CN117599190A - Application of CHCHD3 expression promoter in preparation of myocardial infarction drugs - Google Patents

Application of CHCHD3 expression promoter in preparation of myocardial infarction drugs Download PDF

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CN117599190A
CN117599190A CN202311623303.4A CN202311623303A CN117599190A CN 117599190 A CN117599190 A CN 117599190A CN 202311623303 A CN202311623303 A CN 202311623303A CN 117599190 A CN117599190 A CN 117599190A
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chchd3
myocardial infarction
expression
myocardial
protein
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唐其柱
谢赛阳
邓伟
张彤
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Renmin Hospital of Wuhan University
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Renmin Hospital of Wuhan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Abstract

The invention relates to application of a CHCHD3 expression promoter in preparing a myocardial infarction medicament, which can relieve mitochondrial damage of myocardial tissues after myocardial infarction and improve mitochondrial oxidative phosphorylation. The invention discovers that the specific over-expression of the CHCHD3 gene in the heart can reduce the death rate of mice with myocardial infarction models, so that the heart function of the mice is obviously improved, and meanwhile, the in-vivo over-expression of the CHCHD3 can relieve the mitochondrial damage of myocardial tissues of the mice after myocardial infarction.

Description

Application of CHCHD3 expression promoter in preparation of myocardial infarction drugs
Technical Field
The invention relates to the field of biological medicine, in particular to application of a coiled-coil structural domain protein 3 (CHCHD 3) expression promoter in preparation of medicines for preventing, relieving and/or treating myocardial infarction.
Background
Myocardial infarction (Myocardial Infarction, MI) is a common critical condition in cardiovascular diseases, mainly due to myocardial necrosis caused by acute or chronic ischemia and hypoxia of coronary arteries, clinically mainly with severe and durable poststernal or precordial pressure pain, rest or nitroglycerin can not be completely relieved, with increased serum myocardial enzymatic activity and progressive electrocardiographic changes, arrhythmia, shock or heart failure onset are induced suddenly, and life is often endangered. At present, the treatment method for myocardial infarction mainly comprises medicines, intervention, surgery and the like, but can not fundamentally and effectively repair ischemic necrotic cardiac muscle, a large number of functional cardiac muscle cells undergo apoptosis necrosis after myocardial infarction, and the integrity of the original cardiac structure and function is lost and replaced by fibrous scar tissues due to subsequent heart reconstruction, so that heart failure is finally unavoidable.
Mitochondria are the major organelles of energy supply in cardiomyocytes, and mitochondrial oxidative phosphorylation is an important process for the production of ATP, a direct energy substance. Mitochondrial integrity is an important structural basis for ATP production, and mitochondrial cristae damage is a major contributor to myocardial energy metabolism dysfunction. Energy deficiency is one of the important causes of myocardial remodeling after myocardial infarction, and thus, the resolution of energy metabolism problems and improvement of mitochondrial damage can further prevent myocardial damage after myocardial infarction, so treatment against mitochondrial energy metabolism has great potential in myocardial infarction. At present, main measures for treating myocardial injury and myocardial reconstruction after myocardial infarction are as follows: reperfusion therapy, RAAS inhibitors, including beta blockers, ACEI and ARB drugs, aldosterone receptor antagonists, statins, anti-inflammatory therapy, stem cell transplantation therapy, and the like. However, the treatment effect of myocardial infarction is still not ideal and the death rate is still higher because of the complex pathogenesis, side effects of medicines and the like. Therefore, finding new treatments to alleviate mitochondrial cristae damage to cardiomyocytes and optimize myocardial energy metabolism may be an important strategy to improve myocardial infarction.
