during ischemia partially mimicked the infarct size limitation by IP (26.8+/-3.5%). Thus, transient p38 MAPK activation during ischemic preconditioning mainly mediates the cardioprotection followed by HSP27 phosphorylation and translocation in vivo in the canine heart.     

Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway

Brief intermittent episodes of ischemia and reperfusion, at the onset of reperfusion after a prolonged period of ischemia, confer cardioprotection, a phenomenon termed "ischemic postconditioning" (Postcond). We hypothesized that this phenomenon may just represent a modified form of reperfusion that activates the reperfusion injury salvage kinase (RISK) pathway. Isolated perfused rat hearts were subjected to: (a) 35 minutes of ischemia and 120 minutes of reperfusion, and infarct size was determined by tetrazolium staining; or (b) 35 minutes of ischemia and 7 minutes of reperfusion, and the phosphorylation states of Akt, endothelial NO synthase (eNOS), and p70S6K were determined. Postcond reduced infarct size from 51.2+/-3.4% to 31.5+/-4.1% (P<0.01), an effect comparable with ischemic preconditioning (IPC; 27.5+/-2.3%; P<0.01). Of interest, the combined protective effects of IPC and Postcond were not additive (30.1+/-4.8% with IPC+Postcond; P=NS). Inhibiting phosphatidylinositol 3-kinase (PI3K) at reperfusion using LY or Wortmannin (Wort) during the first 15 minutes of reperfusion completely abolished Postcond-induced protection (31.5+/-4.1% with Postcond versus 51.7+/-4.5% with Postcond+LY, P<0.01; 56.2+/-10.1% with Postcond+ Wort; P<0.01), suggesting that Postcond protects the heart by activating PI3K-Akt. Western blot analysis demonstrated that Postcond induced a significant increase in phosphorylation of Akt, eNOS, and p70S6K in an LY- and Wort-sensitive manner. In conclusion, we show for the first time that ischemic Postcond protects the myocardium by activating the prosurvival kinases PI3K-Akt, eNOS, and p70S6K in accordance with the RISK pathway.     

Dissociation of stress-activated protein kinase (p38-MAPK and JNKs) phosphorylation from the protective effect of preconditioning in vivo

The aim of the present study was to examine and compare the role of the stress-activated protein kinases in ischemic and stretch-induced preconditioning. A model of anesthetized rabbits was used, and the preconditioning protocol included one or three cycles of short ischemia/reperfusion, or short mechanical stretch with acute pressure overload without or with the addition of the stretch blocker gadolinium. Infarct size was determined after 2h reperfusion and p38 MAPK and JNKs phosphorylation was determined after 20 min of prolonged ischemia. Preconditioning stimuli were equally effective in reducing the infarct size (14.2+/-3.4%, 12.9+/-3.0%, 15.9+/-3.3%, P<0.01 vs control). The addition of the stretch channel blocker gadolinium abrogated the effect of stretch preconditioning only, without any effect on ischemic preconditioning. Comparing p38-MAPK and p46/p54 JNKs phosphorylation in the ischemic and non-ischemic regions of the heart at the time of sustained ischemia, activation was observed in the ischemic or mechanically preconditioned groups compared with the control. The addition of gadolinium abolished this activation. The above results indicate that the phosphorylation of p38-MAPK and p46/p54 JNKs is increased in preconditioning but this effect can be dissociated from the protective effect of ischemic preconditioning. Activation of the stress-activated protein kinases may be related to the increased contracture, a characteristic of ischemic preconditioning.     

