Adiponectin Facilitates Postconditioning Cardioprotection through Both AMPK-Dependent Nuclear and AMPK-Independent Mitochondrial STAT3 Activation

Figures

• 

  Figure 1. Adiponectin activated posthypoxic cardiomyocyte nuclear STAT3 and mitochondrial STAT3, respectively, via AMPK-dependent and AMPK-independent pathways. Isolated cardiomyocytes were subjected to hypoxia-reoxygenation (HR) with globular APN (gAd) in the absence or presence of AMPK inhibitor compound C (CC) or STAT3 inhibitor stattic. (a) Cardiomyocyte death assessed by lactate dehydrogenase (LDH) release. (b) Protein expression of cleaved and total caspase-3. (c) Representative images for TUNEL staining and dihydroethidium (DHE) staining. (d) Cell apoptosis assessed by TUNEL staining. (e) Cellular reactive oxygen species production assessed by DHE staining. (f) Representative images for western blotting. (g–i) Protein expression of phosphorylated and total AMPK, STAT3 (Ser727), and STAT3 (Tyr705). Data are mean ± SEM of two independent experiments each performed in triplicate: ∗p < 0.05 and ∗∗p < 0.01.

• 

  Figure 2. Hypoxic postconditioning (HPo) enhanced mitochondrial STAT3 (Ser727) but not nuclear STAT3 (Tyr705) independent of AMPK activation. Isolated cardiomyocytes were subjected to hypoxia-reoxygenation (HR) with hypoxic postconditioning (HPo) in the absence or presence of AMPK inhibitor compound C (CC) or STAT3 inhibitor stattic. (a) Cardiomyocyte death assessed by lactate dehydrogenase (LDH) release. (b) Protein expression of cleaved and total caspase-3. (c) Representative images for TUNEL staining and dihydroethidium (DHE) staining. (d) Cell apoptosis assessed by TUNEL staining. (e) Cellular reactive oxygen species production assessed by DHE staining. (f) Representative images for western blotting. (g–i) Protein expression of phosphorylated and total AMPK, STAT3 (Ser727), and STAT3 (Tyr705). Data are mean ± SEM of two independent experiments each performed in triplicate: ∗p < 0.05 and ∗∗p < 0.01.

• 

  Figure 3. Adiponectin provided additive cardioprotective effects to ischemic postconditioning. Rats were subjected to myocardial ischemia-reperfusion (IR) in the absence or presence of APN (1 × 109 pfu), ischemic postconditioning (IPo), or their combination (APN+IPo). (a, b) Myocardial infarct size determined by TTC staining and expressed as a percentage of the area-at-risk (AAR) served by the occluded artery. (c) Plasma level of creatine kinase- (CK-) MB. (d, e) Cell apoptosis assessed by TUNEL staining. (f–k) mRNA expression of SOD1, Nrf2, HO-1, catalase, PGC-1α, and Nrf1. Data are mean ± SEM, with n = 8 animals per group: ∗p < 0.05 and ∗∗p < 0.01.

• 

  Figure 4. Adiponectin and ischemic postconditioning exerted distinct effects on cardiac AMPK and STAT3 activation in vivo. Rats were subjected to myocardial ischemia-reperfusion (IR) in the absence or presence of APN (1 × 109 pfu), ischemic postconditioning (IPo), or their combination (APN + IPo). (a) Protein expression of nuclear phosphorylation (at Tyr705) and total STAT3. (b) Protein expression of mitochondrial phosphorylated (at Ser727) and total STAT3. (c) Protein expression of phosphorylated and total AMPK. (d) Representative images of western blotting. Data are mean ± SEM, with n = 8 animals per group: ∗p < 0.05 or 0.01.

• 

  Figure 5. STAT3 inhibition abolished while AMPK inhibition partially reduced cardioprotection induced by adiponectin in combination with hypoxic postconditioning. Isolated cardiomyocytes were subjected to hypoxia-reoxygenation (HR) with globular APN (gAd) or hypoxic postconditioning (HPo), or their combination in the absence or presence of AMPK inhibitor compound C (CC) or STAT3 inhibitor stattic. (a) Cardiomyocyte death assessed by lactate dehydrogenase (LDH) release. (b) Protein expression of cleaved and total caspase-3. (c, d) Cell apoptosis assessed by TUNEL staining. (e, f) Cellular reactive oxygen species production assessed by and dihydroethidium (DHE) staining. (g–i) Protein expression of phosphorylated and total of AMPK, STAT3 (Ser727), and STAT3 (Tyr705). Data are mean ± SEM of two independent experiments each performed in triplicate: ∗p < 0.05 and ∗∗p < 0.01.

• 

  Figure 6. Schematic diagram of proposed signaling involved in adiponectin and ischemic postconditioning protection against myocardial ischemia-reperfusion injury via AMPK-dependent and AMPK-independent STAT3 activation. Adiponectin (APN) on the one hand activates AMPK thereby activates STAT3, which increases STAT3 nuclear intension. This enhanced nuclear STAT3 promotes antioxidant gene expression and enhances antioxidant capacity. On the other hand, APN activates STAT3 and increases its mitochondrial intention, which subsequently increases Nrf1 and PGC-1α, thereby reduces reactive oxygen species (ROS) production. These APN-induced increase of antioxidant capacity and reduction of ROS production result in attenuation of myocardial ischemia-reperfusion (IR) injury. While ischemic postconditioning (IPo) activates STAT3 and increases its mitochondrial intension thereby enhances Nrf1 and PGC-1α, it reduces ROS production and eventually attenuates myocardial IR injury. Combinational use of APN and IPo, through activating STAT3, increases its nuclear and mitochondrial retentions, which concomitantly enhances antioxidant capacity and reduces ROS production, jointly lead to attenuation of…