Differential Genes Analysis and Validation of Disulfideptosis in Rat Myocardial Ischemia-Reperfusion Injury Model

摘要

The protocol describes the identification of several novel disulfideptosis-related differential genes associated with myocardial ischemia-reperfusion injury by bioinformatics analysis and experimental validation.

摘要

Myocardial ischemia-reperfusion injury (MIRI) is an additional injury that occurs during the process of restoring heart tissue blood flow after ischemia-induced injury. MIRI seriously affects the efficacy and short-term and long-term prognosis of reperfusion after myocardial infarction. At present, the mechanism of MIRI is not fully clear. Disulfideptosis is a novel mode of cell death, and the relationship between MIRI and disulfideptosis-related genes (DRGs) expression is still unclear. Firstly, this study explores the differentially expressed genes associated with disulfideptosis in MIRI through bioinformatics analysis. Secondly, by constructing a rat model of MIRI, DRGs were further detected. This study identified 12 related genes, including Myh9, SLC7A11, SLC3A2, Myh7b, ACTB, FLNB, Actn1, Actn4, Flnc, Dbn1 and Pdlim1. Myocardial tissue of rats with MIRI shows obvious pathological damage and apoptosis events. The results of immunohistochemistry indicated that MIRI stimulation increased the expression of GLUT1 protein in myocardial tissue but restricted the expression of F-actin protein. In addition, significant differences in the expression of three proteins were validated using external datasets and MIRI rat models. This study demonstrated that DRGs had significant predictive value in MIRI, providing new prospects for exploring biomarkers and potential therapeutic targets of MIRI.

引言

Acute myocardial infarction (AMI) is a severe cardiovascular condition and remains a leading global cause of mortality. Percutaneous coronary intervention has significantly reduced mortality rates in AMI patients¹. However, reperfusion therapy aimed at restoring myocardial blood supply is accompanied by a series of adverse pathological and physiological responses. These processes can result in an increased infarct size, myocardial cell death, sustained ventricular arrhythmias, and sudden death². Myocardial ischemia-reperfusion injury (MIRI) is a complex cardiovascular condition influenced by factors such as cytokines, chemokines, growth factors, oxidative stress, and calcium overload³. Mitigating MIRI remains a significant challenge.

Recently, disulfideptosis has emerged as a novel form of cell death characterized by rapid collapse of the cytoskeletal actin network due to excessive accumulation of disulfides, including cysteine, within cells, resulting from NADPH⁺ depletion. Excessive disulfide accumulation disrupts disulfide bonds among cytoskeletal proteins, leading to actin migration and cell death^4,5. Unlike previously reported forms of cell death such as apoptosis, necrosis, pyroptosis, and ferroptosis, disulfideptosis is initiated by the excessive aggregation of intracellular disulfides and is not antagonized by specific inhibitors of other cell death pathways. As a distinctive cell death, evidence has indicated that cellular glucose deficiency-evoked SLC7A11-overexpressing can trigger disulfidptosis⁴. After MIRI, insufficient insulin secretion can induce hypoglycemia and subsequent disulfidptosis, which may cause further damage to myocardial cells and may be one of the novel mechanisms of MIRI⁵.

In this study, the primary objective was to utilize comprehensive gene expression databases, such as the Gene Expression Omnibus (GEO), to analyze differential gene expression between normal and MIRI samples. We conducted a cross-referential analysis between differentially expressed genes and genes associated with disulfideptosis, with the aim of identifying disulfideptosis-related genes (DRGs) that exhibit differential expression in MIRI. Machine learning algorithms were employed to identify key genes, and we validated the expression patterns of the selected differential genes of interest using an animal model. This approach provides a fresh perspective towards gaining a deeper understanding of the potential mechanisms underlying the onset and progression of MIRI. The primary focus of this research was to investigate a novel cell death mechanism within MIRI, uncover associated differential genes, and conduct preliminary experiments to validate these findings. The ultimate goal was to identify novel therapeutic targets for mitigating MIRI based on this emerging cell death mechanism.

