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CaMKK2 alleviates myocardial ischemia/reperfusion injury by inhibiting oxidative stress and inflammation via the action on the AMPK-AKT-GSK-3β/Nrf2 signaling cascade

  • Original Research Paper
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Abstract

Objective

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) can regulate numerous biological processes and is implicated in diverse pathological processes. Yet its role in myocardial ischemia/reperfusion (MI/R) injury remains unknown. This project explored the possible functions and mechanisms of CaMKK2 in MI/R injury.

Methods

A rat model of MI/R in vivo was established using the left anterior descending coronary artery ligation method. Rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) in vitro to establish a cell model. Overexpression of CaMKK2 was achieved by infecting recombinant adeno-associated virus or adenovirus expressing CaMKK2. Real-time quantitative PCR, immunoblotting, TTC staining, TUNEL assay, ELISA, oxidative stress detection assays, flow cytometry, and CCK-8 assay were carried out.

Results

A decline in CaMKK2 levels was induced by MI/R in vivo or H/R in vitro. Up-modulation of CaMKK2 in rats ameliorated the cardiac injury evoked by MI/R injury accompanied by suppression of cardiac apoptosis, oxidative stress, and proinflammatory response. Rat cardiomyocytes with CaMKK2 overexpression were also protected from H/R damage by inhibiting apoptosis, oxidative stress, and proinflammatory response. CaMKK2 overexpression led to increased phosphorylation of AMPK, AKT, and GSK-3β, and enhanced activation of Nrf2 under MI/R or H/R conditions. Inhibition of AMPK abolished CaMKK2-mediated Nrf2 activation and relevant cardioprotective effect. Restraint of Nrf2 also diminished CaMKK2-mediated relevant cardioprotective effect.

Conclusions

Up-regulation of CaMKK2 provides a therapeutic benefit in the rat model of MI/R injury by boosting the Nrf2 pathway through regulation of AMPK/AKT/GSK-3β, which suggests CaMKK2 as a new molecular target for the treatment of MI/R injury.

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Data availability

The datasets used during the present study are available from the corresponding author upon reasonable request.

Abbreviations

CaMKK2:

Calcium/calmodulin-dependent protein kinase kinase 2

MI/R:

Myocardial ischemia/reperfusion

H/R:

Hypoxia/reoxygenation

AMPK:

Adenosine monophosphate-activated protein kinase

GSK-3β:

Glycogen synthase kinase-3β

Nrf2:

Nuclear factor erythroid 2-related factor 2

RT-qPCR:

Real-time quantitative PCR

AAV:

Adeno-associated virus

LDH:

Lactate dehydrogenase

cTn-I:

Cardiac troponin I

TTC:

Triphenyl tetrazolium chloride

TUNEL:

Terminal-deoxynucleotidyl transferase dUTP-biotin nick end labeling

ROS:

Reactive oxygen species

MDA:

Malondialdehyde

SOD:

Superoxide dismutase

CCK-8:

Cell counting kit-8

ARE:

Anti-oxidant response element

NQO-1:

NADPH quinone oxidoreductase-1

HO-1:

Heme oxygenase-1

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Funding

The work was supported by the Shaanxi Natural Science Foundation Project (2022SF-132), the General Teaching Reform Project of South China University of Technology (2022–10), and the Guangzhou First People’s Hospital Red Cotton Plan Project (2023–06).

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Author notes

  1. Chengliang Li and Jiajia Hao contributed equally to this work and shared the first authorship.

Authors and Affiliations

  1. Department of General Practice, Guangzhou First People’s Hospital, Guangzhou, 510180, China

    Chengliang Li, Jiajia Hao & Huichang Qiu

  2. Healthcare Simulation Center, Department of Research, Education and Information, Guangzhou First People’s Hospital, No.1 Panfu Road, Guangzhou, 510180, China

    Hong Xin

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Contributions

CL and JH designed the work, performed the experiments, and wrote the manuscript. HQ performed data analysis and provided technical support. HX contributed to the conceptualization and reviewed the manuscript.

Corresponding author

Correspondence to Hong Xin.

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Li, C., Hao, J., Qiu, H. et al. CaMKK2 alleviates myocardial ischemia/reperfusion injury by inhibiting oxidative stress and inflammation via the action on the AMPK-AKT-GSK-3β/Nrf2 signaling cascade. Inflamm. Res. 72, 1409–1425 (2023). https://doi.org/10.1007/s00011-023-01756-6

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  • DOI: https://doi.org/10.1007/s00011-023-01756-6

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