p38 MAP kinase is a mediator of ischemic preconditioning in pigs

OBJECTIVE: The role of p38MAPK in ischemic preconditioning (IP) is still equivocal, insofar as the p38MAPK-inhibitor SB203580 abolished IP in rats, rabbits and dogs, but not in pigs. Blockade of p38MAPK prior to the sustained ischemia also generated contradictory findings, insofar as p38MAPK acted as trigger in dogs but as mediator in rats. We have now tested whether the two structurally unrelated p38MAPK-inhibitors, BIX-645 and SB203580, abolished infarct size (IS) reduction by IP in pigs and whether their effects depended on the time of administration. METHODS: Sixty-five enflurane-anesthetized pigs underwent 90 min low-flow ischemia and 120 min reperfusion without or with one preceding cycle of 10 min preconditioning ischemia and 15 min reperfusion. Pigs received BIX-645 (1 mg/kg, i.v.) or SB203580 (10 microM, i.c.) prior to either IP or the sustained ischemia. RESULTS: IS (% TTC-staining) was reduced by IP [4.8+/-3.1(S.E.M.), P<0.05] compared to placebo (25.8+/-5.5). BIX-645 or SB203580 per se had no effect on IS (23.5+/-5.2 and 21.8+/-4.4, respectively). IS reduction by IP was abolished by BIX-645 (26.2+/-6.4 or 25.5+/-4.7) and SB203580 (19.9+/-4.3 or 16.7+/-4.7), given either prior to IP or the sustained ischemia, respectively. The supernatant of homogenized myocardial biopsies taken during the sustained ischemia from preconditioned pigs receiving either BIX-645 or SB203580 inhibited the anisomycin-stimulated ATF-2 phosphorylation in cultured Rat1 fibroblasts. This in vitro inhibition of ATF-2 phosphorylation correlated to the actual IS. CONCLUSION: The attenuation of the IS-reducing effect of IP depends on the effectiveness of blockade of p38MAPK activity. p38MAPK is a mediator of IP in pigs.     

Protein kinase C-dependent activation of P44／42 mitogen-activated protein kinase and heat shock protein 70 in signal transduction during hepatocyte ischemic preconditioning

AIM: To investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (MAPKs) and heat shock protein (HSP)70 signal transduction during hepatocyte ischemic preconditioning. METHODS: In this study we used an in vitro ischemic preconditioning (IP) model for hepatocytes and an in vivo model for rat liver to investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (P44/42 MAPKs) and heat shock protein 70 (HSP70) signal transduction in IP. Through a normal liver cell hypoxic preconditioning (HP) model in which cultured normal liver cells were subjected to 3 cycles of 5 rain of incubation under hypoxic conditions followed by 5 rain of reoxygenation and subsequently exposed to hypoxia and reoxygenation for 6 h and 9 h respectively. PKC inhibitor, activator and MEK inhibitor were utilized to analyze the phosphorylation of PKC, the expression of P44/42 MAPKs and HSP70. Viability and cellular ultrastructure were also observed. By using rat liver as an in vivo model of liver preconditioning (3 cycles of 10-min occlusion and 10-min reperfusion), in vivo phosphorylation of PKC and P44/42MAPKs, HSP70 expression were further analyzed. AST/ALT concentration, cellular structure and ultrastruture were also observed. All the data were statistically analyzed. RESULTS: Similar results were obtained in both in vivo and in vitro IP models. Compared with the control withouts IP (or HP), the phosphorylation of PKC and P44/42 MAPKs and the expression of HSP70 were obviously increased in IP (or HP) treated model in which cytoprotection could be found. The effects of preconditioning were mimicked by stimulating PKC with 4β phorobol-12-myristate 13-acetate (PMA). Conversely, inhibiting PKC with chelerythrine abolished the protection given by preconditioning. PD98059, inhibitor of MEK (the upstream kinase of P44/42MAPKs), also reverted the cytoprotection exerted by preconditioning. CONCLUSION: The results demonstrate that preconditioning induces a rapid activation of P44/421VlAPKs and PKC activation plays a pivotal role in the activation of P44/42 MAPKs pathway that participates in the preservation of liver cells. HSP expression is regulated by signals in PKC dependent P44/42 MAPKs pathway.     

