Current global guidelines for ST-segment elevation myocardial infarction (STEMI) recommend primary percutaneous coronary intervention (PPCI) as the gold standard of treatment [1, 2], and PPCI restores thrombolysis in myocardial infarction flow 3 (TIMI 3) in over 90% of patients. Despite the recovery of the epicardial coronary circulation, however, additional injury caused by PPCI such as microvascular injury, also known as the no-reflow phenomenon, cannot be ignored. Studies have confirmed that up to 40% to 50% of STEMI patients underwent PPCI may experience no-reflow phenomenon, including microvascular obstruction (MVO) and Intramyocardial hemorrhage (IMH) [3, 4]. Among them, ischemic injury of capillaries leads to the occurrence of endothelial gaps and loss of integrity of capillary wall, and extensive erythrocyte extravasation leads to IMH [5, 6]. Studies demonstrated that IMH was closely related to infarct size, MVO and impaired left ventricular (LV) function, and major adverse cardiac events (MACEs) [7, 8, 9, 10, 11, 12]. Therefore, accurate diagnosis of IMH is of great importance in the clinical. According to the recommendations of the guidelines, cardiac magnetic resonance (CMR) imaging is considered the reference diagnostic method for the evaluation of IMH [1, 13]. However, we found some disadvantages of CMR that should not be ignored in the process of practice. First of all, CMR is time-consuming, and it is dangerous for STEMI patients to go for such time-consuming test without electrocardiogram monitoring in acute phase. Second, for those patients with claustrophobia or other contraindications to CMR, it is not feasible to assess IMH by CMR. Finally, CMR is expensive, which will undoubtedly increase the cost of hospitalization for patients. Therefore, considering the above reasons, we tried to find a safe, simple and effective method to predict the presence of IMH. It is well known that myocardial ischaemia/reperfusion injury (MIRI) is a common cause of no-reflow [14], in which inflammatory response plays an important role [15, 16]. Bochaton et al. [17] analyzed 20 consecutive patients with STEMI-PPCI and found that high-sensitivity C-reactive protein (hsCRP) and neutrophils levels were higher in patients with IMH. In addition, although the innate immune response plays an I mportant role in ischaemia-reperfusion injury (IRI), T lymphocytes including T-helper 1 (Th1), Th2, Th17 and regulatory T (Treg) cells, are also involved in the pathogenesis of IRI.

In this study, we focused on the role of Treg cells in MIRI. As far as I know, Treg can be capable of suppressing the innate immune response by inhibiting the macrophage inflammatory phenotype and neutrophil function [18], thereby playing an anti-inflammatory effect in MIRI. Previous studies have reported that Treg can ameliorate IRI in kidney and brain [19, 20]. Recent studies have also reported the protective effect of Treg in mouse MIRI [21, 22]. In this study, we analyzed the circulating Treg level and other common laboratory indicators, and hypothesized that Treg individually or in combination with other indicators, such as left ventricular end-systolic diameter (LVESD), hsCRP and the peak value of Creatine Kinase MB (pCKMB), can be used to predict the presence of IMH in STEMI patients received PPCI.

This study firstly explored the predictive ability of Treg individually or in combination with other 3 biomarkers, including pCKMB, hsCRP and LVESD, for IMH in STEMI patients underwent PPCI. The major findings were these: (1) in STEMI patients received PPCI, IMH group had lower level of Treg frequency than no-IMH group; (2) Treg frequency, pCKMB, hs-CRP and LVESD were independent risk factors for IMH; (3) the discriminatory capability of the 4-biomarker panel (combination of Treg, pCKMB, hsCRP and LVESD) for IMH was stronger than those of the biomarker individually, except Treg; and (4) increased number of abnormal biomarkers led to a significant increase in the risk of IMH (Treg 1.07%, pCKMB $\ge$73.2 ng/mL, hsCRP $\ge$5.20 mg/L, and LVESD $\ge$3.52 cm). Based on the results of this study, we confirmed the effectiveness of Treg individually or in combination with pCKMB, hsCRP and LVESD in predicting IMH. For those patients who cannot undergo CMR examination, Treg undoubtedly is a simple, safe and effective method to predict IMH in clinical practice.

