Is this truly ischemic preconditioning?
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Animals↗
Biomedical subjects
Mihaela M Mocanu
Publications and source records attributed to Mihaela M Mocanu.
A potential role for PTEN in the diabetic heart.
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Animals↗
Failure to protect the myocardium against ischemia/reperfusion injury after chronic atorvastatin treatment is recaptured by acute atorvastatin treatment: a potential role for phosphatase and tensin homolog deleted on chromosome ten?
OBJECTIVES: We sought to ascertain whether chronic oral therapy with atorvastatin protects against ischemia/reperfusion (I/R) injury. BACKGROUND: We have recently shown that acute atorvastatin treatment protects against reperfusion-induced injury by activating the PI3K/Akt/eNOS pathway. However, many patients are on chronic statin therapy, and it is necessary to investigate whether this, in itself, provides a therapeutic advantage. METHODS: Sprague-Dawley rats were orally treated for one day, three days, one week, or two weeks with 20 mg/kg of atorvastatin or vehicle, after which the hearts underwent 35 min of ischemia and 120 min reperfusion (IR). Two additional groups were treated for one or two weeks with atorvastatin and then received a supplementary dose of 40 mg/kg before IR. The risk zone was determined using Evans blue and infarct size (IR%) using triphenyltetrazolium chloride staining. RESULTS: Treatment with atorvastatin for one and three days significantly reduced infarct size versus controls (38.9 +/- 3.1% vs. 56.4 +/- 2.3%; 39.3 +/- 2.4% vs. 61.3 +/- 3.8%, respectively). However, after one or two weeks of treatment, no protection was observed (52.6 +/- 3.8% vs. 58.6 +/- 4.3%; 58.3 +/- 2.7% vs. 52.4 +/- 5.7%, respectively). Surprisingly, a supplementary dose of atorvastatin recaptured the protection in the groups treated chronically (36.2 +/- 2.8% vs. 58.6 +/- 4.3%; 26.8 +/- 1.5% vs. 51.2 +/- 6.7%, at one and two weeks, respectively). Interestingly, we observed an increased level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), the phosphatidylinositol-3 kinase inhibitor, in the chronic treated hearts. CONCLUSIONS: In conclusion, atorvastatin appears to have an acute protective effect that wanes with time associated with an increase in PTEN levels. This waning protection can be recaptured by an acute high dose given immediately before IR. These results may have protential clinical relevance.
Animals↗
Glucagon like peptide-1 is protective against myocardial ischemia/reperfusion injury when given either as a preconditioning mimetic or at reperfusion in an isolated rat heart model.
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Animals↗
Atorvastatin and myocardial reperfusion injury: new pleiotropic effect implicating multiple prosurvival signaling.
We investigated the potential role of atorvastatin, given at reperfusion, to improve survival of the ischemic/reperfused myocardium by activation of p44/42 MAPK and p38 MAPK with its downstream effector, HSP27. We have previously shown that atorvastatin attenuates lethal reperfusion-induced injury via activation of the phosphatidyl inositol 3-kinase (PI3K) prosurvival signaling pathway. In this study we hypothesize that other prosurvival kinases may also be implicated in this protection. Langendorff-perfused mouse hearts were subjected to 35 minutes of global ischemia followed by 30 minutes of reperfusion, and either infarct size or the levels of phosphorylated AKT, p44/42 MAPK, p38 MAPK, and HSP27 were analyzed. Atorvastatin was administered during reperfusion only. We used wortmannin to block PI3K/AKT, U0126 to block p44/42 MAPK, and SB203580 to prevent the phosphorylation of p38 MAPK and HSP27. Atorvastatin significantly reduced infarct size (32.96 +/- 3.4% versus 51.27 +/- 2.79% in controls, P < 0.05). This protection was abrogated by wortmannin (48.38 +/- 4.28%), U0126 (52.58 +/- 7.58), and SB203580 (49.37 +/- 4.16%). Western blot analysis confirmed significant phosphorylation of AKT, p44/42 MAPK, p38 MAPK, and HSP27 following administration of atorvastatin during reperfusion and abrogation of the respective phosphorylation in the presence of their specific inhibitors. Atorvastatin given at reperfusion attenuates lethal reperfusion-induced injury by the phosphorylation of multiple prosurvival pathways involving not only PI3K/AKT but also p44/42 MAPK, p38 MAPK, and HSP27.
