Effect of early Trimetazidine on infarct size in ST-segment-elevation myocardial infarction patients undergoing primary percutaneous coronary intervention

Purpose: Trimetazidine, a metabolic agent with anti-ischemic effects, was reported to reduce reperfusion injury in animal models. In this randomized double-blind placebo-controlled trial, we investigated the effects of trimetazidine on reducing infarction size in patients undergoing revascularization for ST-segment elevation myocardial infarction (STEMI). Methods: Patients with STEMI randomly received trimetazidine (n=87) or placebo (n= 86) in a double-blind manner before primary percutaneous coronary intervention (PCI), and study treatment was maintained for 12 months after the procedure. The primary endpoint was infarction size measured by cardiac magnetic resonance (CMR) after primary PCI. Results: The clinical characteristics of patients (90% male, mean age 57±12 years) in both groups were well-matched on the baseline. Compared with patients in control group, the percentage and weight of infarction size of patients in trimetazidine group were both signi�cantly lower (22.1±11.8% [n =74] vs. 26.9±11.9% [n=74], p=0.010; 28±18g [n =74] vs. 35±19g [n=74], p=0.022), the myocardial microvascular obstruction (MVO) rate measured by CMR was lower in trimetazidine group (29.7% [22/7] vs. 52.7% [39/74], p=0.007), while myocardial salvage index (MSI) was signi�cantly higher in trimetazidine group (48±20% vs. 39±27%, p=0.008). The incidence of readmission due to aggravated heart failure in trimetazidine group was lower than that in the control group without signi�cance (8.0% vs 14.0%, p=0.234). Conclusions: Our study provides suggests that trimetazidine initiated prior to primary PCI, improves myocardial infarct size, MVO and MSI, possibly by reducing reperfusion injury.


Introduction
Reperfusion injury, which occurs in ST-segment elevation myocardial infarction (STEMI) after primary percutaneous coronary intervention (PCI), could further increase myocardial infarct size and lead to poor prognosis.Reperfusion injury has been increasingly concerned on its underlying mechanism and potential treatment.There is growing evidence that trimetazidine (TMZ) applied in patients with acute myocardial ischemia is bene cial in cardioprotection and reducing major cardiac events [1][2].A previous retrospective study showed that administration of TMZ could improve clinical outcomes in patients with acute myocardial infarction (AMI) by signi cantly reducing all-cause mortality in the following 12 months [3].TMZ could protect cardiomyocytes in hypoxia-ischemia via modulating energy metabolism.
Experimental studies performed in animal models of cardiac ischemia/reperfusion injury have revealed that TMZ could reduce myocardial ischemia/reperfusion injury by various mechanisms, mostly due to it facilitating energy from β-oxidation of fatty acid to more e cient glucose oxidation and antioxidant property [4][5][6][7].However, there still lacks research to provide evidence of effects of TMZ on infarct size in STEMI patients who underwent primary PCI [8].Cardiac magnetic resonance (CMR) has emerged as the imaging modality for assessing the cardioprotective e cacy in patients with AMI.Late gadolinium enhancement (LGE) on CMR provides a reliable method to evaluate the infarction size.CMR could also provide the myocardial salvage index (MSI) which demonstrates e cacy of cardioprotective interventions in situations without reduction in absolute infarction size [9][10].In this study, CMR was used to evaluate the effect of early TMZ administration on infarction size and MSI in patients who underwent primary PCI.

Compliance with Ethical Standards
From October 2016 to October 2019, we conducted a prospective, single-center, randomized, double-blind study in the Chinese PLA General Hospital in Beijing.
The study complied with the Declaration of Helsinki, which was approved by the Beijing Ethics Association and the Ethics Committee of the Chinese PLA General Hospital.
Informed consent was obtained from all individual participants included in the study.The trial was registered on ClinicalTrials.gov(registration number: NCT02826616).There is no potential con icts of interest.

Study population
The study population included patients diagnosed with STEMI for the rst time and underwent primary PCI within 12 hours of the onset of chest pain.Diagnosis of STEMI was based on the concurrence of symptoms consistent with STEMI for > 30 minutes; and ST-segment elevation ≥ 1 mm in at least 2 or more inferior leads or ≥ 2 mm in at least 2 or more contiguous precordial leads.Patients were excluded if they met any one of the bellow conditions: already treated with TMZ, history of myocardial infarction, mechanical complications, previous coronary artery bypass grafting (CABG) or PCI, contraindications of CMR, liver or kidney failure, malignant tumor.