Acute myocardial infarction, including non-ST elevated myocardial infarction and STEMI, is the leading cause of cardiovascular disease and mortality worldwide [25]. In STEMI, the coronary artery is often acutely completely blocked, and it is pivotal to open the infarct-related artery (IRA) urgently. Therefore, the preferred treatment strategy for STEMI patients within 12 h of symptom onset is coronary revascularization by PPCI in combination with antithrombotic therapy [1, 2]. However, despite the IRA’s epicardial flow has been restored, a sizable proportion of patients continue to experience hypoperfusion of myocardial tissue after PPCI, called no-reflow phenomenon. IMH is a form of no-reflow phenomenon resulting from the extensive erythrocyte aggregation and extravasation because of the damaged endothelial walls [5, 6]. IMH can be visualized by T2-weighted CMR because breakdown products of hemoglobin are paramagnetic and influence regional magnetic tissue properties [26]. However, not all patients are suitable for CMR to clarify the presence of IMH, such as those with serious condition or contraindications. Therefore, we urgently need to find a simple and effective method to predict the presence of IMH. Carrick et al. [11] revealed that IMH was a better predictor of adverse events after myocardial infarction than MVO. Husser et al. [27] found that the incidence of MACEs was significantly higher in the IMH group than in the non-IMH group. In addition, the Kaplan-Meier analysis showed that the MACE-free survival was significant prolonged in patients without IMH, and IMH is a predictor of poor myocardial remodeling [27]. In addition, Amier et al. [3] also demonstrated that IMH was associated with larger myocardial infarct size, greater MVO range, and lower LVEF. Therefore, in this study, we analyzed various indexes of myocardial enzymes and left ventricular structure, and found that pCKMB and LVESD were independent predictors of IMH. Although the molecular mechanisms involved in the initiation and progression of MIRI are still not completely understood, accumulating evidences have suggested that excessive inflammation plays a predominant role in it [15, 28]. And Carrick et al. [11] also found that IMH was associated with markers of inflammation, including peak monocyte count and peak neutrophil count. In this study, we also found that inflammatory markers were significantly higher in patients with IMH than in patients without IMH, and hsCRP was an independent predictor of IMH. Considering that Treg cells have been verified to playing an anti-inflammatory effect in MIRI by inhibiting the macrophage inflammatory phenotype and neutrophil function in animal experiments [18, 21, 22], we hypothesized that the circulating Treg levels are related to the presence of IMH in STEMI patients. The final results were consistent with our expectation that Treg is a protective factor of IMH and can independently predict IMH occurrence in STEMI patients received PPCI. Moreover, we proposed for the first time that Treg can be combined with pCKMB, hsCRP and LVESD to predict the presence of IMH. These findings not only indirectly indicated that Treg may have a certain protective effect on human MIRI, but also provided us with a simpler method to predict IMH than CMR, especially for those who can’t finish the CMR. Considering that previous studies have reported that adoptive transfer of Tregs is beneficial in kidney, brain, liver and myocardial ischaemia/reperfusion (I/R) injury [19, 20, 29, 30], we can assume that adoptive transfer of Tregs can significantly reduce the occurrence of IMH in STEMI patients received PPCI. In the future, we will do a lot of work to confirm the scientific hypothesis that adoptive transfer of Tregs may be an effective biologic therapy for the prevention or treatment of IMH after myocardial infarction.

Limitations of the study are as follows. First, it was a single-center study with a small sample size. Therefore, the research results obtained need to be further verified by a large sample size and multi-center study. Second, because the laboratory parameters are measured only once, there may be potential bias due to measurement error. Third, this study did not include the follow-up data, and it is not clear whether Treg levels can predict prognosis of STEMI patients. In future work, we will not only continue to expand the sample size to confirm the stability of the conclusions, but also collect the follow-up data to further explore the impact of Treg level on the prognosis.

In a word, the current study firstly showed that Treg individually or in combination with pCKMB, hsCRP, and LVESD can effectively predict the presence of IMH in STEMI-PPCI patients.

IMH, intramyocardial hemorrhage; STEMI, ST-segment elevation myocardial infarction; PPCI, primary percutaneous coronary intervention; Treg, regulatory T cell; pCKMB, the peak value of Creatine Kinase MB; hsCRP, high-sensitivity C-reactive protein; LVESD, left ventricular end-systolic diameter; NRI, net reclassification improvement; IDI, integrated discrimination improvement; PSM, propensity score matching; IPTW, inverse probability weighting; TIMI, thrombolysis in myocardial infarction flow; MVO, microvascular obstruction; LV, left ventricular; MACEs, major adverse cardiac events; CMR, cardiac magnetic resonance; MIRI, myocardial ischaemia/reperfusion injury; IRI, ischaemia-reperfusion injury; Th1, T-helper 1; Th2, T-helper 2; Th17, T-helper 17; CABG, coronary artery bypass graft; LVEF, left ventricular ejection fraction; eGFR, estimated glomerular filtration rate; PBMCs, peripheral blood mononuclear cells; Foxp3, transcription factor Forkhead box P3; FITC, fluorescein isothiocyanate; APC, allophycocyanin; PE, phycoerythrin; SD, standard deviation; AAR, area at risk; ROC, receiver operating characteristic; ACEI/ARB, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker; WBC, white blood cell; FPG, fasting plasma glucose; pMYO, the peak value of myoglobin; pTNI, the peak value of troponin I; pNT-pro BNP, the peak value of N-terminal pro-brain natriuretic peptide; AUC, area under the ROC curve; IRA, infarct-related artery.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

YZ participated in the design of the project, the implementation of basic experiments, the statistics of data and the writing of manuscripts. HG, LL and SYL participated in the acquisition of data and the revision of the manuscript. YH, JSL, BH and DHL participated in the data analysis of CMR and the drafting of the content of CMR. HC and WPL contributed discussion and edited the manuscript. HWL provided funding support, designed the study and revised the discussion section of the manuscript. All authors read and approved the final manuscript.

The study data collections were approved by the Institutional Review Board of Beijing Friendship Hospital affiliated to Capital Medical University (Approval No. DR20190043), and written informed consent was obtained from all patients.

We sincerely thank all staffs who have contributed to this subject.

This work was supported by the National Key R&D Program of China (Grant No.2021ZD0111000), the National Natural Science Foundation of China (Grant No.82070357) and Beijing Key Clinical Subject Program.

The authors declare no conflict of interest.