Classic ischemic but not pharmacologic preconditioning is abrogated following genetic ablation of the TNFalpha gene

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) is known to mimic ischemic preconditioning (IP). However, it is not known whether TNFalpha-preconditioning is mediated by 'established' preconditioning signaling or via novel signaling cascades. Moreover, whether TNFalpha is required to induce the ischemic preconditioning phenotype has not been determined. METHODS: To evaluate the role of TNFalpha, we determined the infarct-sparing effect of IP comparing TNFalpha null (TNFalpha-/-) and wild-type mice. The IP protocol included 4x5 min ischemia/reperfusion (I/R) prior to the index 35 min of global ischemia followed by 45 min of reperfusion in isolated perfused murine hearts. Infarct size was measured as a percentage of cardiac volume. To evoke particular signaling pathways numerous pharmacologic studies were performed. RESULTS: Following IP, infarct size was significantly reduced by 43% in wild-type mice. In contrast, infarct size was not attenuated by IP in the TNFalpha-/- group versus I/R controls (Infarct size-36+/-3%). Interestingly, pharmacologic preconditioning with adenosine (100 microM) and diazoxide (30 microM) mimicked IP in both the wild-type (infarct size-11+/-4% and 18+/-2%) and in TNFalpha-/- mice (infarct size-15+/-4% and 23+/-3%) versus respective I/R controls. Recombinant TNFalpha (0.5 ng/ml) administered for 7 min followed by a 10-min washout mimicked IP in wild-type mice but not in the TNFalpha deficient mouse hearts. The cardioprotective effects of IP, adenosine and TNFalpha were abolished by the co-administration of the putative mitochondrial K(ATP) blocker 5-hydroxydecanoate. CONCLUSIONS: We demonstrate that cardiac TNFalpha production is required for ischemic preconditioning-induced cardioprotection but not necessary in pharmacologic preconditioning with adenosine or diazoxide in TNFalpha-/- mice. Moreover, TNFalpha administration is sufficient to activate preconditioning in wild-type mice. Finally, as 5-hydroxydecanoate abrogates ischemic, adenosine and TNFalpha induced preconditioning, this suggests that diverse signaling pathways converge at the level of mitochondrial K(ATP) channel activation to mediate this cardioprotection.     

Involvement of p44/p42 MAP kinase in insulin-like growth factor-I-induced alkaline phosphatase activity in osteoblast-like-MC3T3-E1 cells

It has been shown that insulin-like growth factor-I (IGF-I) stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated the involvement of the mitogen-activated protein (MAP) kinase superfamily in the IGF-I-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. IGF-I-stimulated alkaline phosphatase activity dose dependently in the range between 1 nM and 0.1 microM. IGF-I induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase but not stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). PD98059 and U0126, specific inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly suppressed the IGF-I-induced alkaline phosphatase activity. On the contrary, SB203580 and PD169316, specific inhibitors of p38 MAP kinase, failed to affect the activity induced by IGF-I. Specific inhibitors for phosphatidylinositol 3-kinase (PI3K)/Akt pathway (LY294002 and wortmannin) also had no significant effect on IGF-I-induced p44/p42 MAP kinase phosphorylation. The phosphorylation of p44/p42 MAP kinase induced by IGF-I was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase among the MAP kinase superfamily plays a role in the IGF-I-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells.     

Remifentanil limits infarct size but attenuates preconditioning-induced infarct limitation

OBJECTIVES: We investigated the influence of the narcotic anesthetic remifentanil on irreversible myocardial ischemic injury. METHODS: New Zealand White rabbits were anesthetized with propofol (0.7-1.8 mg.kg.min) and then subjected to 30 min regional myocardial ischemia and 3 h reperfusion (CON). Some animals also underwent ischemic preconditioning, elicited by either one (IP1) or two (IP2) cycles of 5 min ischemia and 5 min reperfusion, and/or remifentanil, administered either as a transient infusion mimicking the preconditioning protocol (RP2, 10 microg x kg x min) or as a continuous infusion (R, 3-10 microg x kg x min). Rabbits were randomly assigned to experimental groups. Infarct size was assessed with tetrazolium. Results are reported as mean+/-SD. RESULTS: Non-preconditioned infarct size was approximately 50% of the area-at-risk (49.6+/-20.1% CON). Both one and two cycles of ischemic preconditioning markedly reduced infarct size (49.6+/-20.1% CON versus 18.6+/-8.6% IP and versus 7.5+/-7.6% IP2; both p<0.001). Preconditioning with remifentanil modestly reduced infarct size (49.6+/-20.1% CON versus 29.3+/-8.5% RP2; p<0.01). However, sustained administration of remifentanil did not provide protection (49.6+/-20.1% CON versus 43.9+/-16.2% R), and it attenuated the protection offered by preconditioning (49.6+/-20.1% CON versus 35.6+/-20.7% R+IP1, p=NS; and versus 14.5+/-14.5% R+IP2; p<0.05). CONCLUSION: Transient pre-ischemic administration of remifentanil modestly reduces infarct size in propofol-anesthetized rabbits, but continuous administration of remifentanil increases the threshold for ischemic preconditioning-induced infarct limitation.     

