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PTEN Activity Is Modulated During Ischemia and ReperfusionInvolvement in the Induction and Decay of Preconditioning
Zheqing Cai and Gregg L. Semenza

Circulation Research. 2005;97:1351-1359, originally published December 8, 2005
https://doi.org/10.1161/01.RES.0000195656.52760.30

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PTEN Activity Is Modulated During Ischemia and Reperfusion
Zheqing Cai, Gregg L. Semenza

Circulation Research December 2005, 97 (12) 1351-1359; DOI: https://doi.org/10.1161/01.RES.0000195656.52760.30

By Zheqing Cai and Gregg L. Semenza

Figure 1. Analysis of cardiac protection and signal transduction induced by IPC. A, LVDP was measured in Langendorff-perfused rat hearts under the fol...Show More

Figure 1. Analysis of cardiac protection and signal transduction induced by IPC. A, LVDP was measured in Langendorff-perfused rat hearts under the following conditions: nonischemic hearts (NIS); hearts subjected to I-30/R-45 (I/R); hearts subjected to I-15/R-30 before I/R (IPC); and hearts in which 1 μmol/L wortmannin (WT) was perfused (red bar) during the last 15 minutes of IPC (IPC+WT). *P<0.01 compared with I/R or IPC+WT (n=5). B, Lysates were prepared from nonischemic hearts and hearts that were subjected to I-15/R-30 as an IPC stimulus in the presence or absence of WT. Heart lysates were analyzed for p-Akt, total Akt, p-p70, and β-actin by immunoblot assays. C and D, The levels of p-Akt and p-p70 were quantified by densitometric analysis of immunoblots.Show Less
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PTEN Activity Is Modulated During Ischemia and Reperfusion
Zheqing Cai, Gregg L. Semenza

Circulation Research December 2005, 97 (12) 1351-1359; DOI: https://doi.org/10.1161/01.RES.0000195656.52760.30

By Zheqing Cai and Gregg L. Semenza

Figure 2. Effect of ischemia-reperfusion on PTEN activity and protein levels. A, PTEN activity was determined, in the same heart lysates analyzed in F...Show More

Figure 2. Effect of ischemia-reperfusion on PTEN activity and protein levels. A, PTEN activity was determined, in the same heart lysates analyzed in Figure 1B, by a specific PTEN phosphatase assay. B, PTEN protein levels were quantified by immunoblot assay in the same lysates. *P<0.01 compared with nonischemic hearts (NIS) or IPC plus wortmannin (IPC+WT) (n=3).Show Less
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PTEN Activity Is Modulated During Ischemia and Reperfusion
Zheqing Cai, Gregg L. Semenza

Circulation Research December 2005, 97 (12) 1351-1359; DOI: https://doi.org/10.1161/01.RES.0000195656.52760.30

By Zheqing Cai and Gregg L. Semenza

Figure 3. Effect of prolonged reperfusion on PTEN and Akt activities. A, PTEN phosphatase activity was determined in heart lysates prepared at baselin...Show More

Figure 3. Effect of prolonged reperfusion on PTEN and Akt activities. A, PTEN phosphatase activity was determined in heart lysates prepared at baseline and after 15 minutes of ischemia followed by 30, 60, or 120 minutes of reperfusion. B, Lysates were subjected to immunoblot assays. C, Immunoblot data for PTEN and p-Akt were quantified by densitometry (n=3 to 4).Show Less
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PTEN Activity Is Modulated During Ischemia and Reperfusion
Zheqing Cai, Gregg L. Semenza

Circulation Research December 2005, 97 (12) 1351-1359; DOI: https://doi.org/10.1161/01.RES.0000195656.52760.30

By Zheqing Cai and Gregg L. Semenza

Figure 4. Effect of prolonged reperfusion on cardiac protection. A, LVDP was measured in hearts that were nonischemic (NIS); hearts subjected to I-30/...Show More

