Application of sarcolipin in early non-invasive diagnosis of type I ejection fraction preserved heart failure

Abstract

The application discloses application of sarcolipin (Sln) in early noninvasive diagnosis of type I ejection fraction reserved heart failure, and belongs to the technical field of early diagnosis of diseases. The application provides application of a reagent for detecting Sln in preparation of a heart failure early diagnosis kit, and the reagent can be used as a sensitive index for early noninvasive diagnosis of HFpEF. It is found in the application that Sln has the strongest correlation with LVEDP and is negatively correlated with LVEDP, which indicates that the content change of Sln can reflect early diastolic dysfunction of myocardium, and significant down-regulation of Sln indicates early occurrence of HFpEF. Meanwhile, the Sln value can reflect early diastolic dysfunction of myocardium, and the Sln value is more sensitive than BNP.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of early diagnosis of diseases, and particularly relates to application of Sarcolipin in early non-invasive diagnosis of type I ejection fraction preserved heart failure. BACKGROUND

[0002] HFpEF is defined as heart failure with LVEF>50%. The diagnosis of HFpEF is challenging, and the current diagnosis of HFpEF mainly relies on clinical manifestations, echocardiography and natriuretic peptide levels, but the sensitivity and specificity are low. The initial examination of HFpEF should include electrocardiogram, X-ray chest film, transthoracic echocardiography; clinically highly suspected, but unable to be diagnosed as HFpEF by routine transthoracic echocardiography and natriuretic peptide detection, further load echocardiography examination is recommended, and invasive hemodynamic examination can be performed on those who cannot be diagnosed.

[0003] The current diagnosis process of HFpEF is as shown in the following table, but in the case of no clinical symptoms or atypical clinical symptoms, and no obvious indications of transthoracic echocardiography and natriuretic peptide detection, it is difficult for the clinic to diagnose HFpEF. On the one hand, invasive hemodynamic examination is limited by the level of the hospital, and on the other hand, it is often not accepted by patients in the case of no symptoms, so the early diagnosis rate of HFpEF is low. Therefore, it is crucial to find non-invasive biomarkers for early diagnosis of HFpEF, which is conducive to taking treatment measures as soon as possible and slowing down the development of heart failure. Figure 1 SUMMARY

[0004] The application provides application of muscle lipid protein Sarcolipin (Sln) in early non-invasive diagnosis of type I ejection fraction preserved heart failure, and takes the content of Sln as a diagnostic marker of type I HFpEF, so that early diagnosis can be non-invasively completed, and the detection method is simple and has high sensitivity.

[0005] The application provides application of a reagent for detecting Sln in preparation of a kit for early diagnosis of heart failure.

[0006] Preferably, the content of Sln in peripheral blood is detected by a method utilizing ELISA.

[0007] Preferably, the heart failure includes type I ejection fraction preserved heart failure.

[0008] The application further provides application of an ELISA reagent for detecting the content of muscle lipid protein Sln in peripheral blood in preparation of a kit for early diagnosis of type I ejection fraction preserved heart failure.

[0009] Preferably, the content of muscle lipid protein Sln in peripheral blood is significantly down-regulated, indicating that the patient has type I ejection fraction preserved heart failure.

[0010] ​The application further provides an early diagnosis kit for type I ejection fraction preserved heart failure, which comprises an ELISA reagent for detecting the content of myolipid protein Sln in peripheral blood.

[0011] Beneficial effects: The application finds that the expression level of Sarcolipin (Sln) is closely related to myocardial contractile endurance, and the myolipid protein Sln can be secreted into blood, and when the myocardial contractility is slightly reduced in the early stage, the content of myolipid protein Sln in peripheral blood is significantly reduced, even earlier than the invasive hemodynamic examination (such as the detection of left ventricular end-diastolic pressure). Therefore, Sln is a sensitive index for early non-invasive diagnosis of HFpEF, earlier than the invasive hemodynamic examination. The application detects the early diagnosis method of HFpEF-1 type through a mouse model, finds that Sln has the strongest correlation with LVEDP and is significantly negatively correlated. The content of Sln in myocardium is closely related to the change of LVEDP, which indicates that the change of the content of Sln can reflect the early diastolic dysfunction of myocardium, and the significant reduction of Sln indicates the early occurrence of HFpEF. Meanwhile, the correlation between the blood Sln content of a hypertensive patient and myocardial function is analyzed, and it is found that the correlation between the blood Sln of the patient and E / A is significantly negatively correlated (R=0.7048, P<0.001), which indicates that the Sln value can reflect the early diastolic dysfunction of myocardium. Finally, the correlation between the blood Sln content of a hypertensive patient combined with acute myocardial infarction and myocardial LVEDP is analyzed, which again proves that the correlation between the blood Sln of the patient and the left ventricular end-diastolic pressure is significantly negatively correlated (R=-0.6584, P=0.0169), which indicates that the Sln value can reflect the early diastolic dysfunction of myocardium and is more sensitive than BNP.

