Application of microprotein MTLN as intervention target in relieving adriamycin myocardial injury

Abstract

The invention discloses application of microprotein MTLN as an intervention target in relieving adriamycin myocardial injury, and belongs to the technical field of bioengineering. According to the invention, an AAV9 vector is utilized to deliver MTLN-shRNA or an overexpression sequence to a mouse myocardial tissue, so as to regulate and control the cardiolipin level and influence the adriamycin-induced myocardial injury phenotype. Experiments prove that the interference of MTLN can significantly improve the heart function of a mouse suffering from adriamycin myocardial injury and reduce the fibrosis area, and a new strategy is provided for clinical treatment of adriamycin cardiotoxicity.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the application of the microprotein MTLN as an intervention target in alleviating doxorubicin-induced myocardial injury. Background Technology

[0002] Doxorubicin is an anthracycline antibiotic produced by Actinomycetes. As a broad-spectrum anticancer drug, it is widely used to treat various malignant tumors, including breast cancer, leukemia, and gastric cancer. Despite its significant efficacy in antitumor therapy, doxorubicin's dose-dependent cardiotoxicity limits its clinical application. Existing research on the treatment of myocardial injury caused by doxorubicin mainly focuses on the development of protective drugs with single mechanisms such as antioxidation and anti-apoptosis. This approach struggles to completely resolve the myocardial damage side effects of doxorubicin, and the safe and efficient targeted delivery of these drugs to the heart is challenging. This not only affects the efficacy of treating myocardial injury with doxorubicin but also impacts the antitumor therapeutic effect of doxorubicin, hindering the subsequent clinical application of these treatment strategies. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the microprotein Mitoregulin (MTLN) as an intervention target in alleviating doxorubicin-induced myocardial injury, thereby addressing the problems existing in the prior art. This invention achieves the effect of alleviating doxorubicin-induced myocardial injury by targeting and regulating the content of the microprotein MTLN in the myocardium, thereby altering the content of cardiolipin, a key target of doxorubicin-induced myocardial injury. This method of alleviating doxorubicin-induced myocardial injury by regulating the main target of injury can be used in combination with other protective therapies, which will help promote the final clinical application of preventive intervention programs for doxorubicin-induced myocardial injury.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides the use of an inhibitor targeting MTLN in the preparation of a drug for the prevention or treatment of doxorubicin-induced myocardial injury, wherein the amino acid sequence of the MTLN is shown in SEQ ID NO.1.

[0006] Furthermore, the inhibitor is an interfering nucleic acid targeting MTLN, wherein the interfering nucleic acid is selected from: double-stranded siRNA comprising SEQ ID NO.2 and SEQ ID NO.3; and double-stranded siRNA comprising SEQ ID NO.4 and SEQ ID NO.5.

[0007] This invention provides a kit for detecting the risk of myocardial injury from doxorubicin, comprising reagents for detecting the expression level of MTLN in myocardial tissue; the amino acid sequence of the MTLN is shown in SEQ ID NO.1.

[0008] The present invention also provides the application of a reagent for detecting MTLN expression levels in screening drugs that alleviate doxorubicin-induced myocardial injury, wherein the amino acid sequence of the MTLN is shown in SEQ ID NO.1.

[0009] The present invention also provides a biomarker for assessing the risk of doxorubicin-induced myocardial injury, the biomarker being a microprotein MTLN, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] The present invention discloses the following technical effects:

[0011] Doxorubicin's dose-dependent cardiotoxicity limits its clinical application, and existing treatments focus on single mechanisms such as anti-oxidation and anti-apoptosis, with limited efficacy. Cardiolipin is a key target of doxorubicin's cardiotoxicity, and cardiolipin damage is an important upstream event in doxorubicin-induced lipid peroxidation and apoptosis. However, there is currently no effective method to improve doxorubicin-induced myocardial damage by regulating cardiolipin levels. This invention is the first to discover that the microprotein MTLN participates in regulating cardiolipin levels and can be used as an intervention target to alleviate doxorubicin-induced myocardial damage, overcoming the limitations of existing technologies.