研究方案

For this study, nine Sprague-Dawley (SD) male rats, aged 6-8 weeks and weighing 180-220 g, were selected from the Hubei Experimental Animal Research Center [SCXK (Hubei) 20200018]. Rats were kept in specific pathogen free animal houses to acclimatize for 1 week, with a 12 h/12 h light and dark cycle, free drinking and eating. The current study was conducted with approval from the Animal Ethics Committee of The Third Affiliated Hospital of Zunyi Medical University (approval number: (2016)-1-56). All procedures were performed in accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Stringent measures were implemented to minimize the number of animals used and to mitigate their suffering.

1. Differential expression analysis of genes related to MIRI and disulfideptosis

NOTE: RNA sequencing data from MIRI named GSE214122 were selected to screen genes associated with disulfideptosis according to literature report⁶.

1. Open the GEO database, type GSE214122 into the Search box, and click Enter. Download the generated Series Matrix File(s) in TXT format under the Download section.
   NOTE: Before conducting data analysis, it is typically necessary to preprocess the downloaded data. This includes checking for missing values, removing low-quality samples and genes, and performing normalization or standardization on the data. The retrieved GSE214122 dataset contained samples from the sham-operated and MIRI groups.
2. Perform differential gene expression analysis of the samples using the DESeq2 package⁷. Consider genes with |log2FC|>1 (fold change greater than 2) and FDR<0.05 as statistically significant, resulting in the identification of differentially expressed genes associated with MIRI. Generate a heatmap of differential gene expression in step 1.2 using R's pheatmap package⁷.
3. Construct a Venn diagram using R's VennDiagram package⁷ (see Table of Materials). Generate Volcano plots to visualize disulfideptosis-related genes using an EnhancedVolcano package in R⁷.
4. Construct a PPI network connecting the MIRI differentially expressed genes with disulfideptosis genes using the STRING database⁷ (see Table of Materials).

2. Construction of the rat MIRI model

1. Fast the rats overnight before performing anesthesia. Anesthetize rats by intraperitoneal injection of 2% sodium pentobarbital (40 mg/kg) and secure the limbs to a rat board in a supine position. Confirm depth of anesthesia by no response to toe pinching, loss of the righting reflex, and the eyewink reflex. Use vet ointment to prevent dryness while under anesthesia.
2. Disinfect the skin of the neck and chest with iodophor and deiodinate with 75% alcohol. Shave the fur of the neck and the left chest with an electric razor and make a median neck incision approximately 2.5 cm in length with a scalpel (see Table of Materials).
3. Separate trachea with forceps and perform endotracheal intubation with a connection to a small animal ventilator³ (see Table of Materials).
4. Cut 4 cm longitudinally along the left side of the sternum with ophthalmic scissors and bluntly separate the subcutaneous muscles with forceps to expose the pleura between the third and fourth ribs.
5. Make a 4-5 cm incision from the xiphoid process to the middle of the left second intercostal space while holding the scalpel at a 45° angle. Gently and slowly separate the pectoralis major and anterior serratus muscles using ophthalmic forceps to access the intercostal space.
6. Make a 1.5 cm transverse incision between the left third and fourth ribs using ophthalmic scissors. After dissecting the pericardium using ophthalmic forceps, raise the left atrial appendage with forceps to identify the coronary ostium at the aortic root.
7. Ligature the left anterior descending coronary artery between the pulmonary cone and left atrial appendage using a 6-0 suture (see Table of Materials).
8. After 30 min, loosen the suture to allow reperfusion for 120 min, followed by closure of the muscle and skin layers using a 6-0 suture. Apply penicillin (see Table of Materials) by intraperitoneally injecting with 80,000-100,000 units.
9. In the sham group, perform all the procedures as done for the model group, but only thread the left anterior descending coronary artery without ligation or reperfusion.
10. Once the rats regain consciousness, remove the endotracheal tubes. Return the rats to the animal facility for recovery, where they will have free access to food and water. House the rat subjected to surgery in a single cage with an independent ventilation system in a sterile laboratory.
11. During 30 min of ischemia and 120 min of reperfusion, perform echocardiography. The modeling was considered successful if the left ventricular ejection fraction measured less than 50%^1,6.

