Trimetazidine Protects against Hypoxia/Reoxygenation Injury in Cardiomyocytes by Modulating miR-125b-5p/ATG4D Axis

Abstract

Background: Trimetazidine (TMZ) has been shown to have a protective effect against myocardial ischemia/reperfusion (I/R) injury (MIRI) by modulating the levels of microRNA (miRNA). The aim of this study was to investigate whether TMZ could prevent myocardial I/R injury by regulating miR-125b-5p. Methods: A cardiomyocyte injury model was established using hypoxia/reoxygenation (H/R) processing. Cellular function assays were used to determine the viability, apoptosis, miR-125b-5p expression, apoptosis- and autophagy-related protein levels as well as Adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/unc-51-like kinase 1 (ULK1) pathway level in cardiomyocytes under H/R treatment or co-treatment of miR-125b-5p inhibitor and TMZ. A dual-luciferase reporter assay was used to verify the predicted targeting relationship between autophagy-related 4D (ATG4D) and miR-125b-5p. The possible involvement of target genes and the AMPK/mTOR/ULK1 pathway in miR-125b-5p-mediated effects was also analyzed. Results: H/R reduced miR-125b-5p messenger RNA (mRNA) expression and cell viability, while promoting the apoptosis and the protein levels of markers relevant to autophagy and apoptosis (p < 0.01). These effects of H/R were reversed by TMZ, while the reversing effect of TMZ was largely diminished by miR-125b-5p inhibitor (p < 0.01). The targeting relationship between miR-125b-5p and ATG4D has been confirmed. ATG4D overexpression or knockdown rescued the impact of miR-125b-5p mimic/inhibitor upon H/R-treated cells (p < 0.01). H/R facilitated phosphorylated (p)-AMPK and p-ULK1 protein levels yet reduced that of p-mTOR, and this effect was offset by miR-125b-5p mimic but was potentiated by the inhibitor (p < 0.01). The impacts of miR-125b-5p mimic or inhibitor upon the AMPK/mTOR/ULK1 pathway were reversed following the overexpression or knockdown of ATG4D, respectively (p < 0.01). Besides, the impacts of miR-125b-5p mimic or inhibitor upon H/R-treated cells were confirmed to be counteracted in the presence of AMPK/mTOR/ULK1 pathway agonist or inhibitor. Conclusions: Trimetazidine protects cardiomyocytes from H/R injury by modulating miR-125b-5p/ATG4D axis.