[0012] The results of this invention show that the microprotein MTLN affects cardiolipin levels and the severity of doxorubicin-induced myocardial injury. Interfering with the expression level of the microprotein MTLN increases the survival rate of H9C2 cells after doxorubicin treatment and significantly improves cardiac function in mice with doxorubicin-induced myocardial injury. Specifically, this invention significantly improves the survival rate of cardiomyocytes after doxorubicin treatment by interfering with MTLN expression; animal experiments show increased ejection fraction and reduced cardiac fibrosis area. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1(A) shows the amino acid sequence comparison of MTLN among species; (B) shows the immunofluorescence confirmation that MTLN is located in mitochondria (Bar = 50 μm); (C) shows the relative expression level of MTLN in various tissue types; (D) shows the intersection of genes that change in the same direction as MTLN in human, rat, and mouse species, and performs gene ontology function analysis; (E) shows the combined analysis results of cardiac ribosomal sequencing and RNA sequencing data from patients with dilated cardiomyopathy and controls, derived from the literature (van Heesch S, et al. The translational landscape of the human heart. Cell. 2019); (F) shows the GSEA analysis results of the sequencing data.

[0015] Figure 2 (A) shows the Western blot (WB) detection of the MTLN-siRNA interference effect; (B) shows the comparison of the abundance of monomeric cardiolipin in the non-targeted lipid profile detection of MTLN-interfered H9C2 cells and control cells, with data expressed as mean ± standard error; (C) shows the ELISA detection results of cardiolipin levels in MTLN-interfered H9C2 cells and control cells, with each point representing one biological replicate, and data expressed as mean ± standard error. The p-value was calculated using an unpaired t-test, with ***p-value < 0.001; (D) shows the experimental statistical results of cell viability detection using the CCK8 assay, with data expressed as mean ± standard error. The p-value was calculated using an unpaired t-test, with **p-value < 0.01 and ****p-value < 0.0001; (E) shows a representative image of the mitochondrial morphology observation results of H9C2 cells under transmission electron microscopy.

[0016] Figure 3 (A) is a schematic diagram of the animal experiment process; (B) is a representative image of the immunofluorescence experiment results of mouse myocardial tissue sections in each group; (C) is a WB test of mouse myocardial tissue to verify the overexpression / interference effect of AAV9; (D) is the ELISA detection result of cardiolipin level, with each point representing one mouse myocardial tissue sample. The data are expressed as mean ± standard error, and the p-value is calculated by unpaired t-test, with ***p-value < 0.001; (E) is a representative image of the mitochondrial morphology observation results of mouse myocardial tissue under transmission electron microscopy; (F) is the statistical result of left ventricular ejection fraction and short-axis contraction rate of mouse echocardiography, with the data expressed as mean ± standard error. The p-value is calculated by unpaired t-test, with ***p-value < 0.001 and ****p-value < 0.0001; (G) is a representative image of mouse echocardiography; (H) is a representative image of mouse myocardial tissue sections stained with HE and Masson staining, Bar = 50 μm. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Statistical analysis of all experimental data was performed using SPSS and Prism software. Error bars in the statistical graphs are represented as mean ± standard error. To determine statistical differences between groups, the Kolmogorov-Smirnov test was first used to confirm whether the data points conformed to a normal distribution. If the data points conformed to a normal distribution, an unpaired t-test was used; otherwise, the Wilcoxon rank-sum test was used to determine statistical differences between groups. A p-value less than 0.05 indicates a statistical difference between the two groups. The p-values ​​in the statistical graphs are represented by the number of asterisks (*), where * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and so on. “ns” indicates no statistical difference between the two groups.

[0023] The amino acid sequence of the microprotein MTLN is as follows:

[0024] MADVSERTLQLSVLVAFASGVLLGWQANRLRRRYLDWRKRRLQDKLAATQKKLDLA*(SEQ IDNO.1).

[0025] This invention constructs siRNA targeting the microprotein MTLN:

[0026] Forward sequence of rat species: CUACUGCAUGCUGAGCGUUTT (SEQ ID NO.2);

[0027] Rat species reverse sequence: AACGCUCACAUGCAGUAGTT (SEQ ID NO.3);

[0028] Forward sequence of mouse species: GAACAUGGCUUGCUUCAGATT (SEQ ID NO.4);

[0029] Mouse species reverse sequence: UCUGAGAGCAAGCCAUGUUCTT (SEQ ID NO.5).

[0030] In H9C2 cells, MTLN-siRNA was transfected using Lipo8000 (Beyotime Biotechnology), and the decrease in MTLN expression level was confirmed by Western blotting. In mice, MTLN-shRNA and MTLN overexpression sequences were loaded onto adeno-associated virus type 9 (AAV9) containing the cTnT promoter, administered via tail vein injection. Three weeks later, myocardial tissue sections were collected from sacrificed mice for immunofluorescence and Western blotting to confirm successful MTLN intervention.