Opioid-induced cardioprotection occurs via glycogen synthase kinase beta inhibition during reperfusion in intact rat hearts

Glycogen synthase kinase (GSK) inhibition produced by ischemic preconditioning has been previously shown to be regulated through phosphatidylinositol-3 kinase (PI3K). Therefore, we determined whether opioid-induced cardioprotection (OIC) occurs during reperfusion by altering GSK phosphorylation through PI3K and target of rapamycin (TOR). Furthermore, we determined if selective GSK inhibitors, SB216763(SB21) or SB415286(SB41), emulate OIC. Rats were treated with the nonselective opioid agonist, morphine (MOR, 0.3 mg/kg), the delta-selective opioid agonist BW373U86 (BW, 1 mg/kg), or the GSK inhibitors, SB21 (0.6 mg/kg) or SB41(1.0 mg/kg), either 10 minutes before ischemia or 5 minutes before reperfusion. Five minutes before opioid or SB21 treatment, some rats received either the PI3K inhibitor wortmannin (15 microg/kg) or LY294002 (0.3 mg/kg) or the TOR inhibitor rapamycin (3 microg/kg). After 30 minutes of ischemia followed by 2 hours of reperfusion, infarct size was assessed. MOR, BW, SB41, and SB21 reduced infarct size compared with vehicle when administered before ischemia (42.9+/-2.6, 40.3+/-2.3, 46.6+/-1.6, 42.2+/-1.8 versus 60.0+/-1.1%, respectively; P<0.001) and showed similar protection when administered 5 minutes before reperfusion (43.6+/-2.3, 40.2+/-2.6, 44.8+/-2.8, 39.4+/-0.8%, respectively; P<0.001). Wortmannin, LY294002, and rapamycin were found to inhibit OIC; however, they did not abrogate SB21-induced infarct size reduction. At 5 minutes of reperfusion, both MOR and BW increased P-GSKbeta at Ser9 in the ischemic zone compared with vehicle (181+/-20, 178+/-15 versus 75+/-17 DU, respectively; P<0.05), and this effect was abrogated by prior administration of wortmannin or rapamycin in MOR-treated rats. Furthermore, no differences were seen in phosphorylation of GSKalpha (Ser21 or Tyr279) or phosphorylation of GSKbeta (Tyr216). These data indicate that OIC occurs via the phosphorylation of GSKbeta at Ser9 during reperfusion.     

NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase

The A₁/A₂ adenosine agonist 5′-(N-ethylcarboxamido) adenosine (NECA) limits infarction when administered at reperfusion. The present study investigated whether p70S6 kinase is involved in this anti-infarct effect. Adult rat ventricular myocytes were isolated and incubated in tetramethylrhodamine ethyl ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction in TMRE fluorescence serves as an indicator of collapse of the mitochondrial transmembrane potential. Cells were subjected to H₂O₂ (200 μM), which like ischemia induces loss of mitochondrial membrane potential. Fluorescence was measured every 3 min and to facilitate quantification membrane potential was arbitrarily considered as collapsed when fluorescence reached less than 60% of the starting value. Adding NECA (1 mM) to the cells prolonged the time to fluorescence loss (48.0 ± 3.2 min in the NECA group versus 29.5 ± 2.2 min in untreated cells, P < 0.001) and the mTOR/p70S6 kinase inhibitor rapamycin (5 nM) abolished this protection (31.3 ± 3.4 min). Since cyclosporine A offered similar protection, mitochondrial permeability transition pore formation is a likely cause of the H₂O₂-induced loss of potential. The direct GSK-3β inhibitor SB216763 (3 μM) also prolonged the time to fluorescence loss (49.2 ± 2.1 min, P < 0.001 versus control), and its protection could not be blocked by rapamycin (42.2 ± 2.3 min, P < 0.001 versus control). NECA treatment (100 nM) of intact isolated rabbit hearts at reperfusion after 30 min of regional ischemia decreased infarct size from 33.0 ± 3.8% of the risk zone in control hearts to 11.8 ± 2.0% (P < 0.001), and rapamycin blocked this NECA-induced protection (38.3 ± 3.7%). A comparable protective effect was seen for SB216763 (1 μM) with infarct size reduction to 13.5 ± 2.3% (P < 0.001). NECA treatment (200 nM) of intact rabbit hearts at reperfusion also resulted in phosphorylation of p70S6 kinase more than that seen in untreated hearts. This NECA-induced phosphorylation was blocked by rapamycin. These experiments reveal a critical role for p70S6 kinase in the signaling pathway of NECA’s cardioprotection at reperfusion. Returned for 1st revision: 3 November 2005 1st revision received: 3 February 2006 Returned for 2nd revision: 23 February 2006 2nd revision received: 1 March 2006     