[0012] The application further constructs the correlation between the content of Sln and HFpEF, and the normal reference value of blood Sln is 44.2-66.4 ng / L in the distribution range of 5%-95%, and the distribution range of blood Sln in HFpEF patients is 15-36.5 ng / L. Therefore, when the detection value of blood plasma Sln of a patient with hypertension, myocardial ischemia or the like is lower than 51.24 ng / L, it indicates that the heart function may have been slightly damaged, and when the detection value is lower than 36.5 ng / L, it indicates that the patient enters the HFpEF stage. The application further verifies the constructed ROC curve, and it is proved that the method described in the application can complete early non-invasive detection with high sensitivity and high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The current diagnosis flowchart for HFpEF is shown in the figure 1 : One of the following conditions must be met: (1) sinus rhythm: BNP≥35 pg / ml or NT-proBNP≥125 pg / ml; (2) atrial fibrillation: BNP≥105 pg / ml or NT-proBNP≥365 pg / ml; 2: Positive needs to meet one of the following conditions: (1) load echocardiogram E / e' is greater than or equal to 15; (2) in the resting state, PCWP is greater than or equal to 15mmHg or LVEDP is greater than or equal to 16mmHg by cardiac catheterization; (3) load cardiac catheterization, PCWP is greater than or equal to 25mmHg at peak exercise; 1mmHg=0.133kPa;

[0014] Figure 2 The figure is the change rule of the degree of myocardial hypertrophy, myocardial contraction and diastolic function of mice within three months of TAC;

[0015] Figure 3 The figure is the change rule of the Sln content in the myocardium of mice within three months of TAC;

[0016] Figure 4 The figure is the correlation analysis result between the Sln content in the myocardium of mice and the existing detection indexes;

[0017] Figure 5 The figure is the correlation analysis result between the change of the Sln content in the blood of mice within three months of TAC and LVEDP;

[0018] Figure 6 The figure is the correlation analysis result between the Sln content in the blood of hypertensive patients and myocardial function;

[0019] Figure 7 The figure is the correlation analysis result between the Sln content in the blood of hypertensive patients and myocardial function;

[0020] Figure 8 The figure is the result of the 95% confidence interval of the normal value of blood Sln, the 5%-95% distribution interval of the normal reference value of blood Sln, and the 5%-95% distribution interval of blood Sln in HFpEF patients;

[0021] Figure 9 The figure is the ROC curve of the sensitivity of BNP detection and Sln detection. DETAILED DESCRIPTION

[0022] The application provides a reagent for detecting Sln and application of the reagent in preparation of a heart failure early diagnosis kit.

[0023] The heart failure preferably includes type I ejection fraction preserved heart failure (HFpEF), and the HFpEF is classified into five types according to etiology, wherein the HFpEF-1 type is the most common, which is a vascular disease related HFpEF, mainly caused by hypertension or coronary heart disease. The early diagnosis of the HFpEF-1 type is taken as an example for description in the embodiments of the application, but it cannot be identified as the whole protection scope of the application.

[0024] Table 1 HFpEF etiology classification

[0025]

[0026] The application preferably detects the content of Sln in peripheral blood by an ELISA method, and the application does not have special limitations on the detection method of the ELISA, and in the examples, commercially available kits are used for detection, such as Human Myoferlin (SLN) ELISA Kit (CB20214-Hu) and Mouse Myoferlin (SLN) ELISA Kit (CB15599-Mu) purchased from Shanghai Kebio Biotech Co., Ltd.