[0031] Example 1

[0032] This embodiment verifies the basic biological characteristics of the microprotein MTLN. Functional analysis revealed that MTLN may be involved in regulating mitochondrial respiration. Using human heart sequencing data from the literature, it was confirmed that MTLN expression was significantly upregulated in the hearts of patients with dilated cardiomyopathy and was associated with myocardial contractile function, mitochondrial function and fatty acid metabolism.

[0033] Experimental methods:

[0034] (1) The amino acid sequence and comparison process of microprotein MTLN among species is as follows: the amino acid sequences of humans, chimpanzees, pigs, dogs, rabbits, mice and rats are obtained through the publicly available ensemble gene database and sequence comparison is performed using Jalview software;

[0035] (2) Immunofluorescence (to prove that MTLN is located in mitochondria). The specific immunofluorescence procedure is as follows: One day in advance, passage the cells to be used for immunofluorescence onto a glass plate at an appropriate cell density. On the second day, discard the cell culture medium using a suction pump, wash the cells once with PBS, and then immerse the cells in 4% paraformaldehyde fixative and incubate at room temperature for 20 minutes to fully fix them. After fixation, rinse the cells three times with immunofluorescence washing buffer, immersing them fully each time and incubating for at least 5 minutes. After rinsing, add immunofluorescence fixative to immerse the cells and incubate at room temperature for 1 hour. The primary fluorescent antibody is prepared using primary antibody and immunofluorescence staining solution at a concentration of 1:500. After cell fixation, discard the immunofluorescence fixative, immerse the cells in the primary fluorescent antibody, and incubate overnight. On the second day, discard the primary fluorescent antibody, rinse the cells three times with immunofluorescence washing buffer, immersing them fully each time and incubating for at least 5 minutes. The secondary fluorescent antibody is prepared using secondary antibody and immunofluorescence staining solution at a concentration of 1:500. After rinsing, add fluorescent secondary antibody to immerse the cells and incubate at room temperature for 1 hour, covering the entire glass plate with aluminum foil to protect it from light. After secondary antibody incubation, rinse the cells three more times with immunofluorescence washing buffer, ensuring full immersion each time and allowing them to stand for at least 5 minutes. The cell rinsing process after adding fluorescent secondary antibody should be as quick as possible, and the cells should always be protected from light. After rinsing, add DAPI staining solution and incubate at room temperature for 5 minutes, then rinse the cells three more times with immunofluorescence washing buffer, ensuring a minimum standing time of 5 minutes after each full immersion, again protecting the cells from light during the rinsing process. After completing all fluorescence staining and rinsing steps, observe the cells using a laser confocal microscope. When observing cells using a laser confocal microscope, first locate the cell focal plane using a white light field of view, then switch to the target fluorescence channel to find a representative field of view location and capture a fluorescence image. The captured fluorescence images are then processed using ImageJ software. Images from the same field of view but different fluorescence channels are merged for fluorescence colocalization analysis to draw experimental conclusions.

[0036] (3) The procedure for detecting the relative expression level of MTLN in each tissue type is as follows: take heart, muscle, liver, kidney and other tissues from wild-type c57 mice to extract tissue proteins for WB and compare the expression level of MTLN in each tissue type.

[0037] (4) Genes in the human, rat, and mouse species that showed the same trend as MTLN were intersected for gene ontology function analysis. Specifically, co-expression analysis was performed in each of the three species to screen for genes positively correlated with MTLN expression. Pearson correlation analysis was used, with a correlation coefficient r > 0.5 and p < 0.05 as the screening criteria. After obtaining lists of genes in the three species that showed the same trend as MTLN, intersection analysis was performed using R language to obtain the set of genes that were positively correlated in all three species. The intersecting genes were uniformly converted to human gene symbols, and then gene ontology (GO) function enrichment analysis was performed using the clusterProfiler R package. The analysis scope included biological processes, molecular functions, and cellular components. A human annotation database was used, with a significance threshold of Benjamini-Hochberg corrected p < 0.05 and a minimum gene set size of 10. The GO analysis results were visualized using enrichplot and ggplot2.

