Sodium tanshinone IIA sulfonate prevents the adverse left ventricular remodelling: Focus on polymorphonuclear neutrophil‐derived granule components

Abstract Aims The aims of this study were to evaluate the effects of sodium tanshinone IIA sulfonate (STS) on left ventricular (LV) remodelling after for ST‐elevated myocardial infarction (STEMI). Methods and results In this prospective, randomized clinical trial, 101 patients with the ST‐elevated MI (STEMI) and a successful reperfusion were immediately randomized to receive STS (80 mg qd for 7 days) or saline control, along with standard therapy. The primary effectiveness endpoint is the % change in LV end diastolic volumes index (%∆ LVEDVi) as measured by echocardiography from baseline to 6 months. Secondary effectiveness endpoints include 6‐month period for major adverse cardiac events (MACE), including the occurrence of recurrent myocardial infarction, death, hospitalization for heart failure and malignant arrhythmia. The 6‐month changes in %∆ LVEDVi were significantly smaller in the STS group than in the control group [−5.05% vs 3.32%; P < 0.001]. With respect to MACE, there was a significant difference between those who received STS (8.16%) and those patients on control (26.00%) (P = 0.019). Meaningfully, results of parallel tests aimed at mechanistic explanation of the reported clinical effects, revealed a significantly reduced levels of neutrophils‐derived granule components in the blood of STS treated patients. Conclusion We found that short‐term treatment with STS reduced progressive left ventricular remodelling and subsequent better clinical outcome that could be mechanistically linked to the inhibition of the ultimate damage of infarcted myocardium by infiltrating neutrophils.


| INTRODUC TI ON
Along with the advance of the intensive care system and revascularization therapy in particular primary percutaneous coronary intervention (PCI), the prognosis for ST-elevation myocardial infarction (STEMI) has dramatically improved. As the number of survivors has increased, a concomitant increase in pathologic cardiac remodelling patients after MI has become a huge problem. Cardiac remodelling characterized by progressive changes in ventricle volume and impaired global function was associated with poor clinical outcome. 1 Moreover, once remodelling is established with clinical heart failure symptoms, therapies to reverse remodelling and/or improve symptoms have limited effects. Thus, the development of novel and potentially more effective devices or pharmaceutical agents would be to prevent the remodelling from occurring in the early stage of MI setting is urgently needed.
It has been already reported that degradation of extracellular matrix (ECM), occurring after heart infarction, associates with a consequent loss of myocardium tissue integrity that allows the infarct zone to be stretched by intraventricular pressure, thereby inducing the adverse left ventricular (LV) remodelling. Compelling evidence indicates polymorphonuclear neutrophils (PMN) infiltrated in coronary plaques and the infarcted myocardium plays a pivotal role in the pathologic infarct extension and the stunning of potentially viable myocardium. 2 In addition to producing inflammatory chemokines, neutrophils have been demonstrated mediated tissue damage by releasing matrix-degrading enzymes and reactive oxygen species. 3 A number of clinical trials reported the increased ratio of circulating neutrophils to lymphocyte could be recognized as a prognostic factor to predict both major adverse cardiac events and chronic LV remodelling in patients suffered from MI. 4,5 Moreover, abnormal elevations in neutrophil count in blood of patients with acute coronary syndrome undergoing primary percutaneous coronary intervention (PCI) associates with larger infarct sizes and worsening of cardiac systolic and diastolic function. 6 Importantly, promising results obtained from preclinical models studying several pharmacological approaches aimed at interfering with the myocardial neutrophil's recruitment, confirmed their beneficial effects on reducing infarct size and the extent of the ultimate cardiac injury. 7 Hence, suggesting that neutrophils depletion strategies could be a desirable pharmacological strategy in patients with MI at a higher risk of LV remodelling.
The cardioprotective effects of sodium tanshinone IIA sulfonate (STS), a potent pharmacological derivative compound extracted from Salvia miltiorrhizabunge, have been elucidated by a number of studies.
Experimental preclinical data have demonstrated that administering STS therapy in rat in vivo model of ischaemia/reperfusion caused inhibition of the nuclear factor-κB-dependent activated accumulation of neutrophils and consequently protected the heart against myocardial reperfusion injury and reduced the infarct size. 8 Results of the another recently published study, demonstrated that administration of STS in Beagle dogs ameliorated their ischaemia-induced myocardial inflammation, after inhibiting NLRP3 inflammasome-dependent JAK2-STAT3 pathway. 9 Moreover, administration of STS also exerted anti-fibrotic effects on cardiac fibroblasts by downregulating generation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species. 10 Based on these findings, it is conceivable to suggest that STS may play a protective role against post-MI failure by limiting the neutrophils infiltrations and release of PMN-derived granule components.
This study was designed to test whether administration of STS in patients with AMI would attenuate remodelling and reduce the occurrence of major adverse cardiac events (MACE). Importantly, the obtained results clearly demonstrated that treatment of our MI patients with this pharmacological compound, holding the unique structure of phytoestrogen, significantly alleviated the neutrophilmediated myocardial tissue damage and consequently reduced the net harmful post-MI outcome.

