Diagnostic markers for myocardial infarction and therapeutic agents for myocardial infarction

Abstract

The application provides a myocardial infarction diagnostic marker and a myocardial infarction therapeutic drug, and belongs to the technical field of in-vitro diagnosis and biological medicine, in particular, the myocardial infarction diagnostic marker comprises FGF18 protein or a protein mutant with at least 90% sequence identity with the FGF18 protein, and the amino acid sequence of the FGF18 protein is shown as SEQ ID No. 1. The myocardial infarction therapeutic drug comprises FGF18 protein or an FGF18 protein expression promoter. The application firstly proposes FGF18 as the myocardial infarction diagnostic marker and the myocardial infarction therapeutic drug, and provides a new thought for the detection and treatment of myocardial infarction.

Description

Technical Field

[0001] This invention relates to the fields of in vitro diagnostics and biomedical technology, and more specifically, to diagnostic markers for myocardial infarction and drugs for the treatment of myocardial infarction. Background Technology

[0002] Myocardial infarction (MI) is one of the leading causes of death and disability worldwide. Although significant progress has been made in improving the survival rate of myocardial infarction through reperfusion strategies such as percutaneous coronary intervention and drug therapy, these methods mainly focus on restoring blood flow or reducing cardiac load and cannot fundamentally reverse the irreversible death of cardiomyocytes (CM) caused by ischemia. The continuous loss of cardiomyocytes after MI, especially apoptosis-driven cell death, is the core pathological link leading to poor cardiac remodeling and the eventual development of heart failure. Therefore, developing novel treatment strategies that can directly promote cardiomyocyte survival and protect the integrity of myocardial structure is the focus of current research.

[0003] In-depth pathological mechanism studies have shown that mitochondrial dysfunction is a key driver of cell death after myocardial infarction. About 90% of the heart's energy comes from mitochondrial oxidative phosphorylation. Ischemic injury triggers excessive mitochondrial fission, inner membrane rupture, and reactive oxygen species (ROS) bursts, which in turn activate the apoptosis cascade. Optic atrophy protein 1 (OPA1) is a key protein that regulates mitochondrial inner membrane fusion and maintains cristae structural stability. Its downregulation directly leads to mitochondrial functional collapse. However, there are currently no therapeutic drugs in clinical practice that can specifically target and regulate OPA1 expression or effectively restore mitochondrial dynamics.

[0004] In terms of diagnosis and prognostic assessment, although markers such as troponin have been widely used in the acute phase diagnosis of MI, there is still a lack of novel biomarkers in the clinic that can reflect the endogenous repair capacity of myocardial tissue, the functional status of mitochondria, and accurately predict the prognosis of myocardial remodeling. Therefore, finding molecular markers that are closely related to the mechanism of myocardial survival is of great significance for early identification of high-risk patients and guidance of individualized treatment.

[0005] Therefore, there is an urgent need to develop diagnostic biomarkers and therapeutic drugs for myocardial infarction based on new mechanisms, in order to solve the problems of the lack of effective interventions for mitochondrial damage and the limited availability of prognostic biomarkers in existing technologies. Summary of the Invention

[0006] One of the objectives of this invention is to provide a diagnostic biomarker for myocardial infarction and a therapeutic drug for myocardial infarction. The diagnostic biomarker for myocardial infarction includes FGF18 protein or a protein mutant having at least 90% sequence identity with FGF18 protein, the amino acid sequence of which is shown in SEQ ID No. 1.

[0007] Specifically, SEQ ID No.1: MYSAPSACTCLCLHFLLLCFQVQVLAAEENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKG KETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQAELQKPFKYTTVTKRSRRIRPTHPG.

[0008] Preferably, the diagnostic marker for myocardial infarction further includes the FGF18 gene or a gene mutant with 90% sequence identity to the FGF18 gene, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0009] Specifically, SEQ ID No. 2: .

[0010] A second objective of this invention is to provide a diagnostic kit for myocardial infarction, the diagnostic kit comprising an FGF18 protein detection reagent and / or an FGF18 gene expression level detection reagent.

