Endoplasmic reticulum stress-related secretory protein in the preparation of heart source sudden death diagnostic reagent

Abstract

The present application belongs to the technical field of biological medicine, and relates to a new use of endoplasmic reticulum stress related secretory proteins, in particular to the forensic diagnosis application of LMAN2, CAPN-1, VCP serum concentration changes in early myocardial ischemia caused cardiogenic sudden death. The present application also provides a corresponding kit. Experiments prove that the endoplasmic reticulum stress related secretory proteins (LMAN2, CAPN-1, VCP) have good diagnostic value for early myocardial ischemia caused cardiogenic sudden death, and are superior to the currently commonly used diagnostic myocardial injury CK-MB and cTn-I indicators, and are expected to be used as a sensitive diagnostic indicator for early myocardial ischemia. The present application opens up a new clue for the forensic diagnosis of early myocardial ischemia caused cardiogenic sudden death.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to new uses of endoplasmic reticulum stress-related secretory proteins. Specifically, it relates to the application of endoplasmic reticulum stress-related secretory proteins in the preparation of diagnostic reagents for sudden cardiac death, especially the application of changes in serum concentrations of endoplasmic reticulum stress-related secretory proteins LMAN2, CAPN-1, and VCP in the preparation of forensic diagnostic reagents for sudden cardiac death caused by early myocardial ischemia. Background Technology

[0002] Clinical practice shows that sudden cardiac death is a serious threat to human life and health, causing more than 7 million deaths worldwide each year, accounting for more than 80% of all types of sudden death. Myocardial ischemia is a common cause of sudden cardiac death, but forensic medicine does not focus on sudden cardiac death caused by early myocardial ischemia. early myocardial ischemia- induced sudden cardiac death, EMI-SCD Diagnosing sudden cardiac death caused by early myocardial ischemia remains a challenge because these cases typically involve acute myocardial ischemia injury occurring within minutes to hours, during which time the myocardium usually does not exhibit gross or histologically characteristic changes. Sometimes, early myocardial ischemic injury may appear under a light microscope as wavy changes in muscle fibers, shrunken nuclei, strongly eosinophilic myocardial cytoplasm, homogenization of the cytoplasm, and necrosis of the contractile bands. However, these morphological changes can also occur in other diseases and conditions, such as electric shock, amphetamine overdose, and cardiac defibrillation during the dying phase. Therefore, diagnosing sudden cardiac death caused by early myocardial ischemia based on morphological changes lacks specificity.

[0003] Some scholars have suggested using molecular diagnostic biomarkers to compensate for the shortcomings of classical anatomical and histological diagnoses. Studies have shown that immunohistochemical staining for specific molecular biomarkers can aid in the diagnosis of acute myocardial ischemia. These molecular biomarkers can be categorized into complement components, inflammatory regulatory factors, cardiomyocyte proteins, plasma proteins, and stress and hypoxia-inducible factors. Among them, connexin 43 (Cx43), Jun B, apoptosis markers, myoglobin, fibronectin, and C5b-9 have been extensively studied. Cx43, in particular, has good reference value when the interval between ischemic events is less than 30 minutes, while Jun B, apoptosis markers, C5b-9, and myoglobin have higher diagnostic value when ischemic injury lasts for 2 hours. However, it should be noted that the actual utility and applicability of these biomarkers in autopsy samples are not yet clear, and some biomarkers (such as Cx43, Jun B, troponin, and apoptosis markers) have insufficient specificity. Furthermore, immunohistochemical staining is subject to tissue sampling bias and the subjectivity of staining intensity assessment. Using quantitative techniques to detect secreted proteins in body fluids can avoid the aforementioned biases and controversies, and facilitates a faster and non-invasive diagnostic approach, making it a promising diagnostic method. In clinical practice, blood is often drawn from patients to detect myocardial enzyme levels, which can determine the presence of myocardial damage, avoiding the trauma and time-consuming drawbacks of invasive techniques.