| Study design
The study is a randomized, parallel-group, controlled study comparing STS with saline control in patients who survived a STEMI after successful reperfusion. Ethics committees at Guangdong Provincial Hospital of CM reviewed and gave their approval (B2011-41-01). All principles of the Declaration of Helsinki were applied. This trial was registered in clinicaltrials.gov (NCT02524964).

| Participates and intervention
The eligible patients were aged 18-80 years, who presented within 24 hours of the onset of symptoms and signs of STEMI, defined as significant ST-segment elevation in at least two contiguous leads according to the recommendation of the American College of Cardiology and European Society of Cardiology. 11 The patients were not considered for enrolment if they presented with cardiogenic shock, severe heart failure (New York Heart Association class IV and need for intravenous inotropic support), sustained ventricular tachycardia or ventricular fibrillation or had return of spontaneous circulation or had identified contraindication to STS.
Consenting, eligible patients or their legally authorized representative provided written informed consent and then randomized in a ratio of 1:1 to STS or saline control. The randomization was performed with a computerized central Interactive Voice and Web Response System. All principles received guidelines-based therapy for STEMI per the discretion of the attending cardiologist. STS was intravenously injected at 80 mg dose immediately after primary or rescue PCI and then every 24 hours for 7 days.

| Effectiveness endpoints
The primary effectiveness endpoint is the %∆ LVEDVi measured by echocardiography from baseline to 6 months after PCI. Secondary effectiveness endpoints include composite of all-cause death, recurrence of MI documented by symptoms and with a new rise in cardiac biomarkers and hospitalization due to heart failure events adjudicated by an independent clinical events committee using standardized definitions. Additional exploratory proteomics analyses were also performed in hope for better mechanistic explanation of the observed clinical effects of STS treatment.

| Echocardiographic measurement of cardiac remodelling
Resting transthoracic echocardiograms will be performed before randomization and then at 6-month follow-up visit. Two-dimensional echocardiographic studies were recorded and submitted to the independent, blinded laboratory for a centralized assessment of quantitative analysis after quality control. As previous reports, the selected images were digitized to obtain endocardial contours and LV cavity volumes at end diastole and end systole from apical orthogonal views including 4-and 2-chamber. 12 The modified Simpson rule was used to calculate EF value using the standard formula according to American Society of Echocardiography guideline recommendations. All measurements were measured by an experienced sonographer from 5 cardiac cycles and the mean values were considered for analysis.

| Proteomics analysis of peripheral blood samples
The blood samples from both STS (n = 9) and saline control (n = 9) groups after 5-day treatment were prepared and assessed the variable cytokines using a Quantibody Human Cytokine Antibody Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway analysis to discover a relation that was not easily visible from the changes of individual proteins.

| Quantification of the neutrophils-derived granule components in peripheral blood
To further confirm the proteins response to STS treatment, plasma and serum from patients received either STS (n = 48) or saline (n = 48) before and after 5-day treatment were measured using a sandwich enzyme-linked immunosorbent assay (ELISA) procedure following manufacturer's protocols (R&D systems Minneapolis, MN, USA).

| Sample size
The sample size determination is based on the primary endpoint, estimated change in LVEDVi from baseline to 6 months after STS treatment. To have >80% power and detect a treatment difference of 5% in change of LVEDVi between STS and saline control (with allocation ratio of 1:1) using a two-sided significance level of 0.05, a minimum of 46 patients were required in each arm. A therapeutic decrease in LVEDV of 5 mL/m 2 has been shown to be associated with favourable effects on mortality. 14 Additional patients will be recruited to account for potential dropouts.

| Statistical analysis
All pharmacokinetic parameters were summarized as mean ± standard deviation, median (first quartile, third quartile), percent coefficient of variation as appropriate. General linear mixed models were used to perform an intention-to-treat analysis that consists of all randomized patients for the effectiveness analyses. The primary and secondary endpoints were assessed using analysis of covariance with major determinants, including demographic parameters

| Patients and baseline clinical characteristics
Between February 2014 and December 2016, 101 patients were randomized (50 to STS and 51 to saline control). Figure 1 illustrates study enrolment and randomization. There was one patient in the STS group and two in the control group lost to follow-up.
Baseline demographics stratified by treatment arm are shown in   (Table 1).
Overall, all patients displayed thrombolysis in myocardial infarction (TIMI) 3 flow within the infarct-related artery, and there was high adherence to all post-MI guideline-recommended therapies.
In the overall cohort, 65.35% of patients were treated with an angiotensin-converting enzyme inhibitor or an angiotensin II receptor blocker, in comparison with 73.27% of those who received β-adrenergic blocking agents.

| Echocardiographic endpoints
The echocardiographic measurements at 6-month follow-up are shown in Figure 2 Left ventricular end-systolic volume index (LVESVi) slightly increased in the control group while was reduced in the STS group (1.82 ± 1.52 and −2.36 ± 1.33 mL/m 2 , respectively, P < 0.01).
Consequently, a statistically significant difference occurred in LVEF between the two groups at the 6-month follow-up: a significantly higher LVEF (55.81 ± 6.98 and 49.94 ± 7.19%, respectively, P < 0.01) were found in the STS group compared with the control group.