[0011] A third objective of this invention is to provide a drug for treating myocardial infarction, wherein the drug includes FGF18 protein or an FGF18 protein expression promoter.

[0012] Preferably, the FGF18 protein expression promoter comprises a recombinant expression vector carrying the nucleotide sequence shown in SEQ ID No. 2.

[0013] Preferably, the recombinant expression vector is an adeno-associated virus vector or an adenovirus vector.

[0014] Preferably, the recombinant expression vector contains a cTnT promoter that drives the expression of the FGF18 gene.

[0015] The fourth objective of this invention is to provide an application of the aforementioned myocardial infarction treatment drug in the preparation of drugs for the prevention or treatment of myocardial infarction, heart failure, and cardiac remodeling after myocardial infarction.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention establishes for the first time the downregulation pattern of FGF18 expression in myocardial infarction tissue, clarifies its use as a diagnostic marker, and the diagnostic kit developed based on this can rapidly and accurately detect FGF18 expression levels, assisting clinical early diagnosis and prognostic assessment, and making up for the shortcomings of existing markers that cannot reflect myocardial endogenous repair capacity and mitochondrial status.

[0017] The myocardial infarction treatment drug provided by this invention targets FGF18. By increasing the expression level of FGF18 protein, it reduces cardiomyocyte apoptosis, providing a new approach for the development of subsequent myocardial infarction drugs. Attached Figure Description

[0018] Figure 1 The results of detecting the expression levels of FGF18 protein and FGF18 mRNA in the ischemic model group cells and control group cells in Example 1 of this invention; Figure 2 This is a vector map of the recombinant adenovirus vector in Example 1 of the present invention; Figure 3 The figure shows the experimental results of detecting reactive oxygen species using dihydroethylenediamine as a fluorescent probe in Example 1 of this invention. Figure 4 The results of TUNEL apoptotic cell detection in Example 1 of this invention; Figure 5 The results show the mRNA levels of IL-1β, IL-6, IL-8, and TNF-α in each group of cells in Example 1 of this invention. Figure 6 This is a vector map of adeno-associated virus that specifically overexpresses FGF18 in cardiomyocytes in Example 2 of the present invention; Figure 7 The Western Blot results show the expression levels of FGF18 protein in the heart tissues of myocardial infarction model mice and the control sham-operated group mice in Example 2 of this invention. Figure 8 The ultrasound results of left ventricular ejection fraction and left ventricular shortening fraction in mice in Example 2 of this invention; Figure 9 The results of TTC staining of the ischemic area of ​​the heart in each group of mice in Example 2 of this invention; Figure 10 The results of masson staining, WGA staining, and TUNEL staining of mice in each group in Example 2 of this invention are shown. Detailed Implementation

[0019] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the present invention.

[0020] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0022] Example 1 Cell experiments In this experiment, 1-2 day old SD rats were used. After sterilization by immersion in 75% ethanol, the hearts were directly excised under aseptic conditions and placed in pre-cooled DPBS (Ca²⁺ and Mg²⁺-free) culture dishes. After gentle squeezing and rinsing to remove residual blood from the heart chambers, the auricle was removed, and the heart tissue was cut into small pieces of approximately 1 mm³. The tissue pieces were transferred to a digestion solution containing 0.08% trypsin (components: 0.08% trypsin, 0.8% NaCl, 0.03% KCl, 0.035% NaHCO₃, 0.1% D-(+)-Glucose, 0.2% HEPES), and digested in portions for 8 minutes each time on a magnetic stirrer at 37°C until the tissue pieces were completely digested. All digestion solution was collected and centrifuged at 1500 rpm for 3 minutes to obtain cell pellet. Cell pellets were resuspended in preheated (37°C) complete culture medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin), filtered through a 200-mesh cell sieve, and incubated at 37°C in a 5% CO2 incubator for 60 min. The supernatant was then collected, cardiomyocyte density was adjusted, and the cells were seeded into culture dishes. Fresh culture medium was added 24 h after seeding, followed by subsequent experiments.

