Activation of AMPK inhibits inflammatory response during hypoxia and reoxygenation through modulating JNK-mediated NF-κB pathway

Abstract

Background: AMP-activated Protein Kinase (AMPK) is a stress-activated kinase that protects against cardiomyocyte injury during ischemia and reperfusion. c-Jun N-terminal kinase (JNK), a mitogen activated protein kinase, is activated by ischemia and reperfusion. NF-κB is an important transcription factor involved in ischemia and reperfusion injury.

Methods and results: The intrinsic activation of AMPK attenuates the inflammation which occurred during ischemia/reperfusion through the modulation of the JNK mediated NF-κB signaling pathway. Rat cardiac myoblast H9c2 cells were subjected to hypoxia and/or reoxygenation to investigate the signal transduction that occurred during myocardial ischemia/reperfusion. Mitochondrial function was measured by the Seahorse XF24 V7 PS system. Hypoxia treatment triggered AMPK activation in H9c2 cells in a time dependent manner. The inhibition of hypoxic AMPK activation through a pharmacological approach (Compound C) or siRNA knockdown of AMPK α catalytic subunits caused dramatic augmentation in JNK activation, inflammatory NF-κB phosphorylation, and apoptosis during hypoxia and reoxygenation. Inhibition of AMPK activation significantly impaired mitochondrial function and increased the generation of reactive oxygen species (ROS) during hypoxia and reoxygenation. In contrast, pharmacological activation of AMPK by metformin significantly inhibited mitochondrial permeability transition pore (mPTP) opening and ROS generation. Moreover, AMPK activation significantly attenuated the JNK-NF-κB signaling cascade and inhibited mRNA and protein levels of pro-inflammatory cytokines, such as TNF-α and IL-6, during hyopoxia/reoxygenation in H9c2 cells. Intriguingly, both pharmacologic inhibition of JNK by JNK-IN-8 and siRNA knockdown of JNK signaling pathway attenuated NF-κB phosphorylation and apoptosis but did not affect AMPK activation in response to hypoxia and reoxygenation.

Conclusions: AMPK activation modulates JNK-NF-κB signaling cascade during hypoxia and reoxygenation stress conditions. Cardiac AMPK activation plays a critical role in maintaining mitochondrial function and inhibiting the inflammatory response caused by ischemic insults.

Keywords: AMP-activated protein kinase; Hypoxia and reoxygenation; Inflammation.

Conflict of interest statement

Conflict of Interest

None.

Copyright © 2018 Elsevier Inc. All rights reserved.

Figures

Figure 1. The kinetics of AMPK activation and JNK activation in response to hypoxia with or without reoxygenation in H9c2 myoblast cells

(A) Immunoblotting measured the levels of p-AMPK and AMPKα protein expression in normoxia or hypoxia conditions. Values are mean ± SEM. N=3, *pvs. normoxia. (B) Immunoblotting measured the levels of p-AMPK, AMPKα, p-JNK, and JNK protein expression in normoxia, hypoxia or hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. normoxia.

Figure 2. AMPK inactivation augments JNK activation during hypoxia and reoxygenation