| Effects of STS treatment on levels of biomarkers detected by proteomics
As proteomic analysis has been already documented as a powerful tool for the identification of novel mechanisms in response to diverse pharmacologic therapies, we further investigate the mechanism underlying the anti-remodelling effect of STS, that could be linked to alterations of numerous circulating factors, detected by antibodies microarrays in blood of patients treated either with STS or with control saline. Compared with the control patients, 75 proteins with significantly differential expression (P < 0.05) were identified in the STS group ( Figure 3). Among them, 42 proteins were upregulated and 33 were downregulated. Cluster analysis was performed based on the differentially expressed cytokines and displayed as heat maps ( Figure 3A). As shown in Figure 3C, applied KEGG analysis allowed us to identify particular pathways that were significantly affected by differentially expressed interleukins and their interactions with cytokine-cytokine cellular receptors that eventually affected the cellular chemotaxis, proliferation and re-organization of the extracellular matrix. These data encouraged the further quantitative assessment of biologically active factors that can be released from neutrophils and potentially affect myocardium.

| Quantification of the neutrophil granulesderived enzymes
As previous studies already documented that neutrophils play an essential role in tissue remodelling after several tissue injures   Growing evidence from numerous studies already suggested that the initial post-MI inflammatory oxidative stress that also occurs within the remote non-infarcted tissues and circulated leucocytes, associates with the higher release of numerous chemokines and proteolytic enzymes that may infiltrate the adjacent infarcted myocardium, and consequently induce the partial degradation of its extracellular matrix and its pathologic remodelling that ultimately contribute to deterioration of cardiac haemodynamic. Among the other injured tissues-derived proteases, neutrophil elastase, released in the early stages of ischaemia, degrades elastin and induces interleukin-6 secretion to impair cardiac contractility by a nitric oxide-dependent pathway contributing to cardiac damage. Meaningfully, it has been shown that the selective pharmacologic inhibition of neutrophil elastase protects against myocardial stunning after ischaemia/ reperfusion in swine and reduce the infarct size. [19][20][21][22] On the other hand, it has been demonstrated that proteinase 3 exacerbate heart failure through cleaving angiotensinogen into angiotensin, activating proinflammatory factors and degrading extracellular matrix including fibronectin and collagen IV. 23,24 In addition, proteinase 3 levels in the plasma serve as a prognostic risk marker for death or rehospitalization for heart failure in post-MI patients. 24 Similarly, high plasma NGAL levels before PCI has been shown to independently predict all-cause mortality for patients with MI, the underlying mechanisms involves protecting MMP-9 from degradation. 25,26 The evidence of higher total and active MMP-8 levels were shown in patients with F I G U R E 4 Quantification of neutrophils-derived granule components. Levels of neutrophils-derived granule components, including neutrophil elastase, myeloperoxidase, proteinase 3, NGAL, MMP-8 and MMP-9 in patients receiving STS or saline control were assessed by ELISA. NGAL, neutrophil gelatinase-associated lipocalin. MMP, matrix metalloproteinases LV rupture than those without rupture, indicating this neutrophil collagenase may promote infarct rupture in humans. 27 Further studies suggested MMP-8 degrades collagens via cleaving type I α1 and α2

| D ISCUSS I ON
chains and subsequently promotes neutrophils migration, and neutrophils depletion inhibits early collagen degradation as a consequence of the lack MMP-8. 28,29 Infiltrating neutrophils are an early source of MMP-9 after MI both with and without reperfusion in humans and multiple animal models. 30  This is a single-centre experience and represents a small number of patients. LV volumes were obtained by echocardiography whenever possible; while cardiac magnetic resonance may be a more precise method. However, our study population contains homogenous unselected patients with STEMI undergoing PCI, thus mirroring the real-world scenario. Future randomized studies with a large sample size of patients with first onset of AMI with a proximal LAD occlusion are required to overcome these limitations.

| CON CLUS IONS
In conclusion, our study demonstrated that addition of STS to current practice guidelines-recommended therapies further reduces the progressive LV remodelling and improves the clinical outcome after acute MI. As, STS treatment did not reduce a total number of neutrophils detected in peripheral blood, but decreased levels of circulating chemokines and proteases, that could be released from their granules and vesicles, we suggest that the immediate daily IV treatment of MI patients with STS mitigates the early release of numerous factors that would be otherwise attracted by the degradation products of the injured myocardial matrix and ultimately exacerbate remodelling of the post-MI heart. Therefore, at this point we might speculate that this phytomedicine might stabilize integrity of cellular membranes, thereby reducing the net level of neutrophil's exocytosis and rupture of their granules and vesicles.

ACK N OWLED G EM ENTS
The authors gratefully acknowledge the contributions of all participants involved in this trial. This work was financially supported nor the decision to publish.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AUTH O R S' CO NTR I B UTI O N S
SM drafted this manuscript; SM, QC and ST performed the experiments, MZZ made statistical analysis; AH made critical revision of the manuscript and contributed to the rationalization of the study.
All authors read and approved the final manuscript.