Association between Cystatin C and Cardiac Function in Acute Myocardial Infarction Patients: A Real-World Analysis

Background Acute myocardial infarction (AMI), as well as its long-term and short-term complications, is known to present with high morbidity and mortality. Cardiac function deterioration and ventricular remodelling after AMI are known to be correlated to worse long-term outcomes. However, the underlying mechanism remains elusive and there is a shortage of serum prediction markers. This study investigates the relationship between in-hospital Cystatin C (CysC) and cardiac function and subsequent prognosis among AMI patients. Research Design and Methods. We measured admission CysC and cardiac function parameters, including ejection fraction (EF) and pro-BNP value in 5956 patients diagnosed with AMI. Simple and multiregression analyses were performed to investigate the correlation between CysC and cardiac function in AMI patients. Major adverse cardiovascular events (MACE), cardiovascular, and all-cause mortality were documented, and 351 participants with high cystatin (≥1.09 mg/L) and 714 low cystatin (<1.09 mg/L) were investigated for survival analysis during a 48-month follow-up. Results 5956 patients with AMI were enrolled in the initial observational analysis, and 1065 patients of the whole cohort were included in the follow-up survival analysis. The admission CysC level was found to be significantly positively correlated to the pro-BNP level (R square = 0.2142, 95% CI 4758 to 5265, p < 0.0001) and negatively correlated to the EF value (R square = 0.0095, 95% CI -3.503 to -1.605, p < 0.0001). Kaplan-Meier survival analysis revealed significantly increased MACE incidence (HR = 2.293, 95% CI 1.400 to 3.755, p < 0.0001), cardiovascular mortality (HR = 3.016, 95% CI 1.694 to 5.371, p = 0.0002), and all-cause mortality (HR = 3.424, 95% CI 2.010 to 5.835, p < 0.0001) in high-admission CysC cohort with AMI at the end of 4-year follow-up. Conclusions Admission CysC is negatively correlated with cardiac function in AMI patients and acts as a novel predictor for MACE incidence in the whole population. Further studies are needed to investigate the specific mechanism of CysC in the cardiac function deterioration among AMI patients.


Introduction
As one of the leading health-threatening diseases worldwide [1], acute myocardial infarction (AMI) is associated with substantial morbidity and mortality [2]. Despite advances in percutaneous coronary interventions (PCI) and their widespread use, the mortality rate of AMI patients, together with its complications, such as heart failure, severe arrhythmia, myocardial free wall rupture (MFWR), and cardiogenic shock (CS), remain very high [3,4]. Especially, cardiac function deterioration and ventricular remodelling after AMI are known to be correlated with increased rehospitalization rate and worse long-term outcomes [5] and have attracted more and more attention. However, the underlying mechanism remains elusive and there is a shortage of serum prediction markers.
Cystatin C (CysC), a low-molecular-weight (13 kDa) protease inhibitor, is synthesized and released into the blood by all nucleated cells, freely filtered by kidney glomerulus and almost completely reabsorbed and metabolized by the proximal tubule, but not secreted [6]. Due to these properties, even very small changes in the glomerular filtration rate (GFR) may significantly alter serum CysC level, potentially making this basic protein a very sensitive marker of renal filtration [7]. Since it was first described in 1961 by Jorgen Clausen in human cerebrospinal fluid [8], CysC has been thoroughly investigated and is considered a promising biomarker for several diseases, including but not limited to kidney disease and nephropathy-related diabetes [9,10], Alzheimer's disease [11], and breast cancer [12].
CysC plays pleiotropic roles in human vascular pathophysiology, particularly in regulating cathepsins S and K [13]. In vivo and in vitro studies have shown elevated levels of cathepsins and lower levels of CysC, which behaves as a potent cathepsin inhibitor-in atherosclerotic tissue [14]. Correspondingly, several studies investigated the functional role of CysC in cardiovascular disease (CVD). An observational meta-analysis showed a strong dose-dependent relation between cystatin C concentrations and CVD [14]. Rothenbacher and his team found the use of cysC based chronic kidney disease (CKD) may result in more accurate risk estimates and have better prognostic value for CVD than creatinine [15]. Additionally, in high-risk patients after ACS, CysC is a strong predictor of major adverse cardiovascular events (MACE), including death from cardiovascular causes and hospitalization for heart failure [16]. However, the evidence for the relationship between in-hospital CysC and cardiac function and subsequent long-term prognosis among AMI patients remains unclear.
In this retrospective cohort study, we investigate the relationship between admission CysC and cardiac in AMI patients. Subsequently, survival analysis was performed to investigate the effects of admission CysC levels on longterm mortality and morbidity in AMI patients.