Coiled-coil domain protein 3 (CHCHD 3) is a small mitochondrial protein encoded by a nuclear gene, primarily localized to the outer mitochondrial membrane, maintaining stability of the mitochondrial cristae. Previous studies revealed that CHCHD3 and mitochondrial contact sites act directly on SAM50 in the cristae tissue system, maintaining structural stability of the mitochondrial cristae, while increasing expression of mitochondrial complexes, promoting ATP production. Currently, research on CHCHD3 is mainly focused on neurodegenerative and tumorigenic development. Recent research evidence suggests that CHCHD3 is involved in acetylcholine-regulated mitochondrial ridge remodeling to reduce palmitate-induced cardiomyocyte hypertrophy in neonatal rat ex vivo (Xue RQ et al free radio Biol Med.2019Dec; 145:103-117.). However, the influence of CHCHD3 on the mitochondrial structure and function of cardiomyocytes in the hypoxic state is not clear, and furthermore, the study of CHCHD3 in myocardial injury after myocardial infarction has not been reported.
Disclosure of Invention
The invention discovers that CHCHD3 is mainly positioned in mitochondria, and after the CHCHD3 is over expressed by isolated myocardial cells, the damage of the mitochondria of the myocardial cells under the anoxic condition can be obviously reduced, and the oxidative phosphorylation function of the mitochondria can be improved. Animal experiments also prove that in a myocardial infarction model, the death rate of mice with the heart-specific over-expressed CHCHD3 gene is reduced, the heart function is obviously improved, and simultaneously, the mitochondrial damage of myocardial tissues of the mice after myocardial infarction can be relieved by the in-vivo over-expression of the CHD 3.
Based on the findings, the invention provides the following technical scheme:
in a first aspect, the present invention provides the use of CHCHD3 or a chd3 expression enhancer in the manufacture of a medicament for the prevention, alleviation and/or treatment of myocardial infarction.
Further, the medicament for preventing, relieving and/or treating myocardial infarction can relieve mitochondrial damage of myocardial tissue after myocardial infarction and improve mitochondrial oxidative phosphorylation function.
Further, CHCHD3 expression promoters include one or more of proteins, proteolytically targeted chimeras, polynucleotides, small molecule compounds.
Further, the chd3 or chd3 expression promoter allows entry into the cell by one or more of the following: direct naked DNA injection method, liposome coated DNA direct injection method, gold coated DNA gene gun bombardment method, propagation defect bacteria carrying plasmid DNA method, replication defect adenovirus carrying target DNA method, PEG modified protein drug injection method, liposome coated protein intravenous injection method, protein microsphere preparation subcutaneous injection method.
Further, the CHCHD3 expression promoter is a recombinant plasmid containing the CHCHD3 gene and capable of normally expressing the CHCHD3 protein.
Further, the CHCHD3 expression promoter is adenovirus Ad-CHCHD3 carrying a CHCHD3 sequence, and the Ad-CHCHD3 promotes the expression of CHCHD3 protein in myocardial tissue.
In a second aspect, the present invention provides a medicament for preventing, alleviating and/or treating myocardial infarction, which is a preparation prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components to a CHCHD3 expression promoter serving as an active substance.
Further, the CHCHD3 expression promoter is a recombinant plasmid containing the CHCHD3 gene and capable of normally expressing the CHCHD3 protein.
Further, the CHCHD3 expression promoter is adeno-associated virus Ad-CHCHD3 carrying CHCHD3 sequence, and the Ad-CHCHD3 promotes the expression of CHCHD3 protein in myocardial tissue.
In a third aspect, the present invention provides a pharmaceutical composition for preventing, alleviating and/or treating myocardial infarction, comprising 2 or more than 2 kinds of drugs for preventing, alleviating and/or treating myocardial infarction, wherein one of the drugs uses CHCHD3 expression promoting agent as an active substance.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention discovers that the novel function of the CHCHD3 gene, namely, the over-expression of the CHCHD3 gene can improve myocardial injury after myocardial infarction and prevent heart failure.
(2) CHCHD3 can improve myocardial injury after myocardial infarction and provides a new target for preparing medicines for preventing, relieving and/or treating myocardial injury after myocardial infarction.