[0038] (5) Combined analysis of cardiac ribosomal and RNA sequencing data from patients with dilated cardiomyopathy and controls, derived from the literature (van Heesch S, et al. The translational landscape of the human heart. Cell. 2019). The analysis process was as follows: RNA sequencing (RNA-seq) and ribosomal footprint sequencing (Ribo-seq) data were used for quality control and adapter removal of the raw sequencing data using FASTP, and then aligned to the reference genome GRCh38 using STAR. Subsequently, HTSeq was used to compare the alignment results and count the reads at the gene level. Differential expression analysis was performed on RNA-seq and Ribo-seq data using DESeq2 to screen for significantly differentially expressed mitochondrial protein genes (the standard being |log2 Fold Change|≥1, Benjamini-Hochberg corrected p-value <0.05). Then, translation efficiency (TE) was analyzed. Based on FPKM, TE = Ribo expression / RNA expression was calculated to screen for genes with significantly altered TE in cardiomyopathy samples. Gene set enrichment analysis (GSEA) was performed using the gseapy Python package (v1.0.4) and the GO_Biological_Process_2025 database.

[0039] Example 2

[0040] This embodiment verifies the decrease in cardiolipin levels after MTLN interference.

[0041] Experimental subject: H9C2 cells (rat cardiomyocyte line)

[0042] Experimental methods:

[0043] (1) Transfect H9C2 cells with MTLN-siRNA and perform WB detection.

[0044] Specifically, MTLN-siRNA was transfected using SEQ ID NO.2 and SEQ ID NO.3.

[0045] (2) MTLN-interfered H9C2 cells, along with a control group (H9C2 cells transfected with non-specific siRNA), were subjected to cardiolipin ELISA and non-targeted lipid profile detection. The procedures for cardiolipin ELISA and non-targeted lipid profile detection were as follows: Cardiolipin ELISA was performed using the ELISA kit from ELISA company MM-71760R2. For non-targeted lipid profile detection, frozen cell samples were sonicated in pre-chilled methanol, followed by the addition of methyl tert-butyl ether (MTBE) and thorough mixing. Ultrapure water was added to induce phase separation, followed by centrifugation at 4°C (12,000×g, 10 min). The upper organic phase was collected, concentrated, dried, and resuspended in an isopropanol / acetonitrile mixture for mass spectrometry detection. The resuspended samples were injected into a high-resolution liquid chromatography-mass spectrometry system, and data were acquired in both positive and negative ion modes. A C18 reversed-phase column was used for chromatography. Mobile phase A was water / acetonitrile (60:40), and mobile phase B was isopropanol / acetonitrile (90:10), both containing 0.1% formic acid and 10 mM ammonium acetate. Lipid separation was achieved through gradient elution. The acquired mass spectrometry data were imported into a lipidomics analysis platform for peak extraction, noise reduction, alignment, qualitative and quantitative analysis. Structural annotation was performed using the built-in lipid database, and the lipids were categorized and analyzed according to lipid type (e.g., phospholipids, triglycerides, sphingolipids, etc.).

[0046] Example 3

[0047] This embodiment verifies that the expression level of MTLN affects the lipid peroxidation level and the severity of ferroptosis in H9C2 cells after doxorubicin treatment.

[0048] Experimental methods:

[0049] (1) CCK8 assay to detect cell viability: The cells to be tested were packed at 1×10⁶ cells per well. 4Cells were seeded at a density of 100 μL in 96-well plates, with two replicates per group, and each well was replenished with 100 μL of complete culture medium. The plates were incubated overnight at 37°C with 5% CO2 to allow cell adhesion. MTLN-siRNA, MTLN overexpression plasmid, or empty plasmid were transfected according to the experimental groups. After 24 hours, different concentrations of doxorubicin were administered, and the plates were incubated for another 24 hours. 10 μL of Cell Counting Kit-8 reagent (Tongren Chemical, catalog number CK04) was added to each well, and the mixture was gently shaken to avoid air bubbles. The plates were then incubated at 37°C with 5% CO2 for 2 hours. The absorbance of each well was read at 450 nm using a microplate reader, with blank wells (cell-free) used as background values. The results were subtracted before analysis. The experiment was repeated three times, yielding six data points per group. The percentage of cell viability in each treatment group relative to the control group was calculated, and statistical analysis was performed.