(A) H9c2 cells were treated with compound C (10 μM) and JNK-IN-8 (1 μM) under normoxia, hypoxia and hypoxia/reoxygenation. Upper: Immunoblotting showed phosphorylation of AMPK and downstream ACC, and phosphorylation of JNK and downstream c-Jun in H9c2 cells with or without AMPK inhibitor compound C and JNK inhibitor JNK-IN-8 under normoxia, hypoxia, and hypoxia/reoxygenation conditions; Lower: quantitative analysis of the relative levels of p-AMPK, p-ACC, p-JNK, and p-c-Jun in H9c2 cells with or without AMPK inhibitor or JNK inhibitor under normoxia, hypoxia, and hypoxia/reoxygenation. Values are mean ± SEM. N=4, *pvs. normoxia, respectively; †p<0.05 vs. hypoxia vehicle; #p<0.05 vs. hypo/reoxy vehicle. (B) AMPKα1/α2 siRNA knockdown AMPKα catalytic subunit and JNK1/2 siRNA knockdown JNK1/2 in the H9c2 myoblast cells under normoxia, hypoxia, and hypoxia/reoxygenation. Upper: Immunoblotting showed phosphorylation of AMPK and downstream ACC, and phosphorylation of JNK and downstream c-Jun in H9c2 cells with or without AMPKα1/α2 siRNA and JNK1/2 siRNA under normoxia, hypoxia, and hypoxia/reoxygenation conditions; Lower: quantitative analysis of the relative levels of p-AMPK, p-ACC, p-JNK, and p-c-Jun in H9c2 cells with or without AMPKα1/α2 siRNA and JNK1/2 siRNA under normoxia, hypoxia and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. normoxia, repectively; †p<0.05 vs. hypoxia scram; #p<0.05 vs. hypo/reoxy scram.

Figure 2. AMPK inactivation augments JNK activation during hypoxia and reoxygenation

(A) H9c2 cells were treated with compound C (10 μM) and JNK-IN-8 (1 μM) under normoxia, hypoxia and hypoxia/reoxygenation. Upper: Immunoblotting showed phosphorylation of AMPK and downstream ACC, and phosphorylation of JNK and downstream c-Jun in H9c2 cells with or without AMPK inhibitor compound C and JNK inhibitor JNK-IN-8 under normoxia, hypoxia, and hypoxia/reoxygenation conditions; Lower: quantitative analysis of the relative levels of p-AMPK, p-ACC, p-JNK, and p-c-Jun in H9c2 cells with or without AMPK inhibitor or JNK inhibitor under normoxia, hypoxia, and hypoxia/reoxygenation. Values are mean ± SEM. N=4, *pvs. normoxia, respectively; †p<0.05 vs. hypoxia vehicle; #p<0.05 vs. hypo/reoxy vehicle. (B) AMPKα1/α2 siRNA knockdown AMPKα catalytic subunit and JNK1/2 siRNA knockdown JNK1/2 in the H9c2 myoblast cells under normoxia, hypoxia, and hypoxia/reoxygenation. Upper: Immunoblotting showed phosphorylation of AMPK and downstream ACC, and phosphorylation of JNK and downstream c-Jun in H9c2 cells with or without AMPKα1/α2 siRNA and JNK1/2 siRNA under normoxia, hypoxia, and hypoxia/reoxygenation conditions; Lower: quantitative analysis of the relative levels of p-AMPK, p-ACC, p-JNK, and p-c-Jun in H9c2 cells with or without AMPKα1/α2 siRNA and JNK1/2 siRNA under normoxia, hypoxia and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. normoxia, repectively; †p<0.05 vs. hypoxia scram; #p<0.05 vs. hypo/reoxy scram.

Figure 3. AMPK plays a role in maintaining mitochondrial integerity during hypoxia and reoxygenation

(A) Mitochondrial respiration measurements of oxygen consumption rate (OCR) were performed with a Seahorse metabolic analyzer. Oligomycin (1 μM), FCCP (2 μM), and rotenone (0.5 μM) combined with antimycin (0.5 μM) were added sequentially to H9c2 cells treated with or without compound C (10 μM) and JNK-IN-8 (1 μM) under normoxia and hypoxia/reoxygenation conditions. (B) Quantitative analysis of mitochondrial function parameters (basal respiration, maximal respiration, spare capacity, and ATP production) are shown in the bar charts. Values are mean ± SEM. N=5, *pvs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (C) Mitochondrial respiration measurements of OCR were performed with a Seahorse metabolic analyzer. Oligomycin (1 μM), FCCP (2 μM), and rotenone (0.5 μM) combined with antimycin (0.5 μM) were added sequentially to H9c2 cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. (D) Quantitative analysis of mitochondrial function parameters (basal respiration, maximal respiration, spare capacity, and ATP production) are shown in the bar charts. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy scram.