3. Harvest heart tissue³

1. After 120 min of reperfusion, place the rats in the euthanasia container to implement euthanasia by the inhalation of carbon dioxide. Collect the heart tissues of the rat immediately.
2. Remove the rats' abdominal hair with a shaving device and sterilize the anterior chest area with iodine and 75% alcohol. Dissect the abdominal cavity of the rat along the midline of the abdomen with a scalpel.
3. Cut the skin from the xiphoid process upward to the submandibular space with surgical scissors and bluntly peel off the skin and subcutaneous tissue with forceps to both sides to expose the thoracic cage and superficial cervical muscle layer (see Table of Materials).
4. Open the thorax and pleura along the midline with a surgical scissor to expose the heart, clamp the aorta with a vascular clip, and cut the artery with ophthalmic scissors. Take out the heart and dissect into 1 mm³ tissue block in cold saline on ice bath and fix with 4% paraformaldehyde for 48 h (see Table of Materials).

4. Hematoxylin-eosin (H&E) staining

1. Dehydrating and embedding: Dehydrate the heart tissue block by soaking in sequence in 75% alcohol, 85% alcohol, 95% alcohol (I), 95% alcohol (II), 100% alcohol (I), and 100% alcohol (II) for 1 h in each solution. Transparentize with xylene (I) and xylene (II) by dipping for 15 min in each solution. Place the heart tissue block in liquid paraffin (I), paraffin (II), and paraffin (III) for 20-30 min in sequence. Allow the paraffin block to cool naturally until solidification.
2. Slice the paraffin-embedded heart tissue block into 4 µm sections using a paraffin slicing machine (see Table of Materials). Lay sections flat with a soft brush in a 45 °C water bath and then pick up the fully expanded sections with a slide.
3. Dewaxing: Bake the slices in an oven for 30-60 min at 55 °C, and dewax with xylene (I) for 5-10 min and xylene (II) for 5-10 min. Then, wash xylene with absolute ethanol for 1-5 min, 95% ethanol for 1-5 min, and 75% ethanol for 1-5 min, and rinse with tap water for 1-5 min.
4. Staining: Stain with a hematoxylin solution for 5-20 min, rinse with tap water for 5-10 s, stain with an eosin solution for 15-30 s, and wash with 75%- 85% ethanol for 30 s.
5. Dehydrating, transparent, and sealing: Dehydrate with 95% ethanol (I) for 0.5-2 min, and 95% ethanol (II) for 2-5 min, followed by absolute ethanol (I) for 2-5 min, and absolute ethanol (II) for 2-5 min. Transparentize with xylene (I) for 1 min and xylene (II) for 1 min, and then seal the slice by adding two drops of neutral resin onto the slice and place a cover glass. Press the cover glass lightly to fill the slice with neutral resin (see Table of Materials).
6. Observe each slice under an optical microscope at 200x magnification in a double-blind manner by two independent technicians.
   1. Select three random visual fields to evaluate myocardial congestion, hemorrhage, fibrosis, necrosis, and degeneration. Use the following scoring criteria: 0 indicated no lesion; 0-1 indicated lesions were less than 1/4 of the designated area; 1-2 indicated lesions ranged from approximately 1/4−1/2 of the designated area; 2-3 indicated lesions ranged from approximately 1/2−3/4 of the designated area; and 3-4 indicated lesions were greater than 3/4 of the designated area.