Study Design and Participants.
This was a single-center, retrospective cohort study. Consecutive patients admitted to the cardiology department of the First Affiliated Hospital of Xi'an Jiaotong University for AMI between January 2016 and December 2020 were enrolled. The inclusion criteria were confirmed admission diagnosis of AMI, and AMI was defined based on the universal definition criteria by the American Cardiology College [1]. The exclusion criteria were [1] severe noncardiac disease with an expected survival of less than 1 year and unwillingness to participate, [2] patients over the age of 80 years or living far away from the hospital's catchment area, and (3)extremely high CysC level (>5 mg/L). A patient could only be included once. The medical records of the patients were collected from the Biobank of the First Affiliated Hospital of Xi'an Jiaotong University, which contains deidentified data derived from raw medical records. Information about patients' present medication, vascular risk factors, and detailed medical history were obtained via questionnaires. Follow-up information was obtained via telephone and questionnaires by the general practitioner (GP). Patients' MACE, including newonset myocardial infarction, acute heart failure and cardiac death, and cardiovascular and all-cause mortality were documented during follow-up. Written informed consent was obtained from all study participants, with ethnic committee approval at the First Affiliated Hospital of Xi'an Jiaotong University.

Clinical Data Collection.
Detailed medical histories were screened from the patients enrolled. Patient characteristics were collected, including age, sex, disease history, and physical examination. Serum CysC levels of all patients were measured within 3 h of admission, by colloidal gold particleenhanced colorimetric immunoassay (Nescauto GC Cystatin C, Alfresa Pharma, Osaka, Japan) with a Hitachi 7600-110 automatic analyzer. Other biochemical results were evaluated immediately after the patients' admission to the hospital. They were all collected prior to PCI. Echocardiography was performed during hospital treatment.

Statistical Analysis.
All statistical analyses were performed by using SPSS for Mac 22.0 (SPSS Inc., Chicago, IL) or GraphPad 9.0 Prism (GraphPad Software San Diego, CA). Data were presented as frequencies or percentages for categorical variables and mean ± SD for continuous variables, unless otherwise indicated. Simple t-test was used to compare continuous variables which are in the normal distribution. Mann-Whitney U test was used to compare continuous variables which do not conform to the normal distribution. χ 2 test was used to compare categorical variables. One-way ANOVA was used to compare continuous variables of three or more independent (unrelated) groups. Simple linear analysis was used for calculating the correlation between CysC and cardiac function parameters. Kaplan-Meier survival curve analysis was used to represent the proportional risk of MACE, cardiovascular, and all-cause mortality for the admission CysC values in AMI patients. A Cox proportional-hazards model was performed to provide a point estimate HR (hazard ratio) and a two-sided 95% confidence interval. Receiver-operator characteristic (ROC) curve analysis and the area under the ROC curve (AUC) were used to compare the predictive value of MACE, cardiovascular, and all-cause mortality among CysC and other indexes. A value of p < 0:05 was considered statistically significant.

Study Population.
From January 2017 till December 2020, a total of 5973 AMI patients were enrolled in the study and 17 patients with extremely high CysC levels (>5 mg/L) were excluded. According to the universal definition criteria and Cutoff Finder, [17,18] Table 1, and the correlation between admission CysC and other metabolomic indexes is displayed in Table 2. The mean value of CysC was 0:76 ± 0:19 mg/L in low CysC and 1:41 ± 0:47 mg/L in the high CysC cohort. The medication was started at admission. No significant difference in blood pressure/HbA1c/TG at baseline was seen in different CysC groups in AMI patients. Subgroup analysis further indicated that, consistently, circulating pro-BNP and cardiac troponin T (cTnT) was higher in high-admission CysC cohort than controls (pro-BNP: 1222 vs. 577.4, p < 0:001; cTnT 1.340 vs. 1.644, p < 0:001) (Figures 2(d) and 2(e)). We also found that high-

Increased Hospital Mortality in High-Admission CysC
Cohort with AMI. As the most frequently used risk assessment tools, the 'Global Registry of Acute Coronary Events' (GRACE) and the 'Can Rapid risk stratification of Unstable angina patients Suppress Adverse outcomes with Early implementation of the American College of Cardiology/ American Heart Association guidelines' (CRUSADE) scores were recommended in describing the severity and mortality risk of AMI patients and management [19]. The average GRACE and CRUSADE scores were 124.5 and 21.74 in all AMI patients, respectively. Both GRACE and CRUSADE scores were significantly positively correlated to the CysC value ( Fig S1). In subgroup analysis, we found GRACE and CRUSADE scores were significantly higher in highadmission CysC cohort than controls (GRACE score 133.6    (Figure 2(a)). 5038 (84.0%) AMI patients were completely reperfused with thrombolysis in myocardial infarction ðTIMIÞ > 2 after PCI, and 918 (16.0%) AMI patients failed to reperfusion with TIMI ≤ 2 and only received medication treatment and intervention. No reflow or slow flow following PCI is independently associated with increased in-hospital mortality, malignant arrhythmias, and cardiac failure [20]. Interestingly, CysC was significantly higher in group TIMI ≤ 2 compared to group TIMI > 2 (0.93 vs. 1.13, p < 0:0001).