[0027] The application also provides application of an ELISA reagent for detecting the content of myoferlin Sln in peripheral blood in preparation of an early diagnosis kit for type I ejection fraction preserved heart failure.

[0028] In the application, the content of plasma Sln is significantly negatively correlated with LVEDP, which indicates that the detection result of blood Sln is a non-invasive detection index for diagnosing early HFpEF as good as LVEDP; and the correlation between the blood Sln of a patient and E / A and intraventricular diastolic pressure is significantly negative, which indicates that the Sln value can reflect early diastolic dysfunction of myocardium and is more sensitive than BNP. That is, the content of myoferlin Sln in peripheral blood is significantly down-regulated, which indicates that the patient has type I ejection fraction preserved heart failure.

[0029] In the application, the 95% confidence interval of the normal value of plasma Sln is 51.24-60.60 ng / L, and therefore when the detection value of plasma Sln of a patient with hypertension, myocardial ischemia or the like is lower than 51.24 ng / L, it is indicated that the cardiac function may have been slightly impaired, and when the detection value is lower than 36.5 ng / L, it is indicated that the patient enters the HFpEF stage.

[0030] The application also provides an early diagnosis kit for type I ejection fraction preserved heart failure, which comprises an ELISA reagent for detecting the content of myoferlin Sln in peripheral blood.

[0031] In order to further illustrate the application, the application of Sarcolipin in early non-invasive diagnosis of type I ejection fraction preserved heart failure is described in detail in combination with examples below, but they should not be understood as limitations on the protection scope of the application.

[0032] In the application, LVEF: left ventricular ejection fraction; LAVI: left atrial volume index; LVWT: left ventricular wall thickness; E / e': peak velocity of diastolic early blood spectrum of mitral valve / tissue Doppler peak velocity of diastolic early mitral annulus; BNP: B-type natriuretic peptide; NT-proBNP: N-terminal B-type natriuretic peptide precursor; PCWP: pulmonary capillary wedge pressure; LVEDP: left ventricular end-diastolic pressure.

[0033] The experimental methods used in the embodiments of the present application include the following contents:

[0034] 1. Preparing experimental animals required for the invention: mice are placed in a pathogen-free environment, the temperature of the feeding room is maintained at 21-27°C, the humidity is maintained at 50%-60%, and 12 hours of light and 12 hours of darkness are provided.

[0035] 2. Preparing mouse models (aortic constriction) required for the invention (TAC operation): after the mice are anesthetized with 2% isoflurane, they are fixed in a supine position, intubated through the trachea, and connected to a ventilator. After cutting the skin along the left side of the sternum, the chest cavity is opened along the left side of the sternum, and the aortic arch is exposed. A 6-0 silk thread is pre-threaded through the aortic arch, and a 26G cushion needle placed parallel to the blood vessel is ligated with the aortic arch, and then the cushion needle is removed. Then perform echocardiography. Pentobarbital is administered to euthanize the mice, open the chest, remove the heart tissue, wash the blood with saline, and subsequently detect changes in myocardial-related indicators and fluorescence staining.

[0036] 3. Anesthetize the mice with 3% isoflurane and fix them in a supine position on the platform of the ultrasonic imaging system. Maintain the anesthetic state with 1.5% isoflurane. Use a Prospect 3.0 type (purchased from S-Sharp Company in Taiwan, China) small animal ultrasound instrument to obtain cardiac ultrasound images, with a probe center frequency of 20-40 MHz. Record left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVIDd), diastolic interventricular septal thickness (IVSd), and other indicators for statistical analysis.

[0037] 4、ELISA experiment steps: (1) collect human or mouse blood samples, for effective separation of plasma components, the standard centrifugal procedure is used, that is, the blood sample is centrifuged at a speed of 3000 rpm per minute for 20 minutes. After the completion of the centrifugation process, the clear liquid on the upper layer is carefully extracted, which is the required plasma sample for subsequent biochemical experiments. (2) Take out the required board from the aluminum foil bag equilibrated at room temperature for 20 minutes, and the remaining board is sealed with a self-sealing bag and put back at 4°C. (3) Set up standard and sample wells, add different concentrations of standard 50 μL to each standard well; (4) Add 10 μL of sample to be tested to the sample well, then add 40 μL of sample diluent; the blank well is not added. (5) Add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard and sample wells except the blank well, seal the reaction well with a sealing film, and incubate in a 37°C water bath or incubator for 60 min. (6) Discard the liquid, pat dry on the blotting paper, fill each well with washing solution, stand for 1 min, shake off the washing solution, pat dry on the blotting paper, and repeat the plate washing 5 times (or use a plate washer). (7) Add 50 μL of substrate A and B to each well, and incubate at 37°C in the dark for 15 min. (8) Add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm within 15 min. Draw the standard curve: in the Excel worksheet, take the standard concentration as the horizontal coordinate, and the corresponding OD value as the vertical coordinate, draw the standard linear regression curve, and calculate the concentration value of each sample according to the curve equation.