Apelin-13 and apelin-36 exhibit direct cardioprotective activity against ischemiareperfusion injury

Protection against myocardial ischemia-reperfusion (I/R) injury involves activation of phosphatidylinositol-3-OH kinase (PI3K)- Akt/protein kinase B and p44/42 mitogen-activated protein kinase (MAPK), components of the reperfusion injury salvage kinase (RISK) pathway. The adipocytokine, apelin, activates PI3K-Akt and p44/42 in various tissues and we, therefore, hypothesised that it might demonstrate cardioprotective activity. Employing both in vivo (open-chest) and in vitro (Langendorff and cardiomyocytes) rodent (mouse and rat) models ofmyocardial I/R injury we investigated if apelin administered at reperfusion at concentrations akin to pharmacological doses possesses cardioprotective activity. Apelin-13 and the physiologically less potent peptide, apelin-36, decreased infarct size in vitro by 39.6% (p<0.01) and 26.1% (p<0.05) respectively. In vivo apelin-13 and apelin-36 reduced infarct size by 43.1% (p<0.01) and 32.7% (p<0.05). LY294002 and UO126, inhibitors of PI3K-Akt and p44/42 phosphorylation respectively, abolished the protective effects of apelin-13 in vitro.Western blot analysis provided further evidence for the involvement of PI3K-Akt and p44/42 in the cardioprotective actions of apelin. In addition,mitochondrial permeability transition pore (MPTP) opening was delayed by both apelin- 13 (127%, p<0.01) and apelin-36 (93%, p<0.01) which, in the case of apelin-13, was inhibited by LY294002 and mitogen-activated protein kinase kinase (MEK) inhibitor 1. This is the first study to yield evidence that the adipocytokine, apelin, produces direct cardioprotective actions involving the RISK pathway and the MPTP.     

Ischemic preconditioning: a potential role for constitutive low molecular weight stress protein translocation and phosphorylation?

We have investigated whether translocation of constitutive low molecular weight stress proteins (alphaB-crystallin and HSP27) to the myofilament/cytoskeletal compartment occurs during ischemic preconditioning and assessed if this is causally associated with cardioprotection. Triton-insoluble preparations from fresh or aerobically perfused rat hearts (n=4/group) contained relatively little alphaB-crystallin (96 +/- 43 and 43 +/- 36 units respectively) or HSP27 (177 +/- 32 and 101 +/- 26 units respectively). Three preconditioning cycles of (5 min ischemia + 5 min reperfusion) increased the Triton-insoluble crystallin to 864 +/- 61 units (P<0.05) and HSP27 to 1353 +/- 53 units (P<0.05). Two hours of aerobic perfusion following the preconditioning protocol resulted the return of alphaB-crystallin and HSP27 to near control levels (189 +/- 14 units and 252 +/- 24 units, respectively). Stress protein translocation, comparable to that achieved by the IPC protocol was induced by aerobic perfusion with hypercarbic (pH 6.8) perfusion. Thus, three cycles of 5 min hypercarbia + 5 min normocarbia increased alphaB-crystallin to 628 +/- 30 units (P<0.05) and HSP27 to 1353 +/- 53 units. In parallel functional studies, the recovery of LVDP after 35 min ischemia and 60 min of reperfusion was 43 +/- 7% in the ischemic control group, 61 +/- 3% (P<0.05) in the preconditioned group and 42 +/- 6% in the hypercarbic group. Thus, translocation of alphaB-crystallin and/or is not of-itself sufficient to induce cardioprotection. Using a phospho-specific antibody, we have demonstrated that preconditioning not only translocates alphaB-crystallin but also increases its phosphorylation at Ser-59 by 9.7-fold compared to aerobic controls (1616 +/- 402 v 166 +/- 28 units respectively). In contrast, hypercarbia while eliciting a comparable translocation, failed to alter the phosphorylation state of alphaB-crystallin. Preconditioning-induced phosphorylation was significantly attenuated by 50 microM genistein (by 61%), 10 microM SB203580 (by 91%) and 10 microM bisindolylmaleimide (by 68%), but not by 10 microM PD98059 (by 4%). Our findings are consistent with the possibility that ischemic preconditioning may be mediated by phosphorylation and translocation of constitutive low molecular weight stress proteins, particularly alphaB-crystallin.     

