Free fatty acids induce coronary microvascular dysfunction via inhibition of the AMPK/KLF2/eNOS signaling pathway

Zhang,Yanda; Zhao,Jian; Ren,Changzhen; Hu,Bowen; Ding,Ru; He,Zhiqing; Liang,Chun

doi:10.3892/ijmm.2023.5237

Free fatty acids induce coronary microvascular dysfunction via inhibition of the AMPK/KLF2/eNOS signaling pathway

Authors:
- Yanda Zhang
- Jian Zhao
- Changzhen Ren
- Bowen Hu
- Ru Ding
- Zhiqing He
- Chun Liang
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Affiliations: Department of Cardiology, Second Affiliated Hospital of Naval Medical University, Shanghai 200003, P.R. China
Published online on: March 13, 2023 https://doi.org/10.3892/ijmm.2023.5237
Article Number: 34
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Increased levels of serum free fatty acids (FFAs) are closely associated with microvascular dysfunction. In our previous study, a coronary microvascular dysfunction (CMD) model was successfully established via lipid infusion to increase the levels of serum FFAs in mice. However, the underlying mechanisms remained poorly understood. Therefore, the aim of the present study was to explore the mechanism underlying FFA‑induced CMD. A CMD mouse model was established via lipid combined with heparin infusion for 6 h to increase the concentration of serum FFAs. Following the establishment of the model, the coronary flow reserve (CFR), extent of leukocyte activation and cardiac microvascular structures were assessed in the mice. Cardiac microvascular endothelial cells (CMECs) were treated with different concentrations of palmitic acid and cell viability was evaluated. Changes in the expression levels of AMP‑activated protein kinase (AMPK), Krüppel‑like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) were identified by immunohistochemical and western blot analyses. Experiments using AMPK activator, KLF2 overexpression plasmid, small interfering RNAs and nicorandil were subsequently designed to investigate the potential involvement of the AMPK/KLF2/eNOS signaling pathway. These experiments revealed that FFAs could induce CMD in mice, which was characterized by reduced CFR (1.89±0.37 vs. 2.74±0.30) and increased leukocyte adhesion (4,350±1,057.5 vs. 11.8±5.4 cells/mm²) compared with the control mice. CD11b expression and intracellular reactive oxygen species (ROS) levels were increased in CMD model mice compared with control mice. Serum TNF‑α and IL‑6 levels were higher in the model group than in the control group. Transmission electron microscopy revealed that CMECs in heart tissues of model mice were severely swollen. In addition, palmitic acid decreased CMEC viability and increased ROS production in a dose‑dependent manner. Notably, the AMPK/KLF2/eNOS signaling pathway was demonstrated to be suppressed by FFAs both in vivo and in vitro. Activation of this axis with AMPK activator, KLF2 overexpression plasmid or nicorandil restored the CFR in CMD model mice, inhibited oxidative stress and increased CMEC viability. Taken together, the results of the present study demonstrated that FFAs could induce CMD via inhibition of the AMPK/KLF2/eNOS signaling pathway, whereas activation of this pathway led to the alleviation of FFA‑induced CMD, which may be a therapeutic option for CMD.

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