[0023] To simulate an ischemic environment, cells in good condition were mixed and re-seeded. When the cell confluence reached 70%, the medium was replaced with glucose-free and serum-free DMEM, and the cells were placed in a mixed gas environment containing 95% N2 and 5% CO2 (37°C) for hypoxic culture, which was designated as the ischemic model group. The control group cells continued to use normal culture medium and were cultured in a conventional incubator (37°C, 95% air, 5% CO2). After hypoxia intervention for 6 hours, samples were collected from both groups for subsequent indicator detection.

[0024] The expression levels of FGF18 protein and FGF18 mRNA were detected in cells of the ischemia model group and the control group, respectively. The results are as follows: Figure 1 As shown, where, Figure 1 In the figure, A represents the Western blot result of FGF18 protein expression level. Figure 1 In the table, B represents the Real-time quantitative PCR result of FGF18 mRNA expression, CON represents control cells, OGD represents ischemia model cells, and β-Actin represents the internal control. Figure 1 It is evident that, compared to normal cardiomyocytes, the expression levels of FGF18 protein and FGF18 gene were significantly reduced in the ischemic model group, demonstrating the potential of FGF18 as a diagnostic biomarker.

[0025] To evaluate the therapeutic potential of FGF18 protein for myocardial infarction, an experimental group was added to the aforementioned cell experiments: a recombinant adenovirus vector (Ad-FGF18) capable of overexpressing full-length FGF18 was transfected into cells of the partial ischemia model group and the control group. The vector map of the recombinant adenovirus vector is shown below. Figure 2 As shown, the recombinant adenovirus vector was synthesized by Heyuan Biotechnology (Shanghai) Co., Ltd. After successful transfection, cells were cultured and samples were collected for subsequent testing.

[0026] Dihydroethylenediamine was used as a fluorescent probe for the detection of reactive oxygen species. Figure 3 Figure A shows fluorescence images of superoxide levels in ischemic model cells (untransfected and transfected with FGF18 overexpression vector), and control cells (untransfected and transfected with FGF18 overexpression vector). The scale bar is 130 μm. The quantitative results are as follows: Figure 3 As shown in B in the diagram.

[0027] Depend on Figure 3 It is evident that after transfection with the FGF18 overexpression vector, cells overexpress FGF18. This process can effectively reduce several types of reactive oxygen species caused by hypoxia-glucose deprivation, as evidenced by the decrease in fluorescence intensity of dihydroethylenediamine.

[0028] Microscopic images of TUNEL apoptotic cells are shown below. Figure 4 As shown in A, where green represents TUNEL. + Cells, blue represents DAPI, scale bar is 80μm. Figure 4 B in the text shows TUNEL + Quantitative analysis results of cells.

[0029] Depend on Figure 4 It is evident that cardiomyocyte apoptosis increases under hypoxia-glucose deprivation conditions. However, transfection with an FGF18 overexpression vector to increase FGF18 expression can effectively alleviate this phenomenon, demonstrating that FGF18 can significantly reduce cardiomyocyte apoptosis.

[0030] Figure 5 The mRNA levels of IL-1β, IL-6, IL-8 and TNF-α in each group of cells were shown.

[0031] Depend on Figure 5 It is evident that hypoxia-glucose deprivation stimulation significantly upregulated the mRNA levels of IL-1β, IL-6, IL-8, and TNF-α. However, after introducing the FGF18 overexpression vector, the expressed FGF18 significantly inhibited the expression of these factors. In other words, FGF18 can exert a cytoprotective effect in cardiomyocytes by alleviating oxidative stress, apoptosis, and inflammatory responses under ischemic conditions.

[0032] In all the above figures, Vehicle represents untransfected cells, and FGF18 represents cells transfected with the FGF18 overexpression vector.