[0050] (2) Flow cytometry detection of the BODIPY 581 / 591-C11 probe: Cells to be tested were seeded in 6-well plates and transfected with MTLN-siRNA, MTLN overexpression plasmid, or empty plasmid according to experimental groups. After 24 hours, different concentrations of doxorubicin were administered, and the cells were incubated for another 24 hours. A blank control group (without probe) was included. BODIPY 581 / 591-C11 working solution was added to 2 μM (final concentration) in preheated serum-free medium or PBS. The medium was removed, and the cells were gently washed once with PBS. 2 μM of BODIPY C11 working solution was added to each well, and the cells were incubated in the dark for 30 min at 37°C and 5% CO2. After staining, the cells were washed twice with PBS to remove unbound probes. An appropriate amount of trypsin was added to digest the cells (if they were adherent cells), and the cells were collected after digestion was terminated. The cell suspension was transferred to EP tubes and washed once with PBS. The cells were resuspended in PBS to 500 μL. Flow cytometry was used for detection: the green channel (FITC, Ex / Em≈488 / 510nm) represents oxidized BODIPY. The red channel (PE or PI, Ex / Em≈581 / 591nm) represents reduced BODIPY. A blank group was set up for voltage adjustment and compensation settings. At least 10,000 cells / sample were collected. Analysis was performed using Cytek CytExpert software. An increased FITC / PE intensity ratio indicates increased lipid peroxidation (the probe emits green light after oxidation);

[0051] (3) MDA / GSH reagent kit detection. The manufacturers and product numbers of the MDA / GSH reagent kits are: MDA reagent kit (Beyotime Biotechnology, S0131M); GSH reagent kit (Beyotime Biotechnology, S0053).

[0052] (4) The iron staining detection method was as follows: FerroOrange (Dongren Chemical, F374) was used as the cell iron staining probe. Before the experiment, FerroOrange stock solution was prepared to a concentration of 1 mM with anhydrous DMSO and stored at -20℃ in the dark. Before use, it was diluted to a working concentration of 1 μM with HBSS or serum-free medium. The treated adherent cells were gently washed once with PBS, and staining solution containing 1 μM FerroOrange was added. The cells were then incubated in a 37℃, 5% CO2 incubator in the dark for 30 minutes. After staining, the staining solution was removed, the cells were washed once with PBS, and the images were formed on a glass plate using a fluorescence microscope. The TRITC channel was used (excitation 550–560 nm, emission 575–590 nm). Increased fluorescence indicated an increase in iron ions.

[0053] (5) Transmission electron microscopy observation of mitochondrial morphology in H9C2 cells: After H9C2 cell culture, the culture medium was removed, the cells were gently washed once with PBS, and 2.5% glutaraldehyde fixative (prepared with 0.1M phosphate buffer, pH 7.4) was added and fixed at 4℃ for more than 2 hours. After fixation, the cells were washed three times with 0.1M phosphate buffer for 10 minutes each time, then fixed with 1% osmium tetroxide (OsO4) on ice for 15 minutes, and dehydrated twice with pure acetone for 10 minutes each time. The samples were then infiltrated in epoxy resin embedding solution (1:1 acetone and epoxy resin mixture for 2 hours, then transferred to pure resin for overnight infiltration), and transferred to fresh resin the next day and baked at 60℃ for 48 hours to complete the embedding. Ultrathin sections of about 70 nm thickness were cut from the embedded blocks using an ultramicrotome, placed on a copper mesh, stained with 2% uranium acetate for 10 minutes, then stained with 0.5% lead citrate for 5 minutes, washed with distilled water, and air-dried. The ultrastructure of mitochondria in H9C2 cells was observed using transmission electron microscopy (TEM).

[0054] Example 4

[0055] This embodiment verifies that MTLN expression level affects the doxorubicin myocardial injury phenotype.

[0056] Experimental methods:

[0057] (1) Experimental subjects: C57BL / 6 mice

[0058] (2) Experimental treatment and grouping:

[0059] 1) AAV9 was injected via tail vein; AAV9 was purchased from Gemma Biotechnology and uses the myocardial-specific promoter cTnT+intron.

[0060] AAV9 overexpressing MTLN, the overexpression sequence is:

[0061] ATGGCGGACGTGTCTGAGAGGACGCTGCAGGTGTCCGTGCTAGTGGCTTTCGCCTCTGGAGTGGTCCTGGGCTGGCAAGCGAATCGGCTGCGGAGGCGTTACCTAGACTGGAGGAAGCGGAGGCTGCAGGACAAGCTGGCAACGACTCAGAAAAAGCTGGACCTGGCCTGA (SEQ ID NO. 6);

[0062] The siRNA sequence used to interfere with AAV9 in MTLN is:

[0063] Forward sequence: GAACAUGGCUUGCUUCAGATT (SEQ ID NO.4);

[0064] Reverse sequence: UCUGAGAGCAAGCCAUGUUCTT (SEQ ID NO.5).