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Pioglitazone mimics preconditioning in the isolated perfused rat heart: a role for the prosurvival kinases PI3K and P42/44MAPK.
Ischemic preconditioning, the most powerful protection against infarction, activates PI3Kinase (PI3K)/AKT and P42/44MAPK. Pioglitazone, a thiazolidinedione and PPARgamma receptor agonist used in Type II diabetes treatment, has been shown to activate these kinase cascades. We therefore hypothesized that pioglitazone could protect the myocardium when given prior to myocardial ischemia/reperfusion injury. Langendorff perfused rat hearts underwent 40 minutes of stabilization then 35 minutes of regional ischemia and 120 minutes of reperfusion (control) or Pioglitazone (1, 2, 5, and 10 microM)-given before ischemia. Additional groups underwent the same protocol but with either PI3K inhibitors (15 microM LY294002 or 100 nM wortmannin) or P42/44MAPK inhibitors (10 microM U0126 or 10 microM PD98059) given either during stabilization or at reperfusion. Infarct size was determined as a percentage of risk zone (I/R%). Pioglitazone (2 microM) significantly reduced I/R% compared with control (25.4 +/- 3.1 versus 47.3 +/- 3.4; P < 0.05). This protection was abolished by PI3K inhibitors (pioglitazone+LY294002 46.5 +/- 5.0, pioglitazone + wortmannin 48.8 +/- 4.6 versus pioglitazone alone 25.4 +/- 3.1; P < or = 0.05) but not by P42/44MAPK inhibitors (pioglitazone+U0126 30.7 +/- 5.7, pioglitazone + PD98059 28.5 +/- 6.3 versus pioglitazone alone 25.4 +/- 3.1; P < or = 0.05) given in stabilization. However when the inhibitors were given at reperfusion, the protection was abrogated by blocking either pathway (pioglitazone+LY294002 49.8 +/- 3.1, pioglitazone+U0126 48.7 +/- 3.7 versus pioglitazone alone 25.4 +/- 3.1; P < or = 0.05). In conclusion pioglitazone induced significant protection against ischemia/reperfusion injury when administered prior to ischemia. This protection appears to involve PI3K and P42/44MAPK.
Animals↗
Glucagon-like peptide 1 can directly protect the heart against ischemia/reperfusion injury.
Glucagon-like peptide 1 (GLP-1), a gut incretin hormone that stimulates insulin secretion, also activates antiapoptotic signaling pathways such as phosphoinositide 3-kinase and mitogen-activated protein kinase in pancreatic and insulinoma cells. Since these kinases have been shown to protect against myocardial injury, we hypothesized that GLP-1 could directly protect the heart against such injury via these prosurvival signaling pathways. Both isolated perfused rat heart and whole animal models of ischemia/reperfusion were used, with infarct size measured as the end point of injury. In both studies, GLP-1 added before ischemia demonstrated a significant reduction in infarction compared with the valine pyrrolidide (an inhibitor of its breakdown) or saline groups. This protection was abolished in the in vitro hearts by the GLP-1 receptor antagonist exendin (9-39), the cAMP inhibitor Rp-cAMP, the PI3kinase inhibitor LY294002, and the p42/44 mitogen-activated protein kinase inhibitor UO126. Western blot analysis demonstrated the phosphorylation of the proapoptotic peptide BAD in the GLP-1-treated groups. We show for the first time that GLP-1 protects against myocardial infarction in the isolated and intact rat heart. This protection appears to involve activating multiple prosurvival kinases. This finding may represent a new therapeutic potential for this class of drug currently undergoing clinical trials in the treatment of type 2 diabetes.
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Preconditioning the diabetic heart: the importance of Akt phosphorylation.