[0033] Example 2 Animal experiments This study used 6-week-old male C57BL / 6 mice (GemPharmatech Co., Ltd.). A control group and an experimental group were established. Control group mice were normally fed, while experimental group mice were injected via tail vein with adeno-associated virus (AAV9-cTNT-FGF18) that specifically overexpresses FGF18 in cardiomyocytes. The vector map of AAV9-cTNT-FGF18 is shown below. Figure 6 As shown, AAV9-cTNT-FGF18 was synthesized and packaged by Yuan Biotechnology (Shanghai) Co., Ltd., with an injection volume of 5 × 10⁻⁶. 12 RFU was administered per mouse, and the mice were fed for 2 weeks after injection to ensure adequate expression. Subsequently, a portion of the experimental group and control mice underwent permanent ligation of the left anterior descending coronary artery (LAD) to establish a myocardial infarction (MI) model. On the 7th day after MI surgery, the mice were sacrificed under deep anesthesia, and cardiac tissue was collected for experimental analysis. The cardiac tissue was then paraffin-embedded and sectioned. All experimental mice were acclimatized for 4–6 days before the procedure and maintained under stable environmental conditions, including a temperature of 21±2°C, humidity of 50±15%, and a 12-hour light-dark cycle. All animal experimental protocols complied with relevant ethical guidelines and were approved by the Animal Protection and Use Committee (IACUC) of Ningbo University. The experimental report followed the requirements of the ARRIVE guidelines.

[0034] The steps for establishing a mouse model of myocardial infarction are as follows: The skin is incised along the left chest wall, and the pectoralis major and serratus anterior muscles are separated sequentially to expose the 4th intercostal space. The intercostal muscles are incised, and the pleura is carefully lifted, cutting anteriorly to approximately 2 mm from the sternum. The 4th intercostal space is suspended with 7-0 sutures to expand the surgical field, followed by blunt dissection of the pericardium. When ligating the left anterior descending coronary artery (LAD), the LAD originating below the left atrial appendage (which may appear as a white artery) is first identified, and its course is followed to the midline of the left ventricle near the interventricular groove. The ligation site is usually selected in the middle segment of the LAD (approximately 50% of the area), with the needle inserted directly opposite the last great cardiac vein, and the exit point located approximately 2 mm from the interventricular septum. A live knot is applied using 7-0 sutures. After ligation, the distal myocardium is observed for whitening and the heartbeat for weakening to confirm successful ligation. The chest is then closed layer by layer. After weaning the animal off the ventilator, respiratory recovery is observed, and assisted ventilation is prolonged if necessary. The sham surgery group only threaded the sutures without ligating them.

[0035] Postoperatively, anesthesia was maintained using a small animal ventilation anesthesia machine, and cardiac function was assessed using high-resolution color Doppler echocardiography (Vevo 1100, Visual Sonics, Canada). The model's effectiveness was evaluated by measuring aortic arch blood flow and cardiac structural parameters. Key indicators included left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), ejection fraction (EF), and left ventricular fractional shortening (FS). Data for each group were derived from at least five independent measurements.

[0036] Mice whose left anterior descending coronary artery (LAD) was permanently ligated in the control group served as myocardial infarction model mice. Seven days post-surgery, the Western blot results of FGF18 protein expression in the heart tissue of both the myocardial infarction model mice and the control sham-operated group are as follows: Figure 7 As shown in A, the expression levels of the FGF18 gene in the heart tissues of myocardial infarction model mice and the control sham-operated group mice were determined by Real-time quantitative PCR. Figure 7 As shown in B in the figure. Among them, Sham is the control group (sham operation group), MI is the mouse model of myocardial infarction, and β-Actin is the internal control.

[0037] Depend on Figure 7 It is evident that, compared to the sham surgery group, the expression levels of FGF18 protein and mRNA in the myocardial tissue of mice with myocardial infarction were significantly reduced one week after surgery, which is completely consistent with the results of cell experiments.

[0038] Left ventricular ejection fraction and left ventricular shortening fraction were measured by echocardiography in four groups of mice: sham mouse model injected with AAV9-cTNT-FGF18 (AAV-FGF18, Sham), MI mouse model injected with AAV9-cTNT-FGF18 (AAV-FGF18, MI), sham mouse model without AAV9-cTNT-FGF18 injection (AAV-Lacz, Sham), and MI mouse model without AAV9-cTNT-FGF18 injection (AAV-Lacz, MI). The results are as follows: Figure 8 A and Figure 8 As shown in B in the diagram.