PCI Procedures
Eligible patients were randomly assigned to receive TMZ or placebo before primary PCI.Primary PCI was performed by 2 operators using standard techniques.Patients took 300 mg aspirin in the emergency department, followed by 100 mg aspirin per day orally thereafter.A loading dose of 180 mg ticagrelor was administered before catheterization, followed by 90 mg ticagrelor twice daily for the next 12 months.Drug-eluting stents were the stents of choice.All patients were given enoxaparin treatment every 12 hours after primary PCI for 7 days.Blood samples were taken for troponin T testing before and at 6, 12, 24, 48 hours after PCI.The application of thrombectomy and intro-aortic balloon pump (IABP) was at the discretion of the operators.The PCI data analysts were kept blind from grouping.

Experimental Treatment Protocol
All patients were informed of the potential bene ts and risks of the trial before they signed written informed consents and enrolled in the study.
Patients were randomly divided into placebo and TMZ groups by using a computer-generated sequence at a ratio of 1:1.Patients were treated with a dose of 60 mg TMZ (Servier companies) or placebo orally prior to reperfusion by primary PCI and followed by 20 mg TMZ or placebo three times a day for 12 months.Other medications including aspirin, ticagrelor, statins, β-blockers and ACEI/ARB were given in accordance with the European Cardiology Society STEMI guideline [11].

Primary endpoint and clinical Follow-Up
The primary endpoint was the infarction size measured by CMR at 7 days after primary PCI.The secondary endpoint was MSI measured by CMR at 7 days after primary PCI and main adverse cardiac events (MACEs) in following 12 months.We followed up these patients by routine clinical visits and recorded any MACEs until 12 months after the PCI.Follow-up information was obtained at the outpatient clinic.
Good clinical practice training was required for all personnel involved in the trial.MACEs were de ned as stroke, repeat revascularization, and readmission due to acute heart failure, nonfatal myocardial infarction and all-cause death.All end-point events were adjudicated by an independent clinical events committee based on medical record.All were blinded to treatment group.

Cardiovascular magnetic resonance acquisition and analysis
The scan was performed at 7 days after primary PCI so as to assess the nal infarction size, microvascular obstruction (MVO), area at risk (AAR) and MSI.
All participants took a CMR examination on a 1.5T MR Scanner (Achieva; Philips Medical Systems, Best, the Netherlands), using the 32-channel phased-array body coil.
All CMR images were acquired during breath-hold and with ECG triggering.Retrospective ECG gated cine CMR imaging was performed with steady-state free precession (SSFP) sequences using the standard protocol covering short axis and long axis in the 2-and 4-chamber views.The SSFP cine images were acquired continuously from the mitral annulus to the apical level without gaps on the short-axis.Based on the retrospective triggering, 25-30 cardiac phases covering systole and diastole within a cardiac cycle were reconstructed.Infarction size was acquired by the CMR LGE method which acquired the images after 15 min injection of gadolinium (0.1 mmol/kg at 3ml/s).Inversion-recovery CMR LGE images were obtained at end-diastolic on short axis and the inversion time was manually adjusted to null the signal from remote myocardium.Myocardial edema-sensitive black-blood T2-weighted short tau inversionrecovery (STIR) sequence was performed using a fat-saturation triple inversion-recovery sequence.A full stack of LGE and T2 were acquired with about 10 slices of left ventricle (LV) chamber from base to apex, 8 mm apart.Each slice of LGE and T2 owed the same parametric location which was available for the precise analysis afterward.
The CMR data were analyzed by 2 CMR readers using the freely available validated cardiovascular image analysis software CVI42 5.11.2 (Circle Cardiovascular Imaging Inc, Calgary, Canada, version 5.10.1).Cine images on short-axis were used to analyze LV systolic function.The LV function analysis included all slices from end-diastole to end-systole.Left ventricular ejection fraction (LVEF) was calculated by manually tracing the endocardial borders in all phases on an ECG-triggered balanced steady-state-free procession cine sequence by applying multiple slices in the short-axis image covering the entire LV.Speci cally, after manual tracing of the epicardial and endocardial borders, the areas of STEMI were quanti ed in LGE images detected by semi-automated software, by the method of mean + 5SD Ref ROI with manual correction (Fig. 1A-B).We removed the artifact in the remote myocardium which would affect the infarction size calculation in the LGE image.Infarction size was expressed both in grams and in the percentage of the total LV mass.The myocardial AAR was assessed as edema on the CMR scan using T2-weighted STIR sequence (Fig. 1C-D).AAR was de ned as the long signal area on T2-weighted images, and was semi-automated recognized by mean + 2SD Ref ROI.We removed the artifact in the remote non-infarcted myocardium in the T2 STIR image with the manual correction to analyze AAR, and the area of MVO should be manually included in AAR.The MSI was calculated as follows: (AAR, ginfarction size, g)/ AAR, g.MVO was identi ed in LGE images as a subendocardial region with lower enhancement than the surrounding area which was semi-automated discerned via the dark areas inside the infarcted myocardium tissue with manual correction.All the data were validated by inter-and intraobserver analysis of reproducibility method.

