Basic Study
Copyright ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Diabetes. Dec 15, 2023; 14(12): 1862-1876
Published online Dec 15, 2023. doi: 10.4239/wjd.v14.i12.1862
Empagliflozin ameliorates diabetic cardiomyopathy probably via activating AMPK/PGC-1α and inhibiting the RhoA/ROCK pathway
Na Li, Qiu-Xiao Zhu, Gui-Zhi Li, Ting Wang, Hong Zhou
Na Li, Qiu-Xiao Zhu, Gui-Zhi Li, Ting Wang, Hong Zhou, Department of Endocrinology, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, Hebei Province, China
Author contributions: Li N, Zhu QX, Li GZ, Wang T, and Zhou H designed and coordinated the study; Li N, Zhu QX, Li GZ, and Zhou H performed the experiments, and acquired and analyzed the data; Li N interpreted the data; Li N and Zhou H wrote the manuscript; all authors approved the final version of the article.
Supported by Health Commission of Hebei Province, No. 20210196; S & T Program of Hebei, No. 22377726D.
Institutional review board statement: The study was reviewed and approved by the research ethics committee of the Second Hospital of Hebei Medical University Institutional Review Board (Approval No. 2022-AE136).
Conflict-of-interest statement: The authors declare no competing interests for this article.
Data sharing statement: The data of this study are available from the corresponding authors upon request.
ARRIVE guidelines statement: The authors have read the ARRIVE Guidelines, and the manuscript was prepared and revised according to the ARRIVE Guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Hong Zhou, PhD, Chief Physician, Doctor, Department of Endocrinology, The Second Hospital of Hebei Medical University, No. 215, Heping West Road, Shijiazhuang 050000, Hebei Province, China. zhoubs2013@163.com
Received: September 21, 2023
Peer-review started: September 21, 2023
First decision: October 10, 2023
Revised: October 20, 2023
Accepted: November 17, 2023
Article in press: November 17, 2023
Published online: December 15, 2023
Processing time: 83 Days and 20.1 Hours
Abstract
BACKGROUND

Diabetic cardiomyopathy (DCM) increases the risk of hospitalization for heart failure (HF) and mortality in patients with diabetes mellitus. However, no specific therapy to delay the progression of DCM has been identified. Mitochondrial dysfunction, oxidative stress, inflammation, and calcium handling imbalance play a crucial role in the pathological processes of DCM, ultimately leading to cardiomyocyte apoptosis and cardiac dysfunctions. Empagliflozin, a novel glucose-lowering agent, has been confirmed to reduce the risk of hospitalization for HF in diabetic patients. Nevertheless, the molecular mechanisms by which this agent provides cardioprotection remain unclear.

AIM

To investigate the effects of empagliflozin on high glucose (HG)-induced oxidative stress and cardiomyocyte apoptosis and the underlying molecular mechanism.

METHODS

Twelve-week-old db/db mice and primary cardiomyocytes from neonatal rats stimulated with HG (30 mmol/L) were separately employed as in vivo and in vitro models. Echocardiography was used to evaluate cardiac function. Flow cytometry and TdT-mediated dUTP-biotin nick end labeling staining were used to assess apoptosis in myocardial cells. Mitochondrial function was assessed by cellular ATP levels and changes in mitochondrial membrane potential. Furthermore, intracellular reactive oxygen species production and superoxide dismutase activity were analyzed. Real-time quantitative PCR was used to analyze Bax and Bcl-2 mRNA expression. Western blot analysis was used to measure the phosphorylation of AMP-activated protein kinase (AMPK) and myosin phosphatase target subunit 1 (MYPT1), as well as the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and active caspase-3 protein levels.

RESULTS

In the in vivo experiment, db/db mice developed DCM. However, the treatment of db/db mice with empagliflozin (10 mg/kg/d) for 8 wk substantially enhanced cardiac function and significantly reduced myocardial apoptosis, accompanied by an increase in the phosphorylation of AMPK and PGC-1α protein levels, as well as a decrease in the phosphorylation of MYPT1 in the heart. In the in vitro experiment, the findings indicate that treatment of cardiomyocytes with empagliflozin (10 μM) or fasudil (FA) (a ROCK inhibitor, 100 μM) or overexpression of PGC-1α significantly attenuated HG-induced mitochondrial injury, oxidative stress, and cardiomyocyte apoptosis. However, the above effects were partly reversed by the addition of compound C (CC). In cells exposed to HG, empagliflozin treatment increased the protein levels of p-AMPK and PGC-1α protein while decreasing phosphorylated MYPT1 levels, and these changes were mitigated by the addition of CC. Adding FA and overexpressing PGC-1α in cells exposed to HG substantially increased PGC-1α protein levels. In addition, no sodium-glucose cotransporter (SGLT)2 protein expression was detected in cardiomyocytes.

CONCLUSION

Empagliflozin partially achieves anti-oxidative stress and anti-apoptotic effects on cardiomyocytes under HG conditions by activating AMPK/PGC-1α and suppressing of the RhoA/ROCK pathway independent of SGLT2.

Keywords: Empagliflozin; Diabetic cardiomyopathy; AMPK; ROCK; Apoptosis; Oxidative stress

Core Tip: We established a diabetic cardiomyopathy model in db/db mice and treated the mice with empagliflozin for 8 wk, and found that empagliflozin observably improved cardiac function in diabetic mice, which was maybe related to activation of AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and inhibition of the RhoA/ROCK pathway. In order to exclude the effects of metabolic improvement on the heart in vivo, in vitro experiment in high glucose conditions was performed. The results confirmed that the anti-oxidative stress and anti-apoptotic effects of empagliflozin on cardiomyocytes were achieved by activating AMPK/PGC-1α and inhibiting ROCK. Furthermore, the effects were independent of sodium-glucose cotransporter (SGLT)2 inhibition as no SGLT2 expression was detected on cardiomyocytes.