The protective effect of trimetazidine on myocardial ischemia/reperfusion injury through activating AMPK and ERK signaling pathway

Abstract

Introduction: Trimetazidine (TMZ) is an anti-anginal drug that has been widely used in Europe and Asia. The TMZ can optimize energy metabolism via inhibition of long-chain 3-ketoacyl CoA thiolase (3-KAT) in the heart, with subsequent decrease in fatty acid oxidation and stimulation of glucose oxidation. However, the mechanism by which TMZ aids in cardioprotection against ischemic injury has not been characterized. AMP-activated protein kinase (AMPK) is an energy sensor that controls ATP supply from substrate metabolism and protects heart from energy stress. TMZ changes the cardiac AMP/ATP ratio by modulating fatty acid oxidation, thereby triggering AMPK signaling cascade that contributes to the protection of the heart from ischemia/reperfusion (I/R) injury.

Methods: The mouse model of in vivo regional ischemia and reperfusion by the ligation of the left anterior descending coronary artery (LAD) was used for determination of myocardial infarction. The infarct size was compared between C57BL/6J WT mice and AMPK kinase dead (KD) transgenic mice with or without TMZ treatment. The ex vivo working heart perfusion system was used to monitor the effect of TMZ on glucose oxidation and fatty acid oxidation in the heart.

Results: TMZ treatment significantly stimulates cardiac AMPK and extracellular signal-regulated kinase (ERK) signaling pathways (p<0.05 vs. vehicle group). The administration of TMZ reduces myocardial infarction size in WT C57BL/6J hearts, the reduction of myocardial infarction size by TMZ in AMPK KD hearts was significantly impaired versus WT hearts (p<0.05). Intriguingly, the administration of ERK inhibitor, PD98059, to AMPK KD mice abolished the cardioprotection of TMZ against I/R injury. The ex vivo working heart perfusion data demonstrated that TMZ treatment significantly activates AMPK signaling and modulating the substrate metabolism by shifting fatty acid oxidation to glucose oxidation during reperfusion, leading to reduction of oxidative stress in the I/R hearts. Therefore, both AMPK and ERK signaling pathways mediate the cardioprotection of TMZ against ischemic injury. The metabolic benefits of TMZ for angina patients could be due to the activation of energy sensor AMPK in the heart by TMZ administration.

Keywords: AMPK signaling; Cardioprotection; Ischemia/reperfusion; MAPK signaling; Trimetazidine.

Fig. 1

The levels of p-AMPK, p-ACC, p-ERK1/2 and p-AKT. The mice were subjected to 20 minutes of ischemia followed by 15 minutes of reperfusion. (A) Trimetazidine or vehicle (saline) was injected intravenously via the tail vein injection 5 minutes before reperfusion. (B) The effects of trimetazidine on p-AMPK, p-ACC, p-ERK1/2 and p-AKT at basal and ischemia/reperfusion levels in C57BL/6J mice. *p < 0.05 vs. corresponding vehicle group, respectively. †p < 0.05 vs. basal vehicle group.

Fig. 2

The mice were subjected to IP injection with compound C and/or PD98059 an hour before ischemia, followed by 30 min of ischemia and 24 h of reperfusion, trimetazidine (0.5 mg/kg) or vehicle (saline) was injected intravenously via the tail vein 5 minutes before reperfusion. (A) Representative sections of myocardial infarction. (B) The ratio of area at risk (AAR) to the myocardial area (left panel) and the ratio of infarct area to AAR (right panel). Values are means ± SEM. *p < 0.05 vs. corresponding vehicle group, respectively. †p < 0.05 vs. WT + trimetazidine group.

Fig. 3

Histopathologic changes in ischemia/reperfusion (I/R)-injured hearts. Left ventricular sections from representative mice are shown. Vehicle refers to ischemia 20 min and reperfusion 4 h, trimetazidine refers to I/R (20 min/4 h) + trimetazidine (0.5 mg/kg) treatment. The sections are stained with hematoxylin and eosin, demonstrating interstitial hypercellularity and apoptosis.

Fig. 4

AMPK-dependent trimetazidine’s shift metabolism from fatty acid oxidation to glucose oxidation. Values are means ± SEM for 4 dependent experiments. *p < 0.05 vs. corresponding vehicle group. †p < 0.05 vs. C57BL/6J + trimetazidine.

Fig. 5

Contractile properties of cardiomyocytes with or without TMZ treatment. Contractile properties of cardiomyocytes isolated from WT mice with or without TMZ treatment. TMZ facilitates the contractile function of cardiomyocytes both at basal and hypoxia/reoxygenation levels. (A) Resting sarcomere length; (B) peak height; (C) maximal velocity of shortening (−dL/dt); (D) maximal velocity of re-lengthening (+dL/dt); (E) peak shortening (normalized to the resting sarcomere length); (F) time-to-90% peak shortening (TPS90) (G) the intracellular ATP change at basal at hypoxia/reoxygenation levels. After TMZ treatment, intracellular ATP level is decreased both at basal and hypoxia/reoxygenation levels. Means ± SEM, n = 30 cells from 3 mice per group,*p < 0.05 vs. vehicle group, respectively. †p < 0.05 vs. basal group.