Adenosine monophosphate-activated protein kinase is required for pulmonary artery smooth muscle cell survival and the development of hypoxic pulmonary hypertension

Abstract

Human pulmonary artery smooth muscle cells (HPASMCs) express both adenosine monophosphate-activated protein kinase (AMPK) α1 and α2. We investigated the distinct roles of AMPK α1 and α2 in the survival of HPASMCs during hypoxia and hypoxia-induced pulmonary hypertension (PH). The exposure of HPASMCs to hypoxia (3% O2) increased AMPK activation and phosphorylation, and the inhibition of AMPK with Compound C during hypoxia decreased their viability and increased lactate dehydrogenase activity and apoptosis. Although the suppression of either AMPK α1 or α2 expression led to increased cell death, the suppression of AMPK α2 alone increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. It also resulted in the decreased expression of myeloid cell leukemia sequence 1 (MCL-1). The knockdown of MCL-1 or MCL-1 inhibitors increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. On the other hand, the suppression of AMPK α1 expression alone prevented hypoxia-mediated autophagy. The inhibition of autophagy induced cell death in HPASMCs. Our results suggest that AMPK α1 and AMPK α2 play differential roles in the survival of HPASMCs during hypoxia. The activation of AMPK α2 maintains the expression of MCL-1 and prevents apoptosis, whereas the activation of AMPK α1 stimulates autophagy, promoting HPASMC survival. Moreover, treatment with Compound C, which inhibits both isoforms of AMPK, prevented and partly reversed hypoxia-induced PH in mice. Taking these results together, our study suggests that AMPK plays a key role in the pathogenesis of pulmonary arterial hypertension, and AMPK may represent a novel therapeutic target for the treatment of pulmonary arterial hypertension.

Figure 1.

The activation of adenosine monophosphate–activated protein kinase (AMPK) is essential for the survival of human pulmonary artery smooth muscle cells (HPASMCs) during hypoxia. (A and B) HPASMCs were exposed to normoxia (N) (21% O₂) or hypoxia (H) (3% O₂) for 15 and 30 minutes (A) and 24 hours (B). The amounts of phospho-AMPK (pAMPK) at Thr-172 (T172) and total AMPK were determined, and the pAMPK/AMPK ratios were calculated. (C-F) HPASMCs were pretreated with dimethylsulfoxide (DMSO) or 10 μM Compound C (CC) for 1 hour, and then incubated under normoxia (N) or hypoxia (H) for 24 hours for the cell viability assay (C), 8 hours for the lactate dehydrogenase (LDH) assay (D), and for up to 5 hours to detect the cleavage of caspase-3 (E) and for a terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay (F). Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05. **P < 0.01. Tubulin was used as loading control. CTL, control.

Figure 2.

The inhibition of AMPK α₂ induces HPASMC apoptosis during hypoxia. (A) HPASMCs were transfected with small interfering RNA (siRNA) against AMPK α₁ or α₂ before exposure to normoxia (N) or hypoxia (H) for 6 hours, and LDH activity was determined as already described. Scrambled siRNA (si-Neg) was used as control. The amounts of AMPK α₁ and α₂ in the cell lysates were determined by Western blot analysis. β-actin was used as loading control. (B) Wild-type (WT), AMPK α₁–null, and AMPK α₂–null mouse embryo fibroblasts (MEFs) were exposed to normoxia or hypoxia for 8 hours, and then LDH activity was measured. (C) HPASMCs transfected with siRNA against AMPK α₁ or α₂ were incubated during normoxia or hypoxia for 8 hours, and then caspase-3 activity was measured. (D) MEFs were exposed to normoxia or hypoxia for 8 hours, and cell lysates were collected to measure caspase-3 activity. Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05.

Figure 3.

Inhibition of AMPK α₂ decreases the expression of prosurvival protein MCL-1, leading to HPASMC cell apoptosis during hypoxia. (A) HPASMCs were pretreated with dimethylsulfoxide (DMSO) or 10 μM Compound C (CC) for 1 hour, and then incubated under normoxia (N) or hypoxia (H) for 8 hours. The amount of prosurvival myeloid cell leukemia sequence 1 (MCL-1) and B-cell lymphoma-extra large (BCL-XL) and proapoptotic Bcl-2–associated death promoter (BAD) and BH3 interacting-domain death agonist (BID) in the cell lysates were determined by Western blot analysis. (B) Cells were transfected with siRNA against AMPK α₁ or α₂ (si-α1 or si-α2) and then exposed to normoxia (N) or hypoxia (H) for 6 hours. The amount of MCL-1 in the cell lysates was determined as described previously. Tubulin was used as loading control. The MCL-1/tubulin ratios are shown at the top. (C–E) HPASMCs were transfected with si–MCL-1 and exposed to normoxia (N) or hypoxia (H) for 6 hours. (C) The amount of MCL-1 in the cell lysates was determined by Western blot analysis, and the MCL-1/tubulin ratios are shown at the top. LDH activity (D) and caspase-3 activity (E) were determined as described previously. (F and G) HPASMCs were pretreated with various doses of obatoclax mesylate (GX 15-070) (F) and TW-37 (G) for 1 hour, and then incubated under normoxia (NMX) or hypoxia (HPX) for 24 hours. The HPASMC viability was measured as described previously. Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05. **P < 0.01.