Figure 4. Effect of prolonged reperfusion on cardiac protection. A, LVDP was measured in hearts that were nonischemic (NIS); hearts subjected to I-30/R-45 (I/R); subjected to I-15/R-30 before I/R (R-30); and subjected to I-15/R-120 before I/R (R-120) (n=4). B, Heart lysates were prepared and levels of cleaved caspase-3 (c-cas3) and intact PARP were determined by immunoblot assay. C and D, Relative levels of c-cas3 and PARP, respectively, were quantified by densitometric analysis of immunoblots. *P<0.05 compared with nonischemic hearts or R-30 hearts (n=3).Show Less
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PTEN Activity Is Modulated During Ischemia and Reperfusion
Zheqing Cai, Gregg L. Semenza

Circulation Research December 2005, 97 (12) 1351-1359; DOI: https://doi.org/10.1161/01.RES.0000195656.52760.30

By Zheqing Cai and Gregg L. Semenza

Figure 5. Effect of prolonged reperfusion on PTEN and Akt. Hearts were subjected to I-15. Vehicle only (CON) (A) or 1 μmol/L wortmannin (WT) (B) was a...Show More

Figure 5. Effect of prolonged reperfusion on PTEN and Akt. Hearts were subjected to I-15. Vehicle only (CON) (A) or 1 μmol/L wortmannin (WT) (B) was added to perfusates after reperfusion for 15 minutes. After 30 (R-30) or 120 (R-120) minutes of reperfusion, heart lysates were analyzed for p-PTEN, total PTEN, p-Akt, and total Akt by immunoblot assay. C, Total PTEN levels were determined by densitometric analysis of immunoblots. *P<0.01 (n=3). D, Nonischemic heart lysates were subjected to immunoprecipitation (IP) with polyclonal rabbit anti-PTEN or isotype-matched control antibodies followed by immunoblot assay (IB) using an anti-Akt monoclonal antibody.Show Less
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PTEN Activity Is Modulated During Ischemia and Reperfusion
Zheqing Cai, Gregg L. Semenza

Circulation Research December 2005, 97 (12) 1351-1359; DOI: https://doi.org/10.1161/01.RES.0000195656.52760.30

By Zheqing Cai and Gregg L. Semenza

Figure 6. Ischemia-induced dephosphorylation and degradation of PTEN. A, Hearts were subjected to ischemia for the indicated time, and lysates were su...Show More

Figure 6. Ischemia-induced dephosphorylation and degradation of PTEN. A, Hearts were subjected to ischemia for the indicated time, and lysates were subjected to immunoblot assay for PTEN (quantified in graph as band intensity) and β-actin. B, PTEN enzymatic activity was measured in lysates from nonischemic hearts (NIS) or hearts subjected to 30 minutes of ischemia (ISC). *P<0.01 compared with NIS (n=3). C, Immunoblot assay of phosphorylated and total PTEN levels in NIS and hearts subjected to 5 minutes of ischemia (ISC). *P<0.01 compared with NIS (n=3). D, Hearts were perfused with the indicated concentration of okadaic acid for 15 minutes followed by 15 minutes of ischemia. Lysates were subjected to immunoblot assays.Show Less
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PTEN Activity Is Modulated During Ischemia and Reperfusion
Zheqing Cai, Gregg L. Semenza

Circulation Research December 2005, 97 (12) 1351-1359; DOI: https://doi.org/10.1161/01.RES.0000195656.52760.30

By Zheqing Cai and Gregg L. Semenza

Figure 7. Proteasomal degradation of PTEN in the ischemic heart. A, Hearts were perfused with buffer containing the indicated concentration of MG132 f...Show More

