ANP Increases Zn^2⁺ Accumulation During Reperfusion in Ex Vivo and In Vivo Hearts

Abstract

Objective

Atrial natriuretic peptide (ANP) and Zn^2⁺ have been shown to confer cardioprotection against ischemia/reperfusion (I/R) injury. Zn^2⁺ alleviates myocardial hypertrophy and pulmonary hypertension by regulating ANP expression, but its precise role in ANP-mediated cardioprotection remains unclear. This study aimed to investigate whether ANP protects the heart during reperfusion by modulating Zn^2⁺ levels and to explore the underlying mechanisms involved.

Methods

In this study, we utilized an isolated reperfused heart model in rats, as well as wild-type (WT) and ANP knockout (ANP^−/−) mouse models, for in vivo I/R experiments. For clinical investigations, plasma samples were collected from 216 patients with ischemia-related diseases. Evans blue and TTC staining, radioimmunoassay, ICP‒OES, echocardiography, Hydro-Cy3-mediated ROS detection, and Western blotting were employed to evaluate the effect of ANP on Zn^2⁺ homeostasis.

Results

Plasma ANP levels were significantly elevated in patients with ST-elevation myocardial infarction (STEMI), nonST-elevation myocardial infarction (NSTEMI), and heart failure (HF). ANP secretion increased during reperfusion, rather than infarction, both ex vivo and in vivo, promoting Zn^2⁺ accumulation in reperfused tissue. ANP and Zn^2⁺ protected mitochondria and reduced infarct size; these effects were reversed by the Zn^2⁺ chelator TPEN. In WT and ANP^−/− mice, EF% and FS% decreased after reperfusion, with ANP^−/− mice exhibiting significantly worse cardiac function. ANP pretreatment alone improved cardiac function, but combined pretreatment with ANP and TPEN decreased EF% and FS% while increasing LVID. Reperfusion increased ROS levels in both WT and ANP^−/− hearts, which were reduced by ANP pretreatment. I/R injury elevated Zn^2⁺ transporter 8 (ZnT8) expression, an effect that was counteracted by ANP, although this effect was reversed by TPEN. Hypoxia-inducible factor 1-alpha (HIF-1α) expression was elevated in I/R rats and ANP^−/− mice, and it was inhibited by both Zn^2⁺ and ANP pretreatment. However, the HIF-1α inhibitor 2-Me did not reverse the effect of ANP on ZnT8 expression. Additionally, ANP increased PI3K expression in both WT and ANP^−/−I/R mice, but this effect was blocked by the PI3K inhibitor LY294002.

Conclusions

ANP modulates Zn2⁺ homeostasis during reperfusion injury by downregulating ZnT8 through the PI3K signalling pathway, thereby reducing myocardial I/R injury.

© The Author(s), under exclusive licence to Huazhong University of Science and Technology 2025

Funding This work was supported by the National Natural Science Foundation of China (NSFC) (No. 82360065 and No. 81760047).

Data Availability All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Declarations

Conflict of Interest The authors declare that there is no conflict of interest with any financial organization corporation or individual that can inappropriately influence this work.

Ethical Approval and Consent to Participate All animal experiments were meticulously carried out in strict adherence to the detailed guidelines provided by the Yanbian University Animal Care and Use Committee’s Guide for the Care and Use of Laboratory Animals. These guidelines ensure the humane and ethical treatment of the laboratory animals involved in the research (approval number: YD20240510009).

Human Ethics The study protocol was approved by the Ethics Committee of Yanbian University Hospital, Yanji, Jilin Province, China, and informed consent was obtained from all the patients or the family members of the patients before the study.

Consent for Publication Consent for publication was obtained from the participants.

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