[0004] Studies have shown a strong link between prolonged extreme stress and sudden cardiac death. Prolonged exposure to life and work stress, i.e., chronic stress, often triggers sudden cardiac death. In vitro, prolonged stress can be transmitted intracellularly as substructural and molecular changes, most importantly inducing endoplasmic reticulum (ER) stress. ER stress is a cellular response to physiological or pathological stimuli when ER homeostasis is disrupted. Under ischemic conditions, proteins in the ER undergo misfolding, leading to activation of the unfolded protein response (UPR). Sustained UPR further exacerbates ER stress through related signaling pathways, resulting in the release of large amounts of ER stress-related secretory proteins into the bloodstream. This release may precede acute myocardial morphological changes. Therefore, screening for ER stress-related secretory proteins for the diagnosis of early myocardial ischemia-induced sudden cardiac death is theoretically feasible.

[0005] Based on the current state and foundation of existing technology, the inventors of this application intend to provide new uses for endoplasmic reticulum stress-related secretory proteins, especially the application of endoplasmic reticulum stress-related secretory proteins in the preparation of diagnostic reagents for sudden cardiac death. Summary of the Invention

[0006] The purpose of this invention is to provide new applications for endoplasmic reticulum stress-related secretory proteins based on the current state and foundation of existing technologies, specifically involving the application of endoplasmic reticulum stress-related secretory proteins in the preparation of diagnostic reagents for sudden cardiac death. In particular, it relates to the application of changes in serum concentrations of endoplasmic reticulum stress-related secretory proteins LMAN2, CAPN-1, and VCP in the preparation of forensic diagnostic reagents for sudden cardiac death caused by early myocardial ischemia.

[0007] This invention collected 21 cases of sudden cardiac death caused by early myocardial ischemia (survival time <6h) and used 23 cases of non-cardiac sudden death as controls for detection and analysis. The results showed that the serum levels of three endoplasmic reticulum stress-related secretory proteins, LMAN2, CAPN-1, and VCP, were significantly elevated in the group of sudden cardiac death caused by early myocardial ischemia, with LMAN2 increasing by 48.0% (p=0.0022), CAPN-1 by 32.6% (p=0.041), and VCP by 26.2% (p=0.021). Further results showed that the levels of LMAN2, CAPN-1, and VCP did not differ significantly between cases of sudden death occurring during the day and at night; and within 4h post-mortem in the relevant cases, the levels of the above three indicators did not change with the extension of time after death. Meanwhile, the serum levels of LMAN2, CAPN-1, and VCP were not significantly correlated with the age of the deceased or the month of sudden death, indicating that the serum levels of LMAN2, CAPN-1, and VCP were stable and unaffected by mortality factors.

[0008] Furthermore, this invention provides the application of endoplasmic reticulum stress-related secretory proteins in the preparation of diagnostic reagents for sudden cardiac death. Specifically, it relates the application of changes in serum concentrations of endoplasmic reticulum stress-related secretory proteins LMAN2, CAPN-1, and VCP in the preparation of forensic diagnostic reagents for sudden cardiac death caused by early myocardial ischemia.

[0009] More specifically,

[0010] This invention provides a diagnostic kit for sudden cardiac death caused by early myocardial ischemia, the kit containing the detection reagent for endoplasmic reticulum stress-related secretory proteins.

[0011] The kit described herein is useful for diagnosing sudden cardiac death caused by early myocardial ischemia.

[0012] The kit of the present invention contains an ELISA plate coated with protein affinity antibody, endoplasmic reticulum stress-related secretory protein standards, sample diluent, chromogenic solution, stop solution, and instructions for use.

[0013] Using this kit to detect serum LMAN2, CAPN-1, and VCP, the ROC curve analysis results for the diagnosis of EMI-SCD were as follows: the area under the curve (AUC) for serum LMAN2 was 0.8178 (95% CI: 0.6949–0.9407, p=0.0003), the area under the curve (AUC) for CAPN-1 was 0.6988 (95% CI: 0.5429–0.8546, p=0.0241), and the area under the curve (AUC) for VCP was 0.7267 (95% CI: 0.5752–0.8782, p=0.0101).

[0014] At a threshold of 23.63 ng / ml, serum LMAN2 showed a sensitivity of 90.84% ​​and a specificity of 60.87% in diagnosing EMI-SCD. Serum CAPN-1 showed a sensitivity of 76.19% and a specificity of 60.87% in diagnosing EMI-SCD at a threshold of 17.67 ng / ml. Serum VCP showed a sensitivity of 76.19% and a specificity of 65.22% in diagnosing EMI-SCD at a threshold of 3.222 ng / ml.