Statistical analysis
Continuous variables were presented as mean ± SD and compared by the t-test for independent samples.Non-normally distribution variables were presented as a median and interquartile range, and compared by the Wilcoxon rank-sum test.Categorical data were presented as percentages and compared by the χ 2 test or the Fisher exact test when there were less than 5 values in a given cell.All statistical analyses were performed using SPSS statistical software (version 18.0, SPSS, Chicago, Illinois).

Results
A total of 365 patients with STEMI planning for primary PCI were screened for eligibility, of whom 192 patients were not eligible for the following reasons: previous myocardial infarction (36 patients), previous coronary artery bypass grafting (CABG) or PCI (61 patients), renal failure (19 patients), cardiogenic shock (35 patients), pacemakers existing (8 patients), severe infectious (6 patients), malignant tumor (11 patients) and refusal (16 patients).A total of 173 patients with STEMI were randomized (1:1) to receive either TMZ or placebo prior to primary PCI.A total of 148 patients who nally received CMR were eligible for the nal analysis (Figure 2).

Patient characteristics
The mean age of the total study cohort was 57±12 years, and males accounted for 90%.The administrations of statins, β-blockers, and ACEI/ARB had no signi cant difference between the two groups.There was no signi cant difference in time from symptom to balloon and other baseline characteristics between the TMZ and control group (Table 1).TIMI grade before reperfusion wasn't signi cant difference between the two groups.Thrombectomy occurred in 25 patients (28.7%) in the TMZ group and 23 patients (26.7%) in the control group.Compared with the control group, the proportion of patients of TIMI 3 grade after primary PCI was a little higher in TMZ group without signi cance (94.3% vs. 88.4%;p=0.308), which was shown in Table 2.

Major adverse cardiovascular events
Compared with control group, fewer occurrences of cumulative MACEs were observed in TMZ group without signi cance (Figure 4).During the one-year follow-up, the incidence of readmission for acute heart failure was lower in the TMZ group than control group (8.0% vs. 14.0%,p= 0.234).A total of 9 patients died during the follow-up, 4 in TMZ group and 5 in control group.There was no signi cant difference in the incidence of recurrent myocardial infarction or all-cause death (Figure 4, Table 5).