Conflicting evidence exists whether diabetic myocardium can be protected by ischemic preconditioning (IPC). The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is important in IPC. However, components of this cascade have been found to be defective in diabetes. We hypothesize that IPC in diabetic hearts depends on intact signaling through the PI3K-Akt pathway to reduce myocardial injury. Isolated perfused Wistar (normal) and Goto-Kakizaki (diabetic) rat hearts were subjected to 1) 35 min of regional ischemia and 120 min of reperfusion with infarct size determined; 2) preconditioning (IPC) using 5 min of global ischemia followed by 10 min of reperfusion performed one, two, or three times before prolonged ischemia; or 3) determination of Akt phosphorylation after stabilization or after one and three cycles of IPC. In Wistar rats, one, two, and three cycles of IPC reduced infarct size 44.7 +/- 3.8% (P < 0.05), 31.4 +/- 4.9% (P < 0.01), and 34.3 +/- 6.1% (P < 0.01), respectively, compared with controls (60.7 +/- 4.5%). However, in diabetic rats only three cycles of IPC significantly reduced infarction to 20.8 +/- 2.6% from 46.6 +/- 5.2% in controls (P < 0.01), commensurate with significant Akt phosphorylation after three cycles of IPC. To protect the diabetic myocardium, it appears necessary to increase the IPC stimulus to achieve the threshold for cardioprotection and a critical level of Akt phosphorylation to mediate myocardial protection.
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Ischemic preconditioning protects by activating prosurvival kinases at reperfusion.
Pharmacological activation of the prosurvival kinases Akt and ERK-1/2 at reperfusion, after a period of lethal ischemia, protects the heart against ischemia-reperfusion injury. We hypothesized that ischemic preconditioning (IPC) protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion. In isolated perfused Sprague-Dawley rat hearts subjected to 35 min of lethal ischemia, the phosphorylation states of Akt, ERK-1/2, and p70 S6 kinase (p70S6K) were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a biphasic response in Akt and ERK-1/2 phosphorylation during the preconditioning and reperfusion phases after the period of lethal ischemia. IPC induced a fourfold increase in Akt, ERK-1/2, and p70S6K phosphorylation at reperfusion and reduced the infarct risk-to-volume ratio (56.9 +/- 5.7 and 20.9 +/- 3.6% for control and IPC, respectively, P < 0.01). Inhibiting the IPC-induced phosphorylation of Akt, ERK-1/2, and p70S6K at reperfusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 or the MEK-1/2 inhibitor PD-98059 abrogated IPC-induced protection (46.3 +/- 5.8, 49.2 +/- 4.0, and 20.9 +/- 3.6% for IPC + LY-294002, IPC + PD-98059, and IPC, respectively, P < 0.01), demonstrating that the phosphorylation of these kinases at reperfusion is required for IPC-induced protection. In conclusion, we demonstrate that the reperfusion phase following sustained ischemia plays an essential role in mediating IPC-induced protection. Specifically, we demonstrate that IPC protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion.
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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.
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Heat shock protein 27 protects the heart against myocardial infarction.
Heat shock proteins (hsp) represent a group of chaperones which protects the cells against a diversity of stresses. It has been demonstrated that hsp27 is constitutively present in cells where it plays an important role in different cytoprotective processes which ultimately inhibit cell death. We investigated the response of the isolated perfused mouse heart over expressing hsp27 to the ischaemia/reperfusion injury using infarct size as an end point. Our results show for the first time that mice over expressing hsp27 (verified by Western blotting analysis) were found to be protected from lethal ischaemia/reperfusion injury compared to their negative littermates.
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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.
Androstadienes↗
p53 down-regulation: a new molecular mechanism involved in ischaemic preconditioning.