[0015] This invention compares the diagnostic efficacy of the described kit with that of CK-MB and cTn-I, which are currently widely used in clinical practice, in early ischemia:

[0016] The AUC for serum CK-MB in diagnosing EMI-SCD was 0.5590 (95% CI: 0.3851–0.7329, p = 0.5031), and the AUC for cTn-I was 0.5911 (95% CI: 0.4220–0.7602, p = 0.3012). CK-MB showed a diagnostic sensitivity of 85.71% and a diagnostic specificity of 43.48% at a threshold of 143 ng / mL, while cTn-I showed a diagnostic sensitivity of 85.71% and a diagnostic specificity of 34.78% at a threshold of 254.9 ng / L. These results indicate that the kit of this invention has diagnostic sensitivity comparable to CK-MB and cTn-I for EMI-SCD (with the highest sensitivity at 90.84% ​​for LMAN2), but significantly higher diagnostic specificity than both CK-MB and cTn-I, thus meeting the specific diagnostic needs of real-world cases.

[0017] Table 1. Basic information of 21 cases of sudden cardiac death caused by early myocardial ischemia (EMI-SCD)

[0018] Trigg endoplasmic reticulum stress n (%) Male / female Age (mean ± SEM) Survival time (h)(median) PMI (h)(median) alcohol intake 3 (14.29) 3 / 0 45.3±9.0 <0.5-3(<1) <1-4 (<3) quarrel 2 (9.52) 1 / 1 61.0±1.0 <0.5(<0.5) <0.5 (<0.5) Daily activities 4 (19.05) 4 / 0 47.8±5.8 <0.5-1(<0.5) <0.5 (<0.5) physical exertion 1 (4.76) 1 / 0 60.0±0.0 <0.5(<0.5) <0.5 (<0.5) unknown 11 (52.38) 10 / 1 51.2±12.8 <0.5-6(<1) <0.5-4 (<1) Total 21 (100.00) 19 / 2 53.0±10.5 <2.77 (<1) <1.09 (<1)

[0019] Table 2. Comparison of cases of sudden cardiac death caused by early myocardial ischemia with control group cases.

[0020] Category Control (n=23) EMI-SCD (n=21) P value Age (years) 44.6±15.0 53.0±10.5 0.094 Gender, M (%) 65.2% 90.1% 0.11 CK-MB (ng / ml) 95.9±52.3 169.1±80.4 0.18 cTn-I (ng / L) 179.6±64.6 276.2±76.3 0.15 Death time (Daytime%) 95.6% 31.8% <0.0001 Survival time (h) <2.59(±2.48) <2.77(±2.56) 0.31 PMI (h) <0.65(±0.52) <1.09(±0.88) 0.26

[0021] This invention provides the forensic diagnostic application of changes in serum concentrations of endoplasmic reticulum stress-related secreted proteins LMAN2, CAPN-1, and VCP in sudden cardiac death caused by early myocardial ischemia. A corresponding reagent kit is also provided. Experiments have confirmed that endoplasmic reticulum stress-related secreted proteins (LMAN2, CAPN-1, and VCP) have good diagnostic value for sudden cardiac death caused by early myocardial ischemia, and are superior to the currently commonly used clinical indicators CK-MB and cTn-I for diagnosing myocardial injury, showing promise as sensitive diagnostic indicators for early myocardial ischemia. This invention opens up new clues for the forensic diagnosis of sudden cardiac death caused by early myocardial ischemia. Attached Figure Description

[0022] Figure 1 The results showed significantly elevated serum LMAN2, CAPN-1, and VCP levels in EMI-SCD cases. ELISA analysis revealed differences in the levels of three endoplasmic reticulum stress-related secreted proteins, LMAN2 (A), CAPN-1 (B), and VCP (C), between patients with early myocardial ischemia-induced sudden cardiac death (EMI-SCD, referred to as SCD in the figure) and control groups. *, p<0.05; **, p<0.01.