[0038] Example 1

[0039] Correlation analysis of changes in Sln content in mouse blood with LVEDP within three months of TAC

[0040] C57BL / 6J mice were divided into two groups, sham operation group and operation group. The operation group was established by aortic constriction (TAC) to establish a pressure overload heart failure model, and the sham operation group was only opened chest without operation. The mouse peripheral blood was regularly collected after operation, the Sln content in peripheral blood was detected by ELISA, and the left ventricular end-diastolic pressure was detected by carotid artery interventional surgery after blood collection to determine the degree of heart failure in mice. And compare the correlation between Sln and LVEDP.

[0041] The existing measurement standard is used to detect the degree of myocardial hypertrophy, the change rule of myocardial contraction and diastolic function of mice within three months of TAC. The detection indexes include left ventricular end-diastolic diameter (LVIDd), interventricular septal thickness (IVSD), left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), left ventricular ejection fraction (LVEF) and blood BNP concentration. The results are as follows Figures 2 to 4As shown, the expression of Sln significantly decreased with the development of heart failure, and Sln was most relevant to LVEDP, and was significantly negatively correlated. The detection of intraventricular pressure is an invasive hemodynamic examination, and is also the gold standard for early diagnosis of HFpEF. The content of Sln in the myocardium is closely related to the change of LVEDP, which indicates that the change of Sln content can reflect the early diastolic dysfunction of the myocardium, and the significant down-regulation of Sln indicates the early occurrence of HFpEF; and the content of plasma Sln is significantly negatively correlated with LVEDP, which indicates that the detection result of blood Sln is a non-invasive detection index for diagnosing early HFpEF as LVEDP. Figure 5

[0042] Example 2

[0043] Correlation analysis of blood Sln content and myocardial function in patients with hypertension

[0044] 1. Inclusion criteria: 73 patients with diagnosed hypertension were collected, and the cardiac function was grade I-II.

[0045] 2. Exclusion criteria: BNP>100 pg / ml, LVEF<50%, primary cardiomyopathy, dilated cardiomyopathy, valvular heart disease, pericarditis, atrioventricular block, atrial fibrillation, valvular disease, cardiac pacemaker implantation and cardiac surgery history; pulmonary diseases such as pulmonary embolism, pulmonary hypertension, severe coronary heart disease, diabetes, liver and kidney diseases and chronic obstructive pulmonary disease.

[0046] 3. Basic clinical data and laboratory indexes: record gender, age, BMI, cardiac ultrasound indexes and blood pressure. Laboratory indexes: fasting blood glucose (FBS), BNP, creatine kinase isoenzyme (CK-MB), cardiac troponin I (cTnI), serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), etc.

[0047] 4. Statistical method: SPSS22.0 statistical software was used for multiple linear regression analysis to compare the correlation of Sln and other non-invasive indexes.

[0048] In the early stage of HFpEF, it is not easy to take traumatic detection means, and when the diastolic function of the myocardium is weakened, the E / A value of B-ultrasound can reflect the early weakening of the diastolic function of the myocardium. However, with the development of the disease, when the atrium is dilated and the intra-atrial pressure is elevated, the E / A value will reverse from <1 to >1, and then it can no longer reflect the degree of myocardial injury.

[0049] As shown in the results, Figure 6 the correlation between the blood Sln of the patient and E / A was significantly negatively correlated (R=0.7048, P<0.0001), which indicated that the Sln value could reflect the early diastolic dysfunction of the myocardium.

[0050] Example 3​

[0051] 1. Relationship between serum Sln level and LVEDP in patients with hypertension and acute myocardial infarction

[0052] 1. Inclusion criteria: 20 patients with hypertension and acute myocardial infarction were collected, all of whom underwent coronary stent surgery and had cardiac function class I-II.