Ischaemic preconditioning is associated with the activation of prosurvival mechanisms. Here we demonstrate that following a preconditioning protocol, the proapoptotic p53 is inactivated possibly via phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-murine double minute 2 (Mdm2) phosphorylation. Our data show that in preconditioned hearts Mdm2 was significantly phosphorylated, and wortmannin (a PI3K inhibitor) abrogated this effect (Western blotting). Also in preconditioned hearts p53 was shown to be bound to phospho-Mdm2 (co-immunoprecipitation). Furthermore, pifithrin alpha (a p53 inhibitor), administered to isolated perfused hearts prior to ischaemia, significantly attenuated the infarction. In conclusion our results suggest that p53 is implicated in ischaemia/reperfusion injury and that preconditioning counterbalances this effect via PI3K-Akt-Mdm2 phosphorylation.
Androstadienes↗
PI3 kinase and not p42/p44 appears to be implicated in the protection conferred by ischemic preconditioning.
Ischemic preconditioning results in an immediate phase of protection against lethal ischemia/reperfusion injury that is comprised of both irreversible necrosis and programmed cell death, apoptosis. We hypothesized that preconditioning may activate putative anti-apoptotic pathways, through the induction of either phosphatidyl inositol 3-OH kinase (PI3 kinase) or p42/p44 extracellular receptor kinase, attenuating total cell death. Isolated perfused rat hearts were preconditioned with two cycles of 5 min ischemia and 10 min reperfusion. Then they were frozen for Western blot analysis or subjected to 35 min regional ischemia and 120 min reperfusion prior to infarct size assessment. Selective PI3 kinase inhibitors, wortmannin (W, 100 n M) and LY294002 (LY, 15 microM) and the p42/p44 inhibitor, PD 98059 (PD, 10 and 50 microM), were individually infused during the preconditioning protocol. One further group of hearts received both inhibitors (W and PD). The results were expressed as percentage of infarction within the risk zone. Inhibition of PI3 kinase by either W or LY partially abrogated the infarct sparing effect of ischemic preconditioning (I/R%: 44.6+/-2.7 in C, 17.6+/-2.0 in IP, vs 32.2+/-4.2 in W, and 30.9+/-2.6 in LY, P<0.05). Inhibition of ERK phosphorylation however, had no significant effect upon infarct size reduction (17.6+/-2.0 in ischemic preconditioning vs 21.4+/-3.0 in IP+10 microM PD and 15.2+/-1.4 in IP+50 microM PD, P>0.05). Western blot analysis confirmed that PD abrogated the phosphorylation of p42/p44 and LY the phosphorylation of AKT. Combined inhibition with PD+W failed to further attenuate protection (27.6+/-1.3%, P>0.1). These data appear to demonstrate that the PI3 kinase, but not the p42/p44 cascade, is implicated in early ischemic preconditioning.
Androstadienes↗
Adenosine A(3) receptor activation protects the myocardium from reperfusion/reoxygenation injury.
Ischemia-reperfusion induces both necrotic and apoptotic cell death. The ability of adenosine to attenuate reperfusion-induced injury (RI) and the role played by adenosine receptors are unclear. We therefore studied the role of the A(3) receptor (A(3)R) in ameliorating RI using the specific A(3)R agonist 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxi-N-methyl-b-D-ribofuranuronamide (2-Cl-IB-MECA). Isolated rat hearts and cardiomyocytes were subjected to ischemia or simulated ischemia, followed by reperfusion/reoxygenation. The end points were percent infarction/risk zone and annexin-V (apoptosis) and/or propidium iodide positivity (necrosis), respectively. In isolated hearts, 2-Cl-IB-MECA significantly limited infarct size (44.2 +/- 2.7% in control vs. 21.9 +/- 2.4% at 1 nM and 35.8 +/- 3.3% at 0.1 nM, P < 0.05). In isolated myocytes, apoptosis and necrosis were significantly reduced compared with controls (5.7 +/- 2.6% vs. 17.1 +/- 1.3% and 13.7 +/- 2.0% vs. 23.1 +/- 1.5%, respectively, P < 0.0001). In both models, the beneficial effects were abrogated using the A(3)R antagonist MRS-1191. The involvement of A(2a) receptor activation was also examined. This is the first study to demonstrate that A(3)R activation at reperfusion limits myocardial injury in the isolated rat heart and improves survival in isolated myocytes, possibly by antiapoptotic and antinecrotic mechanisms.
Adenosine↗