5. TUNEL assay

1. Dilute proteinase K with phosphate buffer solution (PBS) to 40 µg/mL and add 100 µL of proteinase K to 4 µm sections prepared in step 4.3 to cover the entire sample area and incubate for 10 min at room temperature.
   NOTE: Proteinase K is included in the TUNEL apoptosis detection kit (see Table of Materials).
2. Wash the section 2x with PBS for 5 min each, remove the excess liquid by suction with filter paper, and keep the section moist in a wet box.
   NOTE: Proteinase K must be washed clean to not interfere with subsequent labeling reactions.
3. Uniformly add 50 µL of TUNEL reaction solution containing 2 µL of TdT enzyme and 48 µL of TUNEL reaction buffer and place the section in a wet box to incubate at 37 °C in the dark for 2 h.
   NOTE: TdT enzyme and TUNEL reaction buffer are included in the TUNEL apoptosis detection kit.
4. Discard the TUNEL reaction solution and rinse 2x with PBS for 5 min each. Wash the section 3x for 5 min each with PBS containing 0.1% Triton X-100 and 5 mg/mL BSA.
5. Uniformly add 50 µL of DAPI staining solution and incubate for 10 min at room temperature in the dark. Discard the DAPI staining solution and soak the section in PBS, 3x, 5 min each time.
6. Obtain TUNEL red and DAPI blue signals at excitation and emission wavelengths of 593/614 nm and 364/454 nm, respectively. Capture images under a fluorescence microscope at 200x magnification (see Table of Materials).

6. Immunohistochemical staining

1. Add 200 µL of enhanced endogenous peroxidase blocking buffer to the section in step 4.3 and incubate for 10 min at room temperature in the dark. Wash section 2x with distilled water for 5 min each.
2. Add 1 mL of improved citrate antigen retrieval solution and 49 mL of double-distilled water to the antibody incubation box and gently mix. Immerse the section in the antigen retrieval solution and heat at 95 °C for 20 min before cooling to room temperature (see Table of Materials).
3. Wash the section 2x with PBS for 5 min each. Place the section in the antibody incubation box to incubate with 100 µL of primary antibodies against F-actin (1:100), GLUT1 (1:200), Myh9 (1:200), SLC7A11 (1:200) and SLC3A2 (1:200) overnight at 4 °C.
4. Wash the section 2x with PBS for 5 min each. Add 100 µL of secondary antibody to the section and incubate for 20 min at room temperature.
5. Wash the section 2x with PBS for 5 min each. Stain with 100 µL of DAB working solution for 5 min at room temperature and stop the color reaction by flushing with purified water.
   NOTE: DAB working solution was prepared by mixing DAB chromogenic solution A and DAB chromogenic solution B in a 1:1 ratio, which is included in the DAB horseradish peroxidase color development kit (see Table of Materials).
6. Stain the section with 100 µL of hematoxylin staining solution for 3 min and wash with running water for 3 min (see Table of Materials).
   NOTE: The hematoxylin and eosin staining kit includes a hematoxylin staining solution.
7. Immerse the section in 75% ethanol, 95% ethanol, and 100% ethanol for 2 min in each.
8. Seal the section with neutral resin, cover with a cover slip, and examine the section under an optical microscope at 200x magnification (see Table of Materials). Tissues exhibiting a brown color are positive reactions. Calculate the positive expression rate as the ratio of the stained area to the total area of the field of view. Perform quantitative analysis using Image J software.

7. Western blot detection

NOTE: Lysis buffer and protease inhibitor were included in a BCA protein quantification kit (see Table of Materials).

1. Weigh 0.1 g of fresh myocardial samples from step 3.5 and mix with 1 mL of lysis buffer and 10 µL of protease inhibitor, followed by grinding in a glass grinding container and centrifugating at 10,304 x g for 10 min at 4 °C.
2. Collect the supernatant and perform protein quantification using the BCA method according to the manufacturer's instructions⁸. Boil the supernatant at 95 °C for 5 min.
3. Separate proteins of different molecular weights using 10% separating gel by loading 10 µL sample for protein electrophoresis at 80/120 V.
4. Transfer the proteins on the gel to a PVDF membrane⁹. Subsequently, block the membrane with 5% skim milk powder for 1 h and incubate overnight at 4 °C with Myh9, SLC7A11, SLC3A2, and β-tubulin primary antibodies at a dilution of 1:1000 (see Table of Materials).
5. After washing with PBS 3x for 5 min each, incubate the membrane with secondary antibody (1:5000; see Table of Materials) at room temperature for 2 h, followed by washing with PBS 3x for 5 min each before visualization with a protein visualization instrument (see Table of Materials).
6. Calculate the bands' grayscale ratio to β-tubulin using Image J software^8,9.