(3) The CHCHD3 expression promoter can be used for preparing medicines for preventing, relieving and/or treating myocardial injury after myocardial infarction.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1: A. mRNA expression levels of CHCHDs family molecules in myocardial tissue of normal and clinical myocardial ischemia patients, data derived from GEO public database GSE46224; B. mRNA expression levels of CHCHD3 in myocardial tissue of normal humans and clinical myocardial ischemia patients; C-D, detecting the expression and quantitative analysis result of CHCHD3 by immunohistochemical staining of myocardial tissue of normal people and clinical myocardial ischemia patients; E-F. Protein expression level of CHCHD3 in myocardial tissue of normal and clinical myocardial ischemia patients and quantitative analysis result, beta-Tubulin is used as internal reference.
Fig. 2: c57 mice were pre-and post-coronary left anterior descending ligation for 12 hours, 24 hours, 3 days, 7 days, and 21 days for CHCHD3 protein expression levels and quantitative analysis results, β -Tubulin as an internal reference; mRNA expression level results of CHCHD3 before and 12 hours after left anterior descending coronary artery ligation in c.c57 mice, 24 hours, 3 days, 7 days, and 21 days; D. immunofluorescence showed CHCHD3 expression levels in normal mouse myocardial tissue with cardiomyocytes (cTnT development), endothelial cells (CD 31 development), activated myocardial fibroblasts (α -SMA development) and monocytes (CD 68 development).
Fig. 3: A. normal oxygen and hypoxia (1.0% O) 2 ) Stimulating the treated Neonatal Rat Cardiomyocytes (NRCMs), immunofluorescence detecting co-localization levels of CHCHD3 (green) and mitochondrial fluorescent probe (red) in the cells; B-C normoxic and hypoxic (2%O) 2 ) Protein expression levels of CHCHD3 in stimulated Neonatal Rat Cardiomyocytes (NRCMs) and quantitative analysis results, β -Tubulin served as an internal reference.
Fig. 4: A. schematic representation of a transgenic plasmid containing full length CHCHD3 transcript (nm_ 025336.2) and the α -MHC promoter; B. the heart-specific CHCHD3 gene over-expression identification primer sequence; C. heart-specific CHCHD3 gene overexpression and wild-type mouse gene identification nucleic acid electrophoresis; D. heart-specific CHCHD3 gene overexpression and mRNA levels of CHCHD3 gene in wild-type mouse myocardial tissue; E-F. over-expression of heart-specific CHCHD3 gene and quantitative analysis of CHCHD3 protein expression level in myocardial tissue of wild-type mice, and beta-Tubulin as an internal reference.
Fig. 5: A. heart-specific chd3 gene overexpression (CHCHD 3 cTg) and wild-type mouse post-myocardial infarction survival analysis; heart rupture ratio after myocardial infarction in chchd3 cTg mice and wild type mice; c-d.ttc (2, 3, 5-triphenyltetrazolium chloride) staining showed infarct size 7 days after myocardial infarction in CHCHD3 cTg mice and wild-type mice and quantitative analysis results; pulmonary weight to weight ratio of chchd3 cTg mice and wild type mice 3 weeks after sham surgery or myocardial infarction; chchd3 cTg mice and wild type mice were stained with HE in lung tissue 3 weeks after sham surgery or myocardial infarction, cavitation suggested the occurrence of pulmonary edema.
Fig. 6: serum troponin (cTnT) levels of creatine kinase isoenzyme (CK-MB) in a-b.chchd3 cTg mice and wild-type mice 3 weeks after sham surgery or myocardial infarction; echocardiography of C-e.chchd3 cTg mice and wild-type mice 3 weeks after sham surgery or myocardial infarction, and analysis of left ventricular ejection fraction and overall longitudinal strain.
Fig. 7: chchd3 cTg mice and wild type mice showed mitochondrial morphology in 3 weeks of myocardial tissue following sham surgery or myocardial infarction; chchd3 cTg mice and wild-type mice were quantitatively analyzed for mitochondrial percentage per square micron of mitochondrial area, mitochondrial occupancy, and cristae injury in myocardial tissue 3 weeks after sham surgery or myocardial infarction.