Figure 7. Proteasomal degradation of PTEN in the ischemic heart. A, Hearts were perfused with buffer containing the indicated concentration of MG132 for 15 minutes, followed by 15 minutes of ischemia, and lysates were subjected to immunoblot assays. B and C, Hearts were subjected to ischemia after perfusion with vehicle (ISC) or 1 μmol/L MG132 (ISC+MG) and the levels of total (B) and phosphorylated (C) PTEN were quantified by densitometry. *P<0.01 compared with nonischemic (NIS) or ISC+MG hearts (n=3 each). D, LVDP of NIS hearts and hearts subjected to 15 minutes of ischemia (ISC) (blue bar) and 30 minutes of reperfusion (REP) (red bar) after perfusion of MG132 (gray bar) at a concentration of 0, 2, or 4 μmol/L. *P<0.01 for MG4 or MG2 compared with MG0 (n=3 to 4).Show Less
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PTEN Activity Is Modulated During Ischemia and Reperfusion
Zheqing Cai, Gregg L. Semenza

Circulation Research December 2005, 97 (12) 1351-1359; DOI: https://doi.org/10.1161/01.RES.0000195656.52760.30

By Zheqing Cai and Gregg L. Semenza

Figure 8. Oxidation of PTEN by ischemia-reperfusion or H2O2. A, Lysates were prepared from hearts that were nonischemic (T-15) or exposed to 15 minute...Show More

Figure 8. Oxidation of PTEN by ischemia-reperfusion or H2O2. A, Lysates were prepared from hearts that were nonischemic (T-15) or exposed to 15 minutes of ischemia followed by 0 to 30 minutes of reperfusion (T-30 to T-60). Aliquots were treated with iodoacetamide and analyzed by nonreducing (nonred) PAGE, followed by immunoblot assay to identify reduced (Re) and oxidized (Ox) forms of PTEN or were subjected to standard reducing (Red) PAGE followed by immunoblot assays as indicated. B, Ratio of oxidized:total PTEN and phosphorylated:total Akt at each time point was determined by densitometric analysis of immunoblots. C, Hearts were exposed to ischemia (0 to 20 minutes), followed by 5-minute reperfusion. Cell lysates were analyzed for PTEN, p-Akt, and Akt. D, Hearts were perfused with buffer containing 0 to 10 μmol/L H2O2 for 15 minutes. Hearts were homogenized in lysis buffer containing iodoacetamide. Aliquots were analyzed as described above. Dithiothreitol (DTT) was added to an aliquot of heart lysate to a final concentration of 10 mmol/L as a control for reduced PTEN. E, Akt was analyzed in hearts perfused with buffer alone or buffer containing 0.4 mmol/L 2-mercaptoethanol (2-ME) and subjected to I-15/R-5 or I-15/R-30.Show Less
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PTEN Activity Is Modulated During Ischemia and Reperfusion
Zheqing Cai, Gregg L. Semenza

Circulation Research December 2005, 97 (12) 1351-1359; DOI: https://doi.org/10.1161/01.RES.0000195656.52760.30

By Zheqing Cai and Gregg L. Semenza

Figure 1. Analysis of cardiac protection and signal transduction induced by IPC. A, LVDP was measured in Langendorff-perfused rat hearts under the fol...Show More

Figure 1. Analysis of cardiac protection and signal transduction induced by IPC. A, LVDP was measured in Langendorff-perfused rat hearts under the following conditions: nonischemic hearts (NIS); hearts subjected to I-30/R-45 (I/R); hearts subjected to I-15/R-30 before I/R (IPC); and hearts in which 1 μmol/L wortmannin (WT) was perfused (red bar) during the last 15 minutes of IPC (IPC+WT). *P<0.01 compared with I/R or IPC+WT (n=5). B, Lysates were prepared from nonischemic hearts and hearts that were subjected to I-15/R-30 as an IPC stimulus in the presence or absence of WT. Heart lysates were analyzed for p-Akt, total Akt, p-p70, and β-actin by immunoblot assays. C and D, The levels of p-Akt and p-p70 were quantified by densitometric analysis of immunoblots.Show Less