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Updated: Nov 23, 2025
In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model
Published on: October 27, 2020
Zenglv Fumai Granule protects cardiomyocytes against hypoxia/reoxygenation-induced apoptosis via inhibiting TRIM28
Xiao-Hua Zhang1, Hong-Yu Zhao2, Yu Wang2
1Department of Cardiovascular Diseases, The First Clinical Hospital of Jilin Academy of Chinese Medicine Sciences, Changchun, Jilin 130021, P.R. China.
Abstract:
Myocardial ischemia/reperfusion (MIR) injury, which occurs following acute myocardial infarction, can cause secondary damage to the heart. Tripartite interaction motif (TRIM) proteins, a class of E3 ubiquitin ligases, have been recognized as critical regulators in MIR injury. Zenglv Fumai Granule (ZFG) is a clinical prescription for the treatment of sick sinus syndrome, a disease that is associated with MIR injury. The present study aimed to investigate the effect of ZFG on MIR injury and to determine whether ZFG exerts its effects via regulation of TRIM proteins. In order to establish an in vitro MIR model, human cardiomyocyte cell line AC16 was cultured under hypoxia for 5 h and then under normal conditions for 1 h. Following hypoxia/reoxygenation (H/R) treatment, these cells were cultured with different ZFG concentrations. ZFG notably inhibited H/R-induced cardiomyocyte apoptosis. The expression levels of four TRIM proteins, TRIM7, TRIM14, TRIM22 and TRIM28, were also detected. These four proteins were significantly upregulated in H/R-injured cardiomyocytes, whereas their expression was inhibited following ZFG treatment. Moreover, TRIM28 knockdown inhibited H/R-induced cardiomyocyte apoptosis, whereas TRIM28 overexpression promoted apoptosis and generation of reactive oxygen species (ROS) in cardiomyocytes. However, the effects of TRIM28 overexpression were limited by the action of ROS inhibitor N-acetyl-L-cysteine. In addition, the mRNA and protein levels of antioxidant enzyme glutathione peroxidase (GPX)1 were significantly downregulated in H/R-injured cardiomyocytes. TRIM28 knockdown restored GPX1 protein levels but had no effect on mRNA expression levels. Co-immunoprecipitation and ubiquitination assays demonstrated that TRIM28 negatively regulated GPX1 via ubiquitination. In sum, the present study revealed that ZFG attenuated H/R-induced cardiomyocyte apoptosis by regulating the TRIM28/GPX1/ROS pathway. ZFG and TRIM28 offer potential therapeutic options for the treatment of MIR injury.
Insights
Zenglv Fumai Granule (ZFG) reduces heart damage after myocardial ischemia/reperfusion (MIR) injury by regulating Tripartite Interaction Motif (TRIM) proteins, specifically TRIM28, which impacts the antioxidant enzyme GPX1 and reactive oxygen species (ROS). This study highlights ZFG and TRIM28 as potential therapies for MIR injury.
Area of Science:
- Cardiovascular Biology
- Molecular Medicine
- Pharmacology
Background:
- Myocardial ischemia/reperfusion (MIR) injury causes secondary heart damage after myocardial infarction.
- Tripartite Interaction Motif (TRIM) proteins, E3 ubiquitin ligases, are key regulators in MIR injury.
- Zenglv Fumai Granule (ZFG) is a traditional Chinese medicine used for sick sinus syndrome, linked to MIR injury.
Purpose of the Study:
- To investigate the therapeutic effect of ZFG on MIR injury.
- To determine if ZFG's effects involve the regulation of TRIM proteins.
Main Methods:
- An in vitro MIR model using human AC16 cardiomyocytes subjected to hypoxia/reoxygenation (H/R).
- Treatment of H/R-injured cells with varying concentrations of ZFG.
- Analysis of cardiomyocyte apoptosis, TRIM protein expression (TRIM7, TRIM14, TRIM22, TRIM28), reactive oxygen species (ROS), and glutathione peroxidase 1 (GPX1) levels.
- TRIM28 knockdown and overexpression studies, co-immunoprecipitation, and ubiquitination assays.
Main Results:
- ZFG significantly inhibited H/R-induced cardiomyocyte apoptosis.
- ZFG downregulated the expression of TRIM7, TRIM14, TRIM22, and TRIM28 in H/R-injured cardiomyocytes.
- TRIM28 knockdown reduced apoptosis, while overexpression increased apoptosis and ROS generation; these effects were ROS-dependent.
- TRIM28 negatively regulated GPX1 protein levels via ubiquitination, independent of mRNA levels.
- ZFG treatment restored GPX1 protein levels in H/R-injured cardiomyocytes.
Conclusions:
- ZFG attenuates H/R-induced cardiomyocyte apoptosis through the regulation of the TRIM28/GPX1/ROS pathway.
- TRIM28 plays a critical role in H/R-induced cardiomyocyte apoptosis and oxidative stress.
- ZFG and TRIM28 represent potential therapeutic strategies for managing MIR injury.