[0065] 2) Doxorubicin was administered intraperitoneally at a dose of 5 mg / kg per injection, with a cumulative dose of 20 mg / kg, and the injection was repeated once every 4 days to establish the model.

[0066] NC group: AAV9 was injected via the tail vein, and no additional drug treatment was used;

[0067] DOX group: AAV9 was injected via tail vein as control, and doxorubicin was started via intraperitoneal injection after 3 weeks;

[0068] MTLN-OE+DOX group: AAV9 (MTLN) was injected via the tail vein, and doxorubicin was started via intraperitoneal injection 3 weeks later;

[0069] si-MTLN+DOX group: AAV9 (MTLN-siRNA) was injected via the tail vein, and doxorubicin was started via intraperitoneal injection 3 weeks later;

[0070] At 7 weeks, echocardiographic ejection fraction and short-axis contraction rate were calculated in mice from each treatment group. The day before the examination, depilatory cream was applied to the fur on the front of the mice's chest. After 5 minutes, the fur was wiped away with a gauze moistened with physiological saline. During the examination, the mice were gently held with the left hand to avoid struggling or stress. An M-mode echocardiogram image was acquired using a mouse echocardiogram probe held in the right hand. After capturing M-mode images at three different time points, the mice were returned to their cages for continued rearing. Left ventricular ejection fraction, short-axis contraction rate, and other data were obtained using the measurement and calculation tools built into the mouse echocardiogram machine. All echocardiographic parameters were measured while the mice were awake. Statistical analysis was then performed to draw experimental conclusions.

[0071] (3) WB in mouse myocardial tissue

[0072] Protein extraction from mouse heart tissue samples: Mouse heart tissue samples were kept on ice throughout the protein extraction process. First, an appropriate amount of RIPA lysis buffer containing PMSF and protease inhibitors was added to each sample centrifuge tube, with a final concentration of 1% for both PMSF and protease inhibitors. Then, the mouse heart tissue was minced as finely as possible using ophthalmic scissors; the scissors were cleaned with sterile PBS between different samples to avoid cross-contamination. Next, the samples were transferred to grinding tubes, with two 2mm diameter silica beads added to each tube. A low-temperature tissue homogenizer was used, grinding at 60 Hz for 3 minutes, stopping every minute and allowing the tissue to stand for 1 minute to prevent protein degradation due to temperature rise. After grinding, the samples were immediately transferred to ice, and the grinding beads were discarded. The ground mouse heart tissue samples were allowed to stand on ice for 30 minutes, with a vortexing device for 5 seconds every 10 minutes to ensure complete release of tissue proteins into the liquid system. After standing, all samples were centrifuged at 20,000g at 4°C for 15 minutes. After centrifugation, the supernatant was transferred to a new centrifuge tube for protein concentration determination. The BCA protein concentration assay kit was used to detect tissue protein concentration, and standard proteins at eight concentration gradients were prepared. After adding the reaction solution to a 96-well plate, standard proteins and test samples were added to each well one by one, mixed well, and incubated at 37°C for 30 minutes. After incubation, the absorbance was measured using a microplate reader, and a standard curve was plotted based on the absorbance of the standard proteins and the protein concentration. Based on the standard curve, the protein concentration was calculated using the absorbance of the test samples, and the required volumes of RIPA lysis buffer and protein loading buffer to achieve equal concentrations for all samples were determined. The samples were diluted according to the calculation results, and protein loading buffer was added. After vortexing and mixing, the samples were heated in a 95°C metal bath for 5 minutes to denature them. After boiling and denaturation, the samples were allowed to stand at room temperature until they returned to room temperature before being used for subsequent Western blotting experiments, or they could be stored in a -20°C freezer for no more than 2 months.