Figure 4.

AMPK α₁ facilitates HPASMC survival during hypoxia by promoting autophagy. HPASMCs were either transfected with siRNA for AMPK α₁ or α₂ (A) or pretreated with dimethylsulfoxide (DMSO) or 1 mM 3-methyladenine (3-MA) for 1 hour (B), and then exposed to normoxia or hypoxia for 6 hours. The cleavage of microtubule-associated protein 1 light chain 3B (LC3B) was measured by Western blot analysis. β-actin (A) and tubulin (B) were used as loading controls. The LC3B-II/I ratios are shown at the top (B). After pretreatment with 3-MA and exposure to normoxia or hypoxia, LDH activity (C) and caspase-3 activity (D) were determined. Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05. **P < 0.01.

Figure 5.

Hypertensive human and mouse PASMCs express elevated levels of AMPK phosphorylation. (A) We compared the concentrations of phosphorylated and total AMPK in normal HPASMCs and in HPASMCs isolated from patients with pulmonary arterial hypertension (PAH). Representative blots are shown at the bottom, and the amounts of pAMPK and total AMPK are shown as the pAMPK/AMPK and AMPK/tubulin ratios at the top and middle, respectively. (B) Mice were exposed to normoxia (ambient air, N) or hypoxia (10% O₂, H) for 3 weeks. Whole-lung homogenates were used to determine the concentrations of phosphorylated and total AMPK by Western blot analysis. Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05. Tubulin was used as loading control. (C) The lung sections of mice described in B were immunolabeled with pAMPK and α–smooth muscle actin (SMA) antibodies and 4′6-diamidino-2-phenylindole (DAPI) for study, using fluorescence microscopy.

Figure 6.

The inhibition of AMPK by Compound C prevents hypoxia-induced pulmonary hypertension in mice. Mice were weighed and injected with DMSO or Compound C (CC), 1 day before exposure to normoxia (ambient air, N) or hypoxia (10% O₂, H) for 3 weeks. During the experimental period, mice were injected with DMSO or Compound C once a week, and were weighed weekly. (A) After the completion of exposure, mice were anesthetized, and blood was drawn from the heart and used to measure hematocrit (HCT). (B) Tight ventricular (RV) hypertrophy, expressed as the weight ratio of right ventricle/(left ventricle + septum) (RV/(LV + S)), was determined as described in Materials and Methods. (C) Images of hematoxylin-and-eosin–stained sections of mouse lung were used to calculate the thickness of pulmonary arterial wall. (D) Representative hematoxylin-and-eosin–stained images of mouse lung sections are shown, and the arrows indicate pulmonary arteries. Scale bar at the lower left corner = 100 μm. (E and F) We measured the right ventricular pressure (RVP) in these mice. Representative diagrams of RVP are shown in E, and the quantification of right ventricular systolic pressure (RVSP) is shown in F. Data are presented as means ± SEMs, and are compared with those mice injected with DMSO and exposed to normoxia. The insert in E indicates a duration of 0.2 s. *P < 0.05 and **P < 0.01 (n ≥ 5 for each group). ^#P < 0.05, significant difference between DMSO + H and CC + H groups. n.s., no significance.

Figure 7.

Inhibition of AMPK by Compound C partly reverses hypoxia-induced pulmonary hypertension in mice. We exposed C57BL/6 mice to hypoxia or normoxia for 2 weeks, followed by an injection of CC or DMSO once weekly for another 2 weeks. After the completion of exposure, mice were anesthetized, and RVPs were measured. Representative diagrams of RVP are shown in A, and the quantification of RVSP is shown in B. RV/(LV + S), arterial wall thickness, and HCT values are shown in C, D, and E, respectively. Data are presented as means ± SEMs, and are compared with data for mice injected with DMSO and exposed to normoxia (DMSO + N). *P < 0.05 and **P < 0.01 (n ≥ 5 for each group). ^##P < 0.01, significant difference between DMSO + H and CC + H groups. n.s., no significance.