Fig. 8: schematic of plasmid structure in chchd3 over-expression adenovirus vector; protein level analysis of CHCHD3 after transfection of neonatal rat cardiomyocytes with CHCHD3 over-expression and control empty adenovirus; CHCHD3 overexpression and control mRNA levels of chd3 were quantified after transfection of neonatal rat cardiomyocytes with empty adenovirus.
Fig. 9: chchd3 overexpression and control empty adenovirus transfection neonatal rat cardiomyocytes were transfected under normoxic and hypoxic conditions (1.0% o) 2 ) After culturing for 24 hours in the environment, incubating the mitochondrial fluorescent probe, and performing laser confocal photographing to display the mitochondrial morphology of the myocardial cells; B. thread grainQuantitative analysis results of the volume average length; C-D.CHCHD3 overexpression and control empty adenovirus transfection neonatal rat cardiomyocytes were transfected under normoxic and hypoxic conditions (1.0% O) 2 ) After 24 hours of incubation in the environment, the seahorse cell energy metabolizing instrument detects the quantitative analysis results of the myocardial cell mitochondrial oxygen consumption curve (OCR), basal respiration, ATP production, maximum respiration rate and residual respiration capacity.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The invention provides the use of CHCHD3 or chd3 expression promoters in the manufacture of a medicament for the prevention, alleviation and/or treatment of myocardial infarction.
In some embodiments, the medicament for preventing, alleviating and/or treating myocardial infarction can reduce mitochondrial damage to myocardial tissue following myocardial infarction and improve mitochondrial oxidative phosphorylation.
In some embodiments, CHCHD3 expression promoters include one or more of proteins, proteolytically targeted chimeras, polynucleotides, small molecule compounds.
In some embodiments, the chd3 or chd3 expression enhancer allows entry into the cell by one or more of the following: direct naked DNA injection method, liposome coated DNA direct injection method, gold coated DNA gene gun bombardment method, propagation defect bacteria carrying plasmid DNA method, replication defect adenovirus carrying target DNA method, PEG modified protein drug injection method, liposome coated protein intravenous injection method, protein microsphere preparation subcutaneous injection method.
In some embodiments, the chd3 expression promoter is a recombinant plasmid comprising the CHCHD3 gene and capable of normally expressing the CHCHD3 protein.
In some embodiments, the chd3 expression enhancer is an adenovirus Ad-CHCHD3 carrying a CHCHD3 sequence, the Ad-CHCHD3 enhancing expression of the CHCHD3 protein in myocardial tissue.
The medicine for preventing, relieving and/or treating myocardial infarction is a preparation prepared by taking a CHD3 expression promoter as an active substance and adding pharmaceutically acceptable auxiliary materials or auxiliary components.
In some embodiments, the chd3 expression promoter is a recombinant plasmid comprising the CHCHD3 gene and capable of normally expressing the CHCHD3 protein.
In some embodiments, the CHCHD3 expression enhancer is an adeno-associated virus Ad-CHCHD3 carrying a CHCHD3 sequence, the Ad-CHCHD3 enhancing expression of the CHCHD3 protein in myocardial tissue.
The medicine combination for preventing, relieving and/or treating myocardial infarction provided by the invention comprises 2 or more than 2 medicines for preventing, relieving and/or treating myocardial infarction, wherein one medicine takes CHD3 expression promoter as an active substance.
According to the invention, a mouse (CHD 3-cTg) with over-expressed heart-specific CHCHD3 genes and a wild-type mouse (WT) with identified littermates are taken as experimental objects, and the function of the CHD3 is researched through ligating a myocardial infarction model caused by left anterior descending of a mouse coronary artery, so that the result shows that compared with a normal group, the expression of the CHD3 is obviously down-regulated in the myocardial infarction model of the mouse; compared with littermate wild type mice, the heart-specific CHCHD3 gene over-expression mice have significantly reduced mortality and infarct size after myocardial infarction, reduced weight-to-lung ratio (LW/BW), significantly improved left ventricular ejection fraction and overall longitudinal strain of the heart, and these results indicate that the heart-specific CHCHD3 gene over-expression alleviates the disease course of myocardial infarction.