Increased MACE, Cardiovascular, and All-Cause
Receiver operating characteristic curves were generated, and AUCs were calculated to estimate the predicted values of different biomarkers. The performance of CysC, pro-BNP, uric acid (UA), and creatine (Cre) in predicting MACE, cardiovascular, and all-cause mortality, and MACE was illustrated in Figure 6. CysC showed significant and similar predictive accuracy as compared to pro-BNP. Cre also exhibited a significant predicting value while UA showed no difference in MACE and mortality prediction.

Discussion
In this single-center, retrospective, real-world, populationbased study, we investigate the relationship between inhospital Cystatin C (CysC) and cardiac function and subsequent prognosis among AMI patients. Serum CysC is found to be associated with cardiac function deterioration in patients with AMI. Moreover, high-admission serum CysC level exhibits high incidence of MACE as well as cardiovascular and all-cause mortality rate in AMI patients during 4-year follow-up.
The important implication of the present study is that CysC is identified as a biomarker for cardiac function in AMI patients. Several previous studies investigated the 7 Disease Markers relationship between CysC and heart failure incidents. Via 4 community-based cohorts with 12.5 years of follow-up, Navin Suthahar and their team found CysC was strongly and similarly associated with HF in both sexes [21], as these biomarkers reflect distinct pathophysiological processes [22], and the elevation may indicate cardiovascular or systemic derangement early in the time course of HF progression [23]. Additionally, our result indicated that CysC might be eligible as a potential serum predictor for heart failure in the population after acute myocardial infarction.
Notably, the major outcomes of this study show increased MACE incidence, cardiovascular, and all-cause   Figure 4: Kaplan-Meier survival analysis revealed significantly increased MACE incidence in high-admission CysC cohort with AMI at the end of 4-year follow-up. Kaplan-Meier survival analysis revealed high-admission CysC cohort (CysC ≥ 1:09 mg/L) displayed significantly increased MACE incidence (HR = 2:293, 95% CI 1.400 to 3.755, p < 0:0001) than controls (CysC < 1:09 mg/L). 8 Disease Markers mortality in the high CysC cohort with AMI at the end of 4-year follow-up, indicating that CysC level is a potential independent predictor for cardiac prognosis after AMI. Increasing shreds of evidence have shown that higher CysC is associated with higher cardiovascular risk and mortality rate in patients with non-ST elevated acute coronary syndrome [24], and serum creatinine to cystatin C ratio is associated with major adverse cardiovascular events in patients with obstructive coronary artery disease [25]. In AMI patients, an increased admission CysC level was associated with a higher risk of in-hospital and 1-month death [26]. Besides, circulating cystatin C level on the 12th-14th day after hospital admission predicted the adverse cardiovascular outcome in patients with STEMI [27]. Through a 4-year follow-up study, we further proved that CysC can be included in the risk stratification model to guide the treatment of high-risk AMI patients. Several potential mechanisms may account for the prognostic importance of CysC in AMI patients. First, abnormal CysC value can identify early patients with renal insufficiency before circulating creatinine, which could be linked to atherosclerosis, vascular complications, and increased cardiovascular events [28]. Second, CysC may play an important role in regulating cardiac inflammatory responses [29], contributing to the development of no-reflow and the increased risk of death. Besides, CysC elevation may damage 9 Disease Markers the cardiovascular system by affecting lipid peroxidation, coagulation function, and smooth muscle cell and endothelial cell function [30], and finally, facilitate the vulnerability of atherosclerotic plaque CysC [31].
However, there are several potential limitations in the current work: first, this study is limited in its single-center, retrospective, and observational nature. A future multicenter prospective study with a larger number of patients and a   Figure 6: ROC for predicting MACE, cardiovascular, and all-cause mortality among CysC, pro-BNP, Cre, and UA. Accuracies of CysC, pro-BNP, Cre, and UA for predicting MACE (a), cardiovascular mortality (b), and all-cause mortality(c) presented as areas under the receiver operating characteristic curves, individually. ROC: receiver-operator characteristic; AUC: area under the receiver operating characteristic curve.
10 Disease Markers longer follow-up is required. Also, several parameters, including patient age were not balanced between the high and low CysC cohort. As age might also be a prognostic factor in AMI patients, improving risk stratification independently of age and kidney function would be considered in the further study.
In conclusion, through this retrospective cohort study, we have found that admission CysC is negatively correlated with cardiac function in AMI patients and acts as a novel predictor for MACE incidence in the whole population. Further studies are needed to investigate the specific mechanism of CysC during the cardiac function deterioration of AMI patients.

Data Availability
The authors confirm that the data supporting the findings of this study are available from the corresponding author JS upon reasonable request.