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.