The invention researches the function of CHCHD3 gene in heart diseases, and is specifically embodied in that CHD3 has the function of maintaining and improving heart contraction and simultaneously relieving myocardial injury after myocardial ischemia. Aiming at the function of CHCHD3 for improving myocardial remodeling after myocardial infarction, the CHCHD3 can be applied to the preparation of medicines for preventing, relieving and/or treating myocardial infarction and heart failure.
As a preferred scheme, the CHCHD3 gene is used as a drug target, an in vitro cell model or an animal model of CHD3 gene overexpression is constructed, and the in vitro cell model or the animal model is used for screening drugs and/or biological reagents for preventing, relieving and/or treating myocardial injury and/or heart failure after myocardial infarction, and the aim of preventing, relieving and/or treating myocardial ischemia and then reconstructing and/or heart failure is fulfilled through a genetic engineering technology. The myocardial infarction lesion includes: stable angina pectoris, aortic stenosis and unstable angina pectoris induced myocardial ischemia and heart failure.
According to the invention, through establishing a new-born rat myocardial cell hypoxia model, after transfection of adenovirus containing CHCHD3 (Ad-CHD 3) or a control vector (Ad-CTR), mitochondrial morphology, CHCHD3 expression level and mitochondrial oxidative phosphorylation function of myocardial cells after hypoxia treatment are evaluated by using Mito Tracker Red probes, CHCHD3 immunofluorescence staining, seahorse cell energy metabolizing instrument and other biological technologies. In addition, the invention uses a mouse (CHCHD 3-cTg) with over-expressed heart-specific CHCHD3 gene, uses a wild-type mouse (WT) with identified littermates as a control, constructs a myocardial infarction animal model by ligating the left anterior descending branch of the coronary artery of the mouse, and evaluates the changes of myocardial infarction area, cardiac function, mitochondrial morphology and the like of the mouse after myocardial infarction by utilizing the technologies of small animal ultrasound, 2,3, 5-triphenyltetrazolium chloride (TTC) staining, transmission electron microscopy and the like.
Experimental animals and feeding
Experimental animals: selecting a wild mouse with 10-12 weeks of age, weight of 26.5+/-2.0 g and CHCHD3 gene heart specificity over-expression and littermate as an experimental object;
feeding environment: all experimental mice were housed in the university of martial arts cardiovascular disease institute no specific pathogen (Specific Pathogen Free, SPF) class laboratory animal center, feed conditions: the room temperature is between 22 and 24 ℃, the humidity is between 50 and 70 percent, the lighting time of light and dark alternation is 12 hours, and the food is ingested by free drinking water.
EXAMPLE 1 construction of heart-specific overexpressing mice from the CHCHD3 Gene
CHCHD3 gene heart-specific overexpression heterozygous mice (C57 BL/6J background) were purchased from scotopic biotechnology limited, su zhou, and post-mating screening was performed to obtain CHCHD3 gene heart-specific overexpression homozygous mice for this study, and littermate wild-type mice were used as controls (see fig. 4).
EXAMPLE 2 expression of the CHCHD3 Gene in the hearts of Normal humans and myocardial ischemia patients
Firstly, analyzing mRNA expression levels of CHCHD family molecules in myocardial tissues of normal people and clinical myocardial ischemia patients in a public database GSE46224, and displaying that only CHD3 expression is significantly down-regulated in the myocardial tissues of the myocardial ischemia patients; then, selecting normal human hearts (individuals donated by non-cardiac death) and hearts of myocardial ischemia patients (receptors or heart biopsy tissues replaced by heart transplantation operation patients), performing immunohistochemical staining after making tissue paraffin sections, and displaying that CHD3 is expressed down in the hearts of the myocardial ischemia patients; simultaneously, protein and RNA are respectively extracted for SDS-PAGE-immunoblotting test (Western blot) and real-time quantitative PCR detection, and the result also shows that the CHCHD3 protein and mRNA levels in myocardial tissues of clinical myocardial ischemia patients are obviously reduced (see figure 1).