[0053] 2. Exclusion criteria: LVEF < 50% after coronary stent surgery, and exclusion of primary cardiomyopathy, dilated cardiomyopathy, valvular heart disease, pericarditis, atrioventricular block, atrial fibrillation, valvular disease, cardiac pacemaker implantation and cardiac surgery history; pulmonary embolism, pulmonary hypertension and other pulmonary diseases, severe coronary heart disease, diabetes, liver and kidney diseases and chronic obstructive pulmonary disease.

[0054] 3. The blood samples of the patients were subjected to a standardized centrifugation procedure, i.e. centrifugation of the blood sample at a rate of 3000 revolutions per minute for a duration of 20 minutes. After completion of the centrifugation process, we carefully extracted the clear liquid located on the upper layer, which was the required plasma sample. This plasma sample will be used for subsequent ELISA analysis.

[0055] 4. ELISA was used to detect the contents of BNP and Sln in the plasma of the patients, and the relationships between Sln and BNP and the intraventricular pressure of the patients were analyzed, respectively.

[0056] The results are shown in Figure 7 The correlation between serum Sln and intraventricular diastolic pressure in patients was significantly negative (R = -0.6584, P = 0.0169), indicating that Sln value can reflect early diastolic dysfunction of myocardium and is more sensitive than BNP.

[0057] Table 2 Patient information

[0058]

[0059] Two, determination of normal value of plasma Sln:

[0060] 1. Blood samples from 12 normal male and female subjects were collected. The inclusion criteria were: no cardiovascular and cerebrovascular diseases, no history of diabetes, kidney disease, acute or chronic pulmonary disease, and no gender or age restrictions.

[0061] 2. To effectively separate the plasma component, a standardized centrifugation procedure was used, i.e. centrifugation of the blood sample at a rate of 3000 revolutions per minute for a duration of 20 minutes. After completion of the centrifugation process, the clear liquid located on the upper layer was extracted, which was the required plasma sample. This plasma sample will be used for subsequent biochemical analysis.

[0062] Table 3 Determination of normal value of plasma Sln

[0063]

[0064]

[0065] The results are shown in Table 3 and FIG. 2. Figure 8 The 95% confidence interval of the normal value of blood Sln is 51.24-60.60 ng / L, the 5%-95% distribution interval of the normal reference value of blood Sln is 44.2-66.4 ng / L, and the 5%-95% distribution interval of blood Sln in HFpEF patients is 15-36.5 ng / L. Therefore, when the plasma sIn detection value of patients with hypertension, myocardial ischemia, etc. is lower than 51.24 ng / L, it indicates that the heart function may have been slightly impaired, and when the detection value is lower than 36.5 ng / L, it indicates that it has entered the HFpEF stage.

[0066] ROC curve analysis was performed on the above-mentioned 20 newly diagnosed heart failure patients, and the results are shown in Table 4 and FIG. 3. Figure 9 The AUC value of Sln is higher than that of BNP, and Sln value can reflect early diastolic dysfunction of myocardium and is more sensitive than BNP.

[0067] Table 4 ROC curve sensitivity analysis

[0068]

[0069] III. Accuracy verification

[0070] Standard verification analysis was performed on 3 male and 4 female newly diagnosed heart failure patients. The inclusion criteria are typical symptoms of heart failure and LVEF≥50%. The expression changes of muscle lipid protein Sln were detected in 7 plasma samples.

[0071] Table 5 Standard verification

[0072]

[0073]

[0074] The results show that patients 1, 2, 4, 6, and 7 were diagnosed with heart failure with preserved ejection fraction (HFpEF), and the detection value of blood sIn was lower than 36.5 ng / L, which was consistent with the diagnosis of LVEDP (higher than 16 mmHg) and had higher sensitivity and accuracy than BNP.

[0075] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. Use of an ELISA reagent for detecting the content of muscle lipid protein Sln in peripheral blood in the preparation of an early diagnosis kit for type I ejection fraction preserved heart failure.

2. Use according to claim 1, characterized in that, The significant down-regulation of the content of muscle lipid protein Sln in the peripheral blood indicates that the patient has type I ejection fraction preserved heart failure.

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