Discussion
TMZ is a clinically effective anti-ischemic agent, which reduces fatty acid oxidation while stimulates glucose oxidation by inhibiting long-chain 3-ketoacyl CoA thiolase.It has been reported that TMZ appeared to improve clinical outcomes in patients with AMI by signi cantly reducing all-cause mortality and other MACEs [3,12].Our trial has demonstrated improved outcomes: TMZ treatment initiated prior to primary PCI could reduce infarction size and MVO, and make improvement of MSI in patients with STEMI.Besides, CMR was rstly used to evaluate the effect of TMZ on infarction size and MSI in patients with STEMI in our trial.Other trials have proven that TMZ therapy makes improvement of LVEF, and reduce reperfusion damage for patients with STEMI, especially for non-thrombotic patients [8, [13][14][15][16], and TMZ treatment commenced prior to PCI also made improvements in left ventricular end-diastolic volume and decrease in brain natriuretic peptide (BNP) level in patients with AMI [17], all of which were in line with the results of our clinical trial.
Previous animal experiments suggested TMZ effectively reduced infarction size in animal models [18,19].
Early treatment of TMZ, especially in the acute ischemia-reperfusion phase, could reduce reperfusion myocardial injury through various mechanisms: TMZ modulates the substrate metabolism by shifting fatty acid oxidation to glucose oxidation during reperfusion [2,4,5].TMZ could reduce the intracellular acidosis and deposition of intracellular sodium and calcium, prevent the membrane damage caused by the oxygen free radicals, and reduce white blood cells [20][21][22].TMZ pretreatment could signi cantly inhibit myocardial apoptosis, and its cardiac protective effect appeared to be mediated by the blockade of the mitochondrial apoptotic pathway [23].TMZ treatment signi cantly activates AMPK and ERK signaling pathway, and inhibits MMP-2 and MMP-9 expression, which leads to a reduction of oxidative stress in ischemia-reperfusion hearts [5,21].All of the above mechanisms may contribute to reducing the infarction size for STEMI Some previous researches show no signi cant improvement of TMZ on the prognosis of angina pectoris after recent successful PCI [24].However, a meta-analysis has reported that early TMZ therapy had overall bene ts upon total MACEs in patients with AMI [12].Our study provides additional evidence on the protective effect of the TMZ on relieving reperfusion injury in patients with STEMI after PCI.These inconsistent ndings may be due to the following reason: The bene ts e cacy of TMZ may depend on the timing and dosage of administration, and may also depend on patient status, and TMZ is likely to be more effective in the acute myocardial ischemia phase.Some previous studies did not give an adequate dosage of TMZ before the reperfusion, which might underestimate the cardioprotective effect of TMZ [13].Our study treated STEMI patients with a loading dose of 60mg TMZ before PCI followed by TMZ for a total of 12 months to bene t these patients.It has been reported that pre-procedural oral TMZ administration signi cantly reduced PCI-induced myocardial injury in PCI for stable angina pectoris [25].
Our study has validated that TMZ reduces PCI-induced myocardial injury in primary PCI, and provides a reference for drug administration's dose and duration.
Infarct size is a major determinant of post STEMI mortality, so limiting the extent of infarct size in STEMI is a major therapeutic target [26].MSI also predicts the outcome of STEMI after reperfusion, and has important implications for prognosis [9].It is our hypothesis that TMZ reduces infarct size and improve MSI by ameliorating reperfusion injury.Here, we show that an early therapy of TMZ could signi cantly reduce infarct size, and increase MSI simply by being administered before reperfusion.TMZ did not reduce mortality in our study which might be due to the insu cient sample size, and further evidence is needed to assess potential longer-term clinical bene ts in a larger clinical trial.

Study limitation
There were some limitations in this study.Firstly, not all the CMR exams were performed exactly on the 7th day after PCI in our study, which might affect the nal results of the observed variables due to the rapidly changing pathophysiological processes of the cardiac tissue [27].Secondly, we used CMR data at 7 days post-STEMI as the endpoint.Although scar myocardial size performed at 1 month from the acute episode should be a more reliable measurement, but previous research has taken the infarction size, MVO, AAR and MSI measured with one week as a good alternative [28].Furthermore, 14% of patients enrolled did not undergo the CMR for primary end-point for different reasons, and this attrition rate was as we projected and is similar to those in other STEMI trials using MRI [29].Finally, this study was a singlecenter study, and our conclusion needed further validation in large-scale randomized studies.

Conclusions
This randomized study showed that TMZ initiated prior to primary PCI, reduced myocardial infarct size and MVO, and improved MSI signi cantly, possibly by reducing reperfusion injury, making it a promising treatment for evaluation in larger randomized studies.

Declarations
This work was by grants from the China Cardiovascular Association (2018-CCA-CMVD-04), the Beijing Special fund for Development (SF2020-2-5012) and natural science foundation project (81870178).
Declarations: All authors have reported that they have no relationships relevant to the contents of this paper to disclose.All authors declare that they have no con ict of interest.

Figures 1 Examples
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Figure 2 Flow
Figure 2

Table 1
Baseline characteristics of trimetazidine group and control group

Table 2
Angiographical and procedural characteristics of trimetazidine group and control IABP: intra aortic balloon pumping, TIMI: thrombolysis in myocardial infarction.

Table 3
Troponin T in trimetazidine group and control group after reperfusion

Table 4
Primary endpoints as measured by cardiac magnetic resonance

Table 5
Main adverse cardiac events for one year follow-up