Ischemic postconditioning protects remodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvage kinase pathway

OBJECTIVE: We tested whether ischemic postconditioning (IPostC) is protective in remodeled myocardium. METHODS: Post-myocardial infarct (MI)-remodeled hearts after permanent coronary artery ligation and one kidney one clip (1K1C) hypertensive hearts of male Wistar rats were exposed to 40 min of ischemia followed by 90 min of reperfusion. IPostC was induced by six cycles of 10 s reperfusion interspersed by 10 s of no-flow ischemia. Activation of reperfusion injury salvage kinases was measured using Western blotting and in vitro kinase activity assays. RESULTS: IPostC prevented myocardial damage in both MI-remodeled and 1K1C hearts, as measured by decreased infarct size and lactate dehydrogenase release, and improved function. The reduction in infarct size and the recovery of left ventricular contractility achieved by IPostC was less in 1K1C hearts, but was unchanged in MI-remodeled hearts when compared to healthy hearts. In contrast, the recovery of inotropy was unaffected in 1K1C hearts, but was less in MI-remodeled hearts. Inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway with LY294002 abolished the protective effects of IPostC on both disease models and healthy hearts. Western blot analysis in conjunction with in vitro kinase activity assays identified protein kinase B (PKB)/Akt but not p42/p44 extracellular-signal regulated kinase 1/2 (ERK1/2) as the predominant kinase in IPostC-mediated cardioprotection in remodeled hearts. IPostC increased phosphorylation of the PKB/Akt downstream targets eNOS, GSK3beta, and p70S6K in remodeled hearts. CONCLUSION: Our results offer evidence that IPostC mediates cardioprotection in the remodeled rat myocardium primarily via activation of the PI3K-PKB/Akt reperfusion injury salvage kinase pathway.     

Second window of protection following myocardial preconditioning: an essential role for PI3 kinase and p70S6 kinase

Ischaemic preconditioning (IPC) protects the heart against myocardial infarction acutely as well as several hours later (e.g. 24-48 h). The mechanism of the profound cardioprotection is not completely explored. We hypothesized that PI3K/PDK1/Akt/mTOR/p70S6K-mediated pro-survival pathway is involved in delayed cardioprotection induced by IPC. Under Hypnorm-Diazepam anaesthesia, male New Zealand White rabbits were either sham-operated (SC) or preconditioned by four cycles of 5-min ischaemia and 10-min reperfusion on day 1. Twenty-four hours after recovery, the animals were anaesthetized with sodium pentobarbitone and subjected to 30-min ischaemia followed by 180-min reperfusion. Wortmannin (0.6 mg/kg, i.v.), an irreversible PI3 kinase (PI3K) inhibitor, rapamycin (0.25 mg/kg, i.v.), which prevents the phosphorylation of p70S6 kinase (p70S6K), or DMSO (control vehicle) was given 15 min prior to IPC. IPC significantly reduced infarct size compared to the control group (SC) (31.9 +/- 5.8% (n = 7) vs. 54.9 +/- 2.9% (n = 6), P < 0.05). Wortmannin and rapamycin alone had no effect on infarct size (56.3 +/- 1.6% (n = 6) and 54.7 +/- 3.8% (n = 6), respectively). However, when wortmannin or rapamycin were given prior to IPC the protection was completely abolished (49.9 +/- 2.8% (n = 6), 45.1 +/- 4.6% (n = 7), P < 0.05 vs. IPC). Western blot analysis showed that wortmannin, at a dose of 0.6 mg/kg, and rapamycin, at a dose of 0.25 mg/kg, were sufficient to prevent phosphorylation of Akt and p70S6K, respectively, when the inhibitors were given prior to IPC. We conclude that PI3K/PDK1/Akt/mTOR/p70S6K-signalling pathway plays an essential role in the development of the cardioprotection against infarction in rabbits.     