EXAMPLE 3 mouse myocardial infarction Model (MI) construction
1. The mouse myocardial infarction model is modeled by adopting left anterior descending coronary artery Ligation (LAD), and the model operation flow is as follows:
1.1 preoperative preparation
(1) Anesthesia: mice were first weighed, the amount of needed anesthetic (3% pentobarbital sodium) calculated as 90mg/kg body weight, injected intraperitoneally, and the injection time points recorded. The tail and toe clamping do not have obvious reaction, and the state of the mice is good as the anesthesia success standard (generally, about 10min after injection has no obvious reaction, about 50min after anesthesia has reaction, and about 30min after anesthesia is the optimal operation time).
(2) Preparing an operation area: the skin of the left chest, left side chest, left forelimb and underarm of the mice was dehaired. After shaving, the surgical field is wiped with wet gauze to remove the mouse hair, preferably without affecting the surgical field.
(3) Tracheal cannula: the upper incisors of the mice are fixed on the inclined planes of the V-shaped plates by rubber bands, the tracheal cannula is rapidly and accurately inserted into the trachea through the glottis, then the right lateral position is placed on a heating pad (the heating pad needs to be preheated in advance), and then the tracheal cannula is connected with a breathing machine to fix the mice. If the thoracic cavity fluctuation of the mice is consistent with the frequency of the breathing machine, the successful tracheal intubation is indicated.
2. Left anterior descending coronary artery Ligation (LAD)
Heart-specific overexpression of CHCHD3 gene and littermates wild type mice at 10-12 weeks of age were randomized into CHCHD cTG +sham group (n=10), wt+sham group (n=10), wt+mi group (n=20) and CHCHD cTG +mi group (n=20). (wherein, mice in the Sham surgery (Sham) group were left open with heart exposed and no other treatment, and mice in the MI group were left anterior descending coronary artery ligation surgery after having left open.
The mouse is in a supine position, muscles are passively separated between the third rib and the fourth rib on the left side, after the hemostatic forceps prop open the third rib and the fourth rib and rapidly squeeze out the heart, a 6-0 suture needle with a wire ligates the anterior descending branch of the left coronary artery, the ligature position is 1mm below the left auricle, the needle inserting depth is 0.5mm, the myocardium of the myocardial infarction area below the ligature part turns pale, whether ligature is successful is judged, and whether the electrocardiograph is connected further judges whether the molding is successful. The intraperitoneal injection of penicillin is routinely administered after operation, 10 ten thousand per U per day, for 3 consecutive days. And disinfect the incision skin to prevent infection.
3. Postoperative care
After left anterior descending coronary artery ligation, when the mice breathe spontaneously and have strong reaction with toe, the tracheal intubation is pulled out, and the mice are placed into a feeding cage filled with autoclave sterilized padding, feed and drinking water, and are continuously fed and observed in a feeding room. Mice death was recorded daily for each group.
EXAMPLE 4 myocardial infarction model mouse pathology and molecular biology detection
1. Drawing materials
(1) Working in the early stage: urine cups with 20mL of 10% formaldehyde by volume were prepared in advance and labeled (mouse number, group, type of surgery and date of draw). Placing a culture dish filled with 10% KCl solution at a material sampling position. Opening the analytical balance and zeroing for standby. Mice were sacrificed by re-weighing.