[0039] Depend on Figure 8 It is evident that after injection of AAV9-cTNT-FGF18, the left ventricular ejection fraction and left ventricular shortening fraction in the MI surgical mouse model significantly increased, suggesting that AAV9-cTNT-FGF18 can significantly improve ventricular systolic function in MI model mice.

[0040] The ischemic area of ​​the heart in mice of each group was detected by TTC staining, and the results are as follows: Figure 9 As shown, the scale is 2mm, and it is composed of... Figure 9 It is evident that the ischemic area of ​​the heart in (AAV-FGF18, MI) mice is much smaller than that in (AAV-Lacz, MI) mice, and its ischemic area is only slightly larger than that in the control experiments (AAV-FGF18, Sham) mice and (AAV-Lacz, Sham) mice, proving that the overexpression of FGF18 caused by AAV9-cTNT-FGF18 can significantly reduce the infarct area.

[0041] The results of masson staining, WGA staining, and representative confocal scans of TUNEL and DAPI for each group of mice are as follows: Figure 10 As shown, the scale bar for Masson staining is 2 mm, the scale bar for WGA staining is 30 μm, and the scale bar for representative confocal scans of TUNEL and DAPI is 50 μm. Green represents TUNEL, and blue represents DAPI. Figure 10 It is evident that overexpression of FGF18 induced by AAV9-cTNT-FGF18 can significantly reduce the area of ​​fibrosis. WGA staining results suggest that overexpression of FGF18 induced by AAV9-cTNT-FGF18 can effectively inhibit cardiomyocyte hypertrophy after myocardial infarction. TUNEL staining results show that overexpression of FGF18 induced by AAV9-cTNT-FGF18 can significantly reduce cardiomyocyte apoptosis, which is consistent with the in vitro cell results.

[0042] Figure 8 , Figure 9 , Figure 10 The results demonstrated that FGF18 significantly reduced adverse cardiac remodeling (including infarct size expansion, myocardial fibrosis, and hypertrophy) and promoted cardiac function recovery after myocardial infarction. These results suggest that FGF18, as an important regulator of oxidative stress and remodeling response, may be a promising therapeutic target for cardioprotection after myocardial infarction.

[0043] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. Diagnostic markers for myocardial infarction, characterized in that, The diagnostic markers for myocardial infarction include FGF18 protein or a protein mutant having at least 90% sequence identity with FGF18 protein, the amino acid sequence of which is shown in SEQ ID No.

1.

2. The diagnostic biomarker for myocardial infarction as described in claim 1, characterized in that, The diagnostic markers for myocardial infarction also include the FGF18 gene or a gene mutant with 90% sequence identity with the FGF18 gene, the nucleotide sequence of which is shown in SEQ ID No.

2.

3. A diagnostic kit for myocardial infarction, characterized in that, The myocardial infarction diagnostic kit includes an FGF18 protein detection reagent and / or an FGF18 gene expression level detection reagent.

4. A drug for treating myocardial infarction, characterized in that, The drugs for treating myocardial infarction include FGF18 protein or FGF18 protein expression promoters.

5. The myocardial infarction treatment drug as described in claim 4, characterized in that, The FGF18 protein expression promoter comprises a recombinant expression vector carrying the nucleotide sequence shown in SEQ ID No.

2.

6. The myocardial infarction treatment drug as described in claim 5, characterized in that, The recombinant expression vector is an adeno-associated virus vector or an adenovirus vector.

7. The myocardial infarction treatment drug as described in claim 6, characterized in that, The recombinant expression vector contains a cTnT promoter that drives the expression of the FGF18 gene.

8. The use of the myocardial infarction treatment drug according to any one of claims 4 to 7 in the preparation of drugs for the prevention or treatment of myocardial infarction, heart failure, and cardiac remodeling after myocardial infarction.

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