8. Statistical analysis

1. Express data as the mean ± standard deviation. Analyze data using one-way analysis of variance followed by the least significant difference post hoc test. Perform statistical analysis using a commercial statistical analysis tool (see Table of Materials) and consider p < 0.05 as statistically significant.

结果

Screening of DRGs in MIRI
The GSE214122 dataset from Gene Expression Omnibus included three sham and three MIRI samples data. Using the DESeq2 package in R, 1233 differentially expressed genes (DEGs) were identified between MIRI and sham samples. Based on |log2FC|>2 and FDR<0.05, 417 significantly different genes were further selected using R's pheatmap package (Figure 1A). Then, 15 intersection genes between these 1233 DEGs and 106 DRGs were presented in <...

讨论

Disulfideptosis is closely associated with the actin cytoskeleton, a critical cellular structure essential for maintaining cell shape and viability. Composed of actin filaments, the actin cytoskeleton imparts overall cellular shape and structure. F-actin serves as a marker for the cellular cytoskeleton, and under conditions of glucose starvation, disulfide bonds increase significantly, leading to downregulation of F-actin. This phenomenon primarily affects processes and pathways related to the actin cytoskeleton and cell...

材料

Name	Company	Catalog Number	Comments
75% alcohol	Hunan Tongruijian Pharmaceutical Co. Ltd., Hunan, China	85026
6-0 nylon suture	Shanghai Pudong Jinhuan Medical Supplies Co. Ltd., Shanghai, China	CS002
BCA protein quantification kit	Beyotime Biotechnology, Shanghai, China	P0011
Bull serum albumin	Beyotime Biotechnology, Shanghai, China	ST2254
Centrifuge	Hunan Kaida Scientific Instrument Co. Ltd., Hunan, China	KH19A
DAB horseradish peroxidase color development kit	Beyotime Biotechnology, Shanghai, China	P0203
DAPI staining solution	Beyotime Biotechnology, Shanghai, China	C1006
DESeq2 package		Version 4.1
Electric razor	Kelmerpp	235376
Enhanced endogenous peroxidase blocking buffer	Beyotime Biotechnology, Shanghai, China	P0100B
Filter paper	Nanjing Keruicai Equipment Co., Ltd., Nanjing, China	1.00049E+11
Fluorescence microscope	Nikon	ECLIPSE Ci
GEO database	https://www.ncbi.nlm.nih.gov/geo/
Glass grinder	Shanghai Leigu Instrument Co. Ltd., Shanghai, China	B-013002
GraphPad Prism	GraphPad Software	V8.0
Hematoxylin and eosin staining kit	Beyotime Biotechnology, Shanghai, China	C0105M
Image J software	National Institutes of Health, Bethesda, USA	v1.8.0
Improved citrate antigen retrieval solution (50X)	Beyotime Biotechnology, Shanghai, China	P0083
Iodophor	Folca, Shenzhen, China	1.00077E+11
Optical microscope	Nikon	ECLIPSE Ci
Phosphate buffer solution	Beyotime Biotechnology, Shanghai, China	C0221A
Primary antibodies against GLUT1	Proteintech Group, Inc, Wuhan, China	21829-1-AP
Primary antibodies against MYH9	Proteintech Group, Inc, Wuhan, China	11128-1-AP
Primary antibodies against SLC3A2	Wuhan Lingsi Biotechnology Co., Ltd., Wuhan, China	LJS-D-7468
Primary antibodies against SLC7A11	Proteintech Group, Inc, Wuhan, China	26864-1-AP
Protein visualization instrument	Thermo Fisher Scientific Inc.	iBright CL750
Rat board	Zhengzhou Haopai Biotechnology Co. Ltd., Zhengzhou, China	JPB-E
Scalpel	Shanghai Lianhui Medical Supplies Co., Ltd., Shanghai, China	1.00471E+13
Secondary antibody	Wuhan Boster Biological Technology, Ltd., Wuhan, China	BA1054
STRING database	https://cn.string-db.org/	Version 12.0
Triton X-100	Beyotime Biotechnology, Shanghai, China	ST1722
TUNEL apoptosis detection kit	Proteintech Group, Inc., Wuhan, China	PF00009