(2) Drawing materials: the ophthalmic curved forceps clamp the vascular pedicle below the auricle, shear the heart and rapidly place the heart in 10% KCl solution by mass fraction. After the heart is stopped in diastole, the heart is placed on sterilized gauze, liquid in a heart cavity is lightly squeezed, surface liquid is dipped, the heart is weighed and recorded, and the heart is placed in a corresponding urine cup and fixed for 48 hours for pathological detection. In addition, the heart tissue of the mice is taken at 12 hours, 24 hours, 3 days, 7 days and 21 days after operation respectively, placed in a freezing tube, transferred to-80 ℃ through liquid nitrogen for preservation, and the obtained fresh myocardial tissue is used for molecular biological detection of proteins, RNA and the like within 3 months.
(3) Correlation measurement and calculation: the heart and lungs of the mice were removed, trimmed, and the filters blotted dry, weighed and recorded. Skin at the hind limb tibia of the mice was cut, and tibia length was measured and recorded. The ratio of lung weight to body weight (LW/BW) and the ratio of heart weight to tibia length (HW/TL) were calculated.
(4) CHCHD3 expression in hearts of sham surgery and MI groups: wild-type C57 mice Sham group and MI group were selected from hearts 12 hours, 24 hours, 3 days, 7 days and 21 days after surgery, and the heart extracted proteins were subjected to SDS-PAGE-immunoblotting (Western blot) and detected in combination with an antibody specifically recognizing CHCHD3 protein, and the CHCHD3 expression was measured, and β -Tubulin was used as an internal reference. The results are shown in figure 2, where CHCHD3 protein begins to decrease at 12 hours after MI surgery, with significant downregulation for 3 days, 7 days and 21 days.
2. Pathology detection
2.1TTC (2, 3, 5-triphenyltetrazolium chloride) staining: taking out fresh heart, quick-freezing in a refrigerator at-20deg.C for 20 min to harden heart, cutting heart into five equal parts with blade perpendicular to heart longitudinal axis, placing cut heart tissue in 2% TTC staining solution, placing in a constant temperature water bath at 37deg.C for 20-30min, taking out heart slice, and taking photos.
2.2 preparation of Paraffin sample sections
The main operation procedure comprises: trimming heart or lung tissue, embedding frame treatment, running water flushing, dehydration, transparency, wax dipping, embedding, slicing, spreading, airing or baking for later use.
2.3 hematoxylin-eosin staining (HE) specific procedure
(1) Paraffin sections dewaxed to water: the paraffin sections of the lung tissue are sequentially put into xylene I for 15min, xylene II for 15min, absolute ethanol for 5min, 95% ethanol for 5min, 85% ethanol for 5min, 80% ethanol for 5min, 70% ethanol for 5min (without a shaking table), tap water for 5min, PBS for 3 times, and each time for 5min.
(2) Hematoxylin-stained nuclei: soaking the processed slice in hematoxylin dye solution for 5min, washing with double distilled water to remove residual dye solution, differentiating the differentiating solution (1% ethanol hydrochloride) for several seconds, and washing with tap water for 10min.
(3) Eosin-stained cytoplasm: and (3) transferring the washed slices into eosin dye solution, soaking for 3min, and washing with tap water for 2min.
(4) Gradient ethanol dehydration: the slices are sequentially transferred into 95% ethanol I2 min, 95% ethanol II 4min, absolute ethanol I5 min, absolute ethanol II 5min, xylene I10 min and xylene II 10min for dehydration and transparency.
(5) Sealing piece: and taking out the transparent and successfully dehydrated slice from the xylene solution, slightly airing, and sealing the neutral resin.
(6) And (5) performing microscopic examination by a Leica normal optical microscope, and collecting and analyzing images.
2.4 myocardial tissue transmission electron microscopy
After the cervical dislocation of the mice is killed, hearts are taken out within 3 minutes, a culture dish filled with an electron microscope fixing solution can be prepared in advance before sampling, small myocardial tissues are immediately put into the culture dish after being taken off in vitro, a scalpel is used for cutting the small myocardial tissues into small blocks in the fixing solution of the culture dish, the volume of the sampled tissues is controlled to be 1.0mm multiplied by 1.0mm square, mechanical damage such as forceps extrusion is avoided during sampling, the blades are sharp to avoid contusion tissues, the myocardial tissues are immediately put into the electron microscope fixing solution inner chamber Wen Biguang for fixing for 2 hours after being taken off, then the myocardial tissues are transferred to 4-DEG for preservation, then an electron microscope detection sample is prepared, and the mitochondrial morphology of the myocardial tissues is observed through an electron microscope.