Involvement of both phosphatidylinositol 3-kinase and p44/p42 mitogen-activated protein kinase pathways in the short-term regulation of pyruvate kinase L by insulin

Pyruvate kinase L (PK-L) is a key regulatory enzyme of the hepatic glycolytic/gluconeogenic pathway that can be dephosphorylated and activated in response to insulin. However, the signaling cascades involved in this insulin effect have not been established. In this work we have investigated the potential involvement of phosphatidylinositol 3-kinase (PI 3-K) and p44/p42 mitogen-activated protein kinase (MAPK) pathways in the short-term modulation of PK-L by insulin in primary cultures of rat hepatocytes. Wortmannin, at a concentration of 100 nM, caused a marked inhibition of the PI 3-K/protein kinase B pathway, which became complete at 500 nM wortmannin. Likewise, wortmannin at 100 and 500 nM, elicited partial and total inhibitions of insulin-mediated activation of PK-L, respectively. However, this PI 3-K inhibitor also reduced insulin-mediated phosphorylation of p44/p42 MAPK in cultured rat hepatocytes, indicating that both the PI 3-K and MAPK pathways could be involved in PK-L activation by insulin. Three facts appear to reinforce this hypothesis: 1) the selective and complete inhibition of the PI 3-K/protein kinase B pathway by LY294002 (50 microM) was accompanied by a partial blockade of insulin-induced PK-L activation; 2) when signaling through the MAPK cascade was selectively suppressed by the presence of PD98059 (50 microM), a 50% reduction of insulin-induced activation of PK-L was observed; and 3) the effect of PD98059 (50 microM) on PK-L activation was reinforced by the additional presence of 100 nM wortmannin. We also observed that the blockade of p70 S6-kinase by rapamycin did not affect the activation of PK-L by insulin. From these findings it can be concluded that both PI 3-K and MAPK pathways, but not p70 S6-kinase, are involved in the short-term activation of PK-L by insulin in rat hepatocytes.     

Role of microtubules in ischemic preconditioning against myocardial infarction

OBJECTIVE: The role of microtubules in ischemic preconditioning (PC) was investigated in isolated perfused rabbit hearts. METHODS: Myocardial infarction was induced by 30-min global ischemia and 2-h reperfusion, and infarct size was expressed as a percentage of the left ventricle (%IS/LV). Using separate groups of rabbits, ventricular biopsies were taken before and after PC for determination of protein kinase C (PKC) translocation and p38-mitogen-activated protein kinase (p38MAP kinase) activation. To depolymerize microtubules, we used two structurally different agents, colchicine (50 microM) and nocodazole (1 microM). RESULTS: PC with two cycles of 5-min ischemia/5-min reperfusion significantly reduced infarct size from 60.1+/-5.0% to 20.0+/-5.0%. Although neither colchicine nor nocodazole modified infarct size in nonpreconditioned hearts, these agents abolished the infarct size-limiting effects of PC (%IS/LV=56.1+/-6.0% and 53.5+/-2.5%, respectively). Colchicine prevented translocation of PKC-epsilon and p38MAP kinase activation by PC. PKC translocation by infusion of 1-oleyl-2-acetyl-sn-glycerol in nonischemic hearts was also prevented by colchicine. CONCLUSION: Microtubules play a crucial role in the development of anti-infarct tolerance by PC as a mechanism supporting translocation of activated PKC.