[0073] Western blotting (WB) assay: First, remove the denatured protein samples from the -20°C freezer and re-boil them in a 95°C metal bath, shaking constantly while heating to ensure uniform heating. After the samples have returned to room temperature, centrifuge at 1000g for 1 minute, allowing all samples to return to the bottom of the centrifuge tube. After preparing all the protein samples, assemble the protein electrophoresis gels into the electrophoresis tank, filling the space between the two gels with MOPS electrophoresis buffer, ensuring a stable surface and no leakage. Then, remove the sample combs from the gels and use a WB pipette tip to rinse away any residual gel from each well. Next, add the protein markers or the protein samples to be tested sequentially according to the pre-planned loading order. Control the loading speed and use gentle movements to avoid sample diffusion and cross-contamination between wells. After loading, perform electrophoresis at a constant voltage of 80 volts for 30 minutes to ensure the protein samples are neatly transferred into the stacking gel. Then, perform electrophoresis at a constant voltage of 120 volts for 60 minutes. During electrophoresis, cut an appropriately sized PVDF membrane and activate it in methanol for 10 minutes. After activation, immerse the PVDF membrane in transfer buffer for rehydration. After electrophoresis, remove the glass plate covering the protein electrophoresis gel, allowing the gel to immerse in the transfer buffer. Assemble the transfer apparatus in the following order: transfer clamp, sponge pad, transfer filter paper, PVDF membrane, protein electrophoresis gel, transfer filter paper, sponge pad, and transfer clamp. Place the apparatus in the transfer tank, taking care to prevent air bubbles from entering the transfer clamp and ensuring the electrodes are correctly oriented. The transfer conditions are a constant current of 300 mA for 40 minutes. Note that the transfer voltage should not be lower than 80 volts, otherwise the transfer effect will be poor. If the transfer voltage is too low, it indicates that the transfer buffer has been used too many times and should be prepared fresh. During transfer, place the transfer tank in a 4°C refrigerator and cover it with crushed ice; excessively high temperatures will severely affect the transfer effect. After transfer, the PVDF membrane can be immersed in Ponceau S staining solution to observe the approximate protein distribution. Once the electrophoresis and transfer are confirmed to be successful, wash the PVDF membrane three times with 1×TBST, placing it on a horizontal shaker and shaking rapidly at 100 rpm for 5 minutes at room temperature. After rinsing off the Ponceau S staining solution, block the protein using Western blotting (WB) blocking buffer. Blocking conditions for the PVDF membrane are room temperature and slow shaking at 50 rpm for 1 hour on a horizontal shaker. Cut the blocked PVDF membrane into multiple portions according to experimental needs and incubate each portion overnight with the corresponding primary protein antibody. The next day, remove the PVDF membrane from the primary protein antibody container and wash it three times with 1×TBST, placing it on a horizontal shaker and shaking rapidly at 100 rpm for 10 minutes at room temperature. Then, incubate the PVDF membrane with the corresponding secondary protein antibody (generally diluted 1:10000). Secondary antibody incubation conditions are room temperature and slow shaking at 50 rpm for 1 hour on a horizontal shaker.After the secondary antibody incubation was complete, the PVDF membrane was washed three times with 1×TBST. During washing, the PVDF membrane was placed on a horizontal shaker and shaken rapidly at 100 rpm for 10 minutes at room temperature. After completing all the above steps, the PVDF membrane was placed in a Western blotting instrument, a chemiluminescent substrate was added, and a thin plastic plate was used to cover it to ensure the chemiluminescent substrate was evenly distributed on the PVDF membrane. The exposure time was selected according to the experimental requirements, and background images of the PVDF membrane and band images under various exposure time conditions were captured for analysis to draw experimental conclusions.

[0074] (4) HE and Masson staining of mouse myocardial tissue sections: After echocardiography, mice in each treatment group were sacrificed and tissue samples were collected. The cut mouse hearts were weighed using an electronic balance and then macroscopic images were taken. After imaging, the mouse hearts were placed in tissue fixation solution or tissue RNA preservation solution or directly placed in a -80℃ ultra-low temperature freezer for storage, depending on the needs of subsequent experiments. The mouse hearts fixed in the tissue fixation solution were removed and rinsed continuously with running water for 12 hours. After rinsing, the mouse hearts were dehydrated in a gradient manner. Then, the mouse hearts were cleared with xylene and paraffin was used for paraffin embedding. Finally, the paraffin-embedded tissue blocks were placed in a special metal device for embedding and embedded with paraffin. The mouse heart tissue embedding operation was completed when the paraffin was completely solidified. The paraffin-embedded mouse hearts can be used immediately to prepare paraffin sections or stored at room temperature for later use. Paraffin sections were prepared using a paraffin microtome with a section thickness of 4μm. The prepared paraffin sections can also be stored for a long time at room temperature.