[0023] Figure 2 In EMI-SCD cases, serum LMAN2, CAPN-1, and VCP concentrations were not affected by death time, postmortem interval (PMI), age of the deceased, or month of death. The study showed the correlation analysis results between serum LMAN2, CAPN-1, and VCP levels and death time (A, E, F), postmortem interval (PMI, B, F, J), age of the deceased (C, G, K), and month of death (D, H, L) in cases of sudden cardiac death caused by early myocardial ischemia.

[0024] Figure 3 Receiver operating characteristic (ROC) analysis compared the diagnostic efficacy of LMAN2, CAN-1, VCP (A) and CK-MB, cTnI (B) in early myocardial ischemia (C), showing that serum LMAN2, CAPN-1 and VCP were superior to CK-MB and cTnI in the diagnosis of EMI-SCD and could be used for the diagnosis of EMI-SCD. Detailed Implementation

[0025] The specific experimental materials, methods, and steps in this embodiment of the invention are as follows:

[0026] 1. Case Collection

[0027] All cases were collected from January 2018 to January 2021, and were routine forensic examinations conducted by the Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, and the Shanghai Municipal Public Security Bureau. Detailed records were kept of basic case information (age, gender, medical history), the process of death (prodromal symptoms, survival time, location of death, etc.), and the postmortem time interval (PMI, the time between death and medical examination). Cases with unknown PMI or a PMI longer than 24 hours were excluded to avoid postmortem effects caused by autolysis. Survival time, i.e., the time interval from the onset of symptoms to death, was recorded and calculated. This project focuses on cases of early myocardial ischemia; therefore, cases with a survival time of less than 6 hours were included.

[0028] A total of 44 cases were collected, divided into an EMI-SCD group (n=21) and a control group (n=23). The diagnosis of the EMI-SCD group was based on histological evidence of early ischemia (coagulative necrosis with homogeneous and strongly eosinophilic cytoplasm, and wavy changes in myofibrils), excluding gross cardiac pathology and any other potential non-cardiac causes of death. Age-matched cases without gross or microscopic cardiac pathological signs were selected as the control group. In the control group, 23 cases primarily died from mechanical injuries (n=20, 86.96%), such as traffic accidents (n=9, 39.13%), sharp object injuries (n=9, 39.13%), and falls from heights (n=2, 8.70%). The remaining 3 control group cases died from carbon monoxide poisoning (n=1, 4.35%) and gastric reflux asphyxia (n=2, 8.70%). Tables 1 and 2 record the basic information of the two groups of cases.

[0029] 2. Serum sample preparation

[0030] All cases were examined immediately after death to minimize the effects of postmortem autolysis and putrefaction. Five ml of whole blood was aspirated from the right ventricular chamber of each case. After allowing the whole blood to clot naturally at room temperature for 30 minutes, it was centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant serum sample was collected and placed in a sterile tube, stored at -80°C until use. All samples used in this study were anonymized after collection. Samples with significant hemolysis, hemoglobin contamination, or hyperlipidemia were excluded.

[0031] 3. Antibodies

[0032] Anti-human cTn-I antibody, anti-human CK-MB antibody, anti-human LMAN2 antibody, anti-human CAPN-1 antibody, and anti-human VCP antibody used for ELISA detection were purchased from a biotechnology company and coated in enzyme-labeled wells. All primary antibodies used in this experiment were validated by the manufacturer. According to the manufacturer's instructions, due to the use of specific monoclonal antibodies in this system, cross-reactivity with other non-specific analytes and spectral effects from other biological substances and drugs are negligible.

[0033] 4. Enzyme-linked immunosorbent assay (ELISA)

[0034] The two-site sandwich method is employed, which includes: binding monoclonal antibodies to corresponding dendritic molecules on the ELISA plate to separate the target analytes (LMAN2, CAPN-1, and VCP), adding horseradish peroxidase (HRP)-conjugated secondary monoclonal antibodies to form an antibody-antigen labeled antibody sandwich, and eluting unbound labeled antibodies.

[0035] In this study, all serum samples were thawed and diluted at a ratio of 1:5. 50 μL of each diluted sample or standard at different concentrations was added to a 96-well ELISA plate and incubated with 100 μL of HRP-labeled secondary antibody at 37°C for 1 h. The plates were then washed five times with unbound antibody. Subsequently, 100 μL of enzyme substrate was added per well, and the plates were incubated at 37°C in the dark. 50 μL of stop solution was added to stop the reaction. Absorbance was measured at 450 nm using a microplate reader (Biotek, Winooski, VT, USA) to analyze the signal intensity. The analyte concentrations in each sample were determined by comparing the results with the calibration curve.