Figure 3. AMPK plays a role in maintaining mitochondrial integerity during hypoxia and reoxygenation

(A) Mitochondrial respiration measurements of oxygen consumption rate (OCR) were performed with a Seahorse metabolic analyzer. Oligomycin (1 μM), FCCP (2 μM), and rotenone (0.5 μM) combined with antimycin (0.5 μM) were added sequentially to H9c2 cells treated with or without compound C (10 μM) and JNK-IN-8 (1 μM) under normoxia and hypoxia/reoxygenation conditions. (B) Quantitative analysis of mitochondrial function parameters (basal respiration, maximal respiration, spare capacity, and ATP production) are shown in the bar charts. Values are mean ± SEM. N=5, *pvs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (C) Mitochondrial respiration measurements of OCR were performed with a Seahorse metabolic analyzer. Oligomycin (1 μM), FCCP (2 μM), and rotenone (0.5 μM) combined with antimycin (0.5 μM) were added sequentially to H9c2 cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. (D) Quantitative analysis of mitochondrial function parameters (basal respiration, maximal respiration, spare capacity, and ATP production) are shown in the bar charts. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy scram.

Figure 4. AMPK rgulates opening of mitochondrial permeability transition pore (mPTP) and production of reactive oxygen species (ROS) during hypoxia and reoxygenation

(A) H9c2 myoblast cells were treated with AMPK inhibitor compound C (10 μM) or JNK inhibitor JNK-IN-8 (1 μM) under normoxia and hypoxia/reoxygenation conditions. The opening of mitochondrial permeability transition pore (mPTP) was measured with the MitoProbe transition pore assay kit. Values are mean ± SEM. N=5, *pvs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (B) The mPTP opening in H9c2 myoblast cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy scram. (C) The levels of reactive oxygen species (ROS) in H9c2 cells treated with compound C (10 μM) or JNK-IN-8 (1 μM) under normoxia or hypoxia and reoxygenation conditions. Values are mean ± SEM. N=6, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (D) The ROS production in H9c2 cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=6, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy scram.

Figure 5. AMPK modulates inflammatory NF-κB signaling activation and proinlammatory cyokine expression during hypoxia and reoxygenation

(A) Immunoblotting measured the levels of p-p65 and p65 protein expression in H9c2 myoblast cells treated with compound C (10 μM) or JNK-IN-8 (1 μM) under normoxia and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *pvs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (B) Immunoblotting measured the levels of p-p65 and p65 protein expression in H9c2 myoblast cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy scram. (C) The total RNA was isolated and subjected to real time RT-PCR to analyze the products of pro-inflammatory cytokines, TNF-α and IL-6. 18S rRNA was used as an internal control. Real-time mRNA levels of TNFα and IL-6 were measured in H9c2 cells treated with compound C (10 μM) or JNK-IN-8 (1 μM) under normoxia and hypoxia/reoxygenation conditions. The secreted TNF-α and IL-6 in the culture medium were measured with ELISA kit. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (D) Real-time RT-PCR measured the mRNA levels of TNFα and IL-6 in H9c2 cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. The secreted TNF-α and IL-6 in the culture medium were measured with ELISA kit. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy scram.

Figure 5. AMPK modulates inflammatory NF-κB signaling activation and proinlammatory cyokine expression during hypoxia and reoxygenation

(A) Immunoblotting measured the levels of p-p65 and p65 protein expression in H9c2 myoblast cells treated with compound C (10 μM) or JNK-IN-8 (1 μM) under normoxia and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *pvs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (B) Immunoblotting measured the levels of p-p65 and p65 protein expression in H9c2 myoblast cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy scram. (C) The total RNA was isolated and subjected to real time RT-PCR to analyze the products of pro-inflammatory cytokines, TNF-α and IL-6. 18S rRNA was used as an internal control. Real-time mRNA levels of TNFα and IL-6 were measured in H9c2 cells treated with compound C (10 μM) or JNK-IN-8 (1 μM) under normoxia and hypoxia/reoxygenation conditions. The secreted TNF-α and IL-6 in the culture medium were measured with ELISA kit. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (D) Real-time RT-PCR measured the mRNA levels of TNFα and IL-6 in H9c2 cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. The secreted TNF-α and IL-6 in the culture medium were measured with ELISA kit. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy scram.