[0075] HE staining of mouse myocardial tissue sections: Before staining, the paraffin sections were dewaxed using xylene and ethanol. Each dewaxing step with xylene took 10 minutes, while each dewaxing step with different concentrations of ethanol took 5 minutes. After dewaxing, the sections were stained with hematoxylin and eosin. The stained sections were then dehydrated with ethanol of different concentrations. Finally, neutral resin was used for mounting, taking care to avoid air bubbles that could affect subsequent observation. HE-stained sections were observed under an optical microscope, with each section viewed from multiple fields of view. Representative fields of view were selected for photographing, and scale bars were added for later inter-group comparisons. Experimental conclusions were drawn by observing and comparing HE-stained images from different groups at the same scale.

[0076] Masson staining of mouse myocardial tissue sections: Paraffin sections used for Masson staining were baked before dewaxing, followed by dewaxing with turpentine oil. The first step was staining with Weigert hematoxylin for 20 minutes. After the first staining, the sections were rinsed in running water for 10 minutes. Then, the second staining step was performed with Van Gieson's solution for 1 minute. After all staining steps were completed, the sections were dehydrated with ethanol of different concentrations, mounted with neutral resin, pressed, and dried before microscopic observation. Masson-stained sections were observed under an optical microscope. Different regions of the heart were selected for comprehensive observation of each section. Representative areas were photographed, and scale bars were added for subsequent inter-group comparisons. Experimental conclusions were drawn by observing and comparing Masson-stained images of the same heart region at the same scale.

[0077] Figure 1 This invention presents the amino acid sequences of the microprotein MTLN across species, demonstrating the high conservation of MTLN amino acid sequences among species and indicating good clinical translation potential for related mouse experimental results. Immunofluorescence confirmed the location of MTLN in mitochondria, and gene ontology analysis proved its association with mitochondrial function. Western blot analysis of various mouse tissues demonstrated relatively high expression of MTLN in the heart. Analysis of human cardiac sequencing data from the literature revealed significantly upregulated MTLN expression in the hearts of patients with dilated cardiomyopathy and heart failure, and MTLN is associated with cardiac contractile function, fatty acid metabolism, and mitochondrial function.

[0078] Figure 2 The results showed that the interference effect of the present invention on MTLN-siRNA was detected by Western blotting, proving that the siRNA designed in this invention is effective in intervening in MTLN. The present invention also demonstrated, through non-targeted lipid profile data, that the interference of MTLN led to an upward trend in the level of monolysocardiolipin (MLCL), a central phospholipid deacylation product in H9C2 cells. The present invention further demonstrated, through ELISA detection of cardiolipin levels in MTLN-interfered H9C2 cells and control cells, that the interference of MTLN could induce a downregulation of cardiolipin levels. The present invention also demonstrated, through CCK8 assay and mitochondrial electron microscopy, that MTLN can be used to alleviate doxorubicin-induced myocardial injury at the cellular level.

[0079] Figure 3The results show that, at the animal level, immunofluorescence and Western blotting confirmed the successful intervention of AAV9 on MTLN expression levels in mouse hearts. Statistical results from the cardiolipin ELISA kit confirmed that changes in MTLN expression levels do indeed induce changes in cardiolipin levels. Echocardiography, electron microscopy, and section staining results demonstrated that MTLN expression levels affect the doxorubicin-induced myocardial injury phenotype. This invention demonstrates that MTLN participates in regulating myocardial cardiolipin levels and can serve as an intervention target for alleviating doxorubicin-induced myocardial injury.

[0080] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of an inhibitor targeting the MTLN in the preparation of a drug for preventing or treating doxorubicin-induced myocardial injury, characterized in that, The amino acid sequence of the MTLN is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The inhibitor is an interfering nucleic acid that targets MTLN, and the interfering nucleic acid is selected from: double-stranded siRNA containing SEQ ID NO.2 and SEQ ID NO.3; and double-stranded siRNA containing SEQ ID NO.4 and SEQ ID NO.

5.

3. A kit for detecting the risk of myocardial injury from doxorubicin, characterized in that, It includes reagents for detecting the expression level of MTLN in myocardial tissue; the amino acid sequence of the MTLN is shown in SEQ ID NO.

1.

4. The application of a reagent for detecting MTLN expression levels in screening drugs to alleviate doxorubicin-induced myocardial injury, characterized in that, The amino acid sequence of the MTLN is shown in SEQ ID NO.

1.

5. A biomarker for assessing the risk of doxorubicin-induced myocardial injury, characterized in that, The biomarker is microprotein MTLN, and the amino acid sequence of microprotein MTLN is shown in SEQ ID NO.1.

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