[0036] 5. Statistical Analysis

[0037] Unless otherwise stated, data are presented as mean ± standard error (SEM). Parametric Student's t-test or nonparametric Mann-Whitney test was used for comparisons between the two groups. Linear regression analysis was used to examine the relationship between a pair of parameters (including age and month of death) and LMAN2, CAPN-1, and VCP. Receiver operating characteristic (ROC) curve analysis was used to assess the sensitivity and specificity of differentiating the two groups using serum protein thresholds; the area under the curve (AUC) was calculated and analyzed. These analyses were performed using GraphPad Prism 8.0 (La Giolla, CA, USA). A two-tailed p-value < 0.05 was considered statistically significant.

[0038] Example 1. Serum LMAN2, CAPN-1, and VCP were significantly elevated in EMI-SCD cases.

[0039] This invention investigated the changes in serum levels of LMAN2, CAPN-1, and VCP in EMI-SCD. ELISA analysis showed that serum LMAN2 levels in the EMI-SCD group increased by 48.0% compared to the control group (p=0.0022, e.g., ...). Figure 1As shown in A), CAPN-1 increased by 32.6% (p=0.041, as shown in A). Figure 1 As shown in B), VCP increased by 26.2% (p=0.021, as shown in B). Figure 1 As shown in Figure C), LMAN2, CAPN-1, and VCP were significantly elevated in the serum of EMI-SCD cases.

[0040] Example 2. Serum LMAN2, CAPN-1, and VCP concentrations in EMI-SCD cases were not affected by factors such as death time, postmortem interval (PMI), age of the deceased, and month of death.

[0041] This invention analyzes the correlation between serum LMAN2, CAPN-1, and VCP and parameters such as death time, postmortem interval (PMI), age of the deceased, and month of death.

[0042] Serum LMAN2 levels were slightly higher in the nighttime group (15.4%) compared to the daytime group (p=0.51). Figure 2 A) The PMI < 1h group was slightly higher than the 1h < PMI < 4h group (4.9% increase), but the difference was not statistically significant (p = 0.82). Figure 2 B). Linear regression analysis showed that serum LMAN2 levels were not correlated with the age of the deceased (p=0.77, R2=0.0046). Figure 2 C), not related to the month of death (p=0.86, R2=0.0016, Figure 2 D).

[0043] Serum CAPN-1 levels were 33.8% higher in the nighttime group compared to the daytime group (p=0.25). Figure 2 E), the PMI < 1h group was slightly lower than the 1h < PMI < 4h group (3.3% difference, p = 0.89), Figure 2 F). Linear regression analysis showed that serum CAPN-1 levels were not correlated with the age of the deceased (p=0.68, R2=0.0092, Figure 2 G), was not correlated with the month of death (p=0.43, R2=0.033, Figure 2 H).

[0044] Serum VCP levels were slightly higher in the nighttime group (11.3%) compared to the daytime group (p=0.56). Figure 2 I), the percentage of people with PMI < 1h who were slightly higher (4.6% higher) than those with PMI < 4h who were lower (p = 0.79). Figure 2 J). Linear regression analysis showed that serum VCP levels were not correlated with the age of the deceased (p=0.89, R2=0.0011, J). Figure 2K), was not correlated with the month of death (p=0.62, R2=0.014, Figure 2 L).

[0045] Experimental results showed that serum LMAN2, CAPN-1, and VCP levels remained stable regardless of time of death, postmortem interval (PMI), age of the deceased, and month of death.

[0046] Example 3. Serum LMAN2, CAPN-1, and VCP showed better diagnostic efficacy for EMI-SCD than CK-MB and cTn-I.