Figure 6. AMPK inhibits while JNK promotes apoptosis of H9c2 cells during hypoxia and reoxygenation

(A) Apoptotic cells were detected by staining with TUNEL and DAPI, as described in Methods. Apoptosis in H9c2 myoblast cells treated with compound C (10 μM) or JNK-IN-8 (1 μM) under normoxia and hypoxia/reoxygenation conditions. (B) Apoptosis in H9c2 myoblast cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. (C) Quantitative analysis of apoptosis in H9c2 cells treated with compound C or JNK-IN-8 under normoxia and hypoxia/reoxygenation conditions. Value are mean ± SEM. N=3, *pvs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (D) Quantitative analysis of apoptosis in H9c2 cells treated with AMPKα1/α2 siRNA or JNK1/2 siRNA under normoxia and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy scram.

Figure 7. AMPK activator inhibits JNK activation by hypoxia and reoxygenation and modulates inflammaroty response during hypoxia and reoxygenation

(A) Immunoblotting showed the phosphoryaltion of AMPK α catalytic subunit at Thr172 site in H9c2 cells treated with AMPK activator metformin (5 mM) under normoxia, hypoxia, and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. vehicle, respectively; †p<0.05 vs. normoxia vehicle. #p<0.05 vs. normoxia metformin. (B) Immunoblotting showed the phosphoryaltion of JNK and p65 in H9c2 cells treated with AMPK activator metformin (5 mM) under normoxia, hypoxia, and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (C) The mPTP opening in H9c2 myoblast cells treated with or without metformin. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (D) The ROS production in H9c2 myoblast cells treated with or without metformin. Values are mean ± SEM. N=6, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (E) The total RNA was isolated and subjected to real time RT-PCR to analyze the products of pro-inflammatory cytokines, TNF-α and IL-6 in H9c2 myoblast cells treated with or without metformin. 18S rRNA was used as an internal control. The secreted TNF-α and IL-6 in the culture medium were measured with ELISA kit. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle.

Figure 7. AMPK activator inhibits JNK activation by hypoxia and reoxygenation and modulates inflammaroty response during hypoxia and reoxygenation

(A) Immunoblotting showed the phosphoryaltion of AMPK α catalytic subunit at Thr172 site in H9c2 cells treated with AMPK activator metformin (5 mM) under normoxia, hypoxia, and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. vehicle, respectively; †p<0.05 vs. normoxia vehicle. #p<0.05 vs. normoxia metformin. (B) Immunoblotting showed the phosphoryaltion of JNK and p65 in H9c2 cells treated with AMPK activator metformin (5 mM) under normoxia, hypoxia, and hypoxia/reoxygenation conditions. Values are mean ± SEM. N=3, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (C) The mPTP opening in H9c2 myoblast cells treated with or without metformin. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (D) The ROS production in H9c2 myoblast cells treated with or without metformin. Values are mean ± SEM. N=6, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle. (E) The total RNA was isolated and subjected to real time RT-PCR to analyze the products of pro-inflammatory cytokines, TNF-α and IL-6 in H9c2 myoblast cells treated with or without metformin. 18S rRNA was used as an internal control. The secreted TNF-α and IL-6 in the culture medium were measured with ELISA kit. Values are mean ± SEM. N=5, *p<0.05 vs. normoxia, respectively; †p<0.05 vs. hypo/reoxy vehicle.