[0047] CK-MB and cTn-I are commonly used clinical biomarkers for diagnosing myocardial ischemia, both possessing high sensitivity and specificity; however, their diagnostic efficacy in forensic samples is unknown. This invention compares the diagnostic efficacy of serum LMAN2, CAPN-1, and VCP with clinical CK-MB and cTnI; and uses ROC analysis to analyze three endoplasmic reticulum stress-related proteins (…). Figure 3 A) and CK-MB, cTn-I ( Figure 3 B) Area under the curve (AUC), diagnostic sensitivity, and specificity;

[0048] The results showed that the AUC of serum LMAN2 was 0.8178 (95% CI: 0.6949–0.9407, p = 0.0003). Figure 3 C), CAPN-1 was 0.6988 (95% CI: 0.5429–0.8546, p = 0.0241), and VCP was 0.7267 (95% CI: 0.5752–0.8782, p = 0.0101). Figure 3 C), serum CK-MB was 0.5590 (95% CI: 0.3851-0.7329, p=0.5031), and the AUC of cTn-I was 0.5911 (95% CI: 0.4220-0.7602, p=0.3012).

[0049] At a threshold of 23.63 ng / ml, serum LMAN2 showed a sensitivity of 90.84% ​​and a specificity of 60.87%. Figure 3 C); Serum CAPN-1, at a threshold of 17.67 ng / ml, showed a diagnostic sensitivity of 76.19% and a specificity of 60.87% (C). Figure 3 C); at a serum VCP threshold of 3.222 ng / ml, the sensitivity was 76.19% and the specificity was 65.22% (C). Figure 3 C); CK-MB showed a diagnostic sensitivity of 85.71% and a diagnostic specificity of 43.48% at a threshold of 143 ng / mL. Figure 3(C) The diagnostic sensitivity of cTn-I at a threshold of 254.9 ng / L was 85.71%, and the diagnostic specificity was 34.78%. Figure 3 C);

[0050] Experimental results showed that serum LMAN2 had the best AUC, while serum CK-MB and cTn-I had significantly lower AUC levels than the three endoplasmic reticulum stress-related proteins. Figure 3 C) The diagnostic sensitivity of CK-MB and cTn-I is comparable to that of the three endoplasmic reticulum stress-related proteins, but their diagnostic specificity is significantly lower. Studies have shown that serum LMAN2, CAPN-1, and VCP are superior to CK-MB and cTn-I in the diagnosis of EMI-SCD.

Claims

1. Application of endoplasmic reticulum stress-related secretory proteins in the preparation of diagnostic reagents for sudden cardiac death; wherein the endoplasmic reticulum stress-related secretory proteins are either LMAN2 or VCP, or a combination thereof.

2. The application according to claim 1, characterized in that, The sudden cardiac death mentioned above refers to sudden cardiac death caused by early myocardial ischemia.

3. The application according to claim 1, characterized in that, The diagnostic reagent is used to detect the concentration of endoplasmic reticulum stress-related secretory proteins in serum samples from cases. If the concentration level exceeds a certain threshold, it indicates that the sudden cardiac death is caused by early myocardial ischemia.

4. The application according to claim 3, characterized in that, The case described is a forensic medical case involving suspected early myocardial ischemia leading to sudden cardiac death.

5. The application according to claim 3, characterized in that, The serum levels of the endoplasmic reticulum stress-related secretory proteins LMAN2, CAPN-1, and VCP were significantly elevated in early myocardial ischemia-induced sudden cardiac death.

6. The application according to claim 5, characterized in that, The specified threshold values ​​are: LMAN2 threshold of 23.63 ng / ml, CAPN-1 threshold of 17.67 ng / ml, and VCP threshold of 3.222 ng / ml.

7. A reagent kit for diagnosing sudden cardiac death caused by early myocardial ischemia, characterized in that, The kit contains reagents for measuring endoplasmic reticulum stress-related secreted proteins in serum, including anti-human LMAN2 antibody, anti-human CAPN-1 antibody, and anti-human VCP antibody.

8. The kit for diagnosing sudden cardiac death caused by early myocardial ischemia according to claim 7, characterized in that, The kit is used to determine the concentration of endoplasmic reticulum stress-related secreted proteins in serum. When the concentration level exceeds a threshold, it indicates early myocardial ischemia leading to sudden cardiac death.

9. The kit for diagnosing sudden cardiac death caused by early myocardial ischemia according to claim 8, characterized in that, The threshold values ​​are: LMAN2 threshold of 23.63 ng / ml, CAPN-1 threshold of 17.67 ng / ml and VCP threshold of 3.222 ng / ml.

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