Example 5 echocardiography detection of mouse cardiac function
1. Early preparation
(1) Preparing an anesthesia machine: firstly connecting an oxygen bottle with an air inlet port on an anesthesia machine, unscrewing a sealing cover of a medicine adding port on the anesthesia machine, rapidly adding isoflurane to a safety scale, and then screwing the sealing cover. The total valve on the oxygen bottle is unscrewed, the knob of the flow control valve is adjusted, and the air outlet pressure is maintained to be 0.2-0.3mPa.
(2) Preparing a mouse to be tested: after the mice to be detected are rapidly anesthetized by isoflurane, the left chest area is shaved, the head of the mice after treatment is stretched into the anesthetic catheter sleeve head, and the stable anesthetic state of the mice is maintained by 1.5-2.0% isoflurane.
2. Cardiac function detection
The mice take a left lateral lying position or a supine position, and an ultrasonic couplant is uniformly smeared on the shaved area. And (3) adopting a high-frequency ultrasonic diagnostic apparatus with the frequency of 15MHz, selecting a standard left ventricular papillary muscle short axis section, measuring the left ventricular end diastole inner diameter, the left ventricular end systole inner diameter, the left ventricular ejection fraction and the short axis shortening rate, switching to the long axis section, measuring the whole longitudinal strain of the heart and the like, and evaluating the ventricular wall motion condition.
FIG. 6 shows the results of heart function tests after modeling CHCHD3-cTG and left anterior descending coronary artery ligation in wild type mice. Compared with wild-type sham mice of the same nest, wild-type mice exhibit reduced cardiac contractile function and reduced local movement of the ventricular wall at 3 peripheral post-surgery, mainly manifested by reduced left ventricular ejection fraction and overall longitudinal strain, while CHCHD3-cTG mice exhibit increased left ventricular ejection fraction and overall longitudinal strain, suggesting that heart-specific chd3 overexpression may significantly improve cardiac dysfunction in mice following myocardial infarction.
EXAMPLE 6CHCHD3 under normoxic or hypoxic conditions (1.0% O) 2 ) Expression in stimulated cardiomyocytes
Isolation and culture of neonatal Sprague-Dawley (1-3 days) cardiomyocytes, culturing the primary cardiomyocytes for 48h, changing the solution (the specific procedure of primary neonatal SD rat cardiomyocyte culture is described in example 7 below), starving the cardiomyocytes with serum-free DMEM/F12 for 12h to synchronize the cells, and then administering normal oxygen and low oxygen environment (1.0% O) 2 Three gas incubator) for 48 hours, performing SDS-PAGE-immunoblotting (Western blot) on the extracted protein of the myocardial cells, detecting the specific recognition of the antibody of the CHCHD3 protein, and measuring the expression of the CHCHD3, and taking beta-Tubulin as an internal reference; simultaneously, the expression and subcellular localization of CHCHD3 in cells are evaluated by immunofluorescence. The results of the assay are shown in FIG. 3, in which CHD3 expression is significantly down-regulated in cardiomyocytes stimulated by hypoxia stimulation, while CHD3 localization in mitochondria is significantly reduced.
Example 7 Effect of CHCHD3 overexpression (Ad-CHCHD 3) on hypoxia-stimulated primary cardiomyocyte mitochondrial morphology and function
1. Primary neonatal SD rat cardiomyocyte culture
(1) 10 Sprague-Dawley rats, which were born for 1-3 days, were sterilized with 75% alcohol below the neck, the hearts were removed with ophthalmic scissors and micro-forceps, and placed in a glass plate containing 10mL DMEM/F12 solution. And taking the other one, and repeating the process.
(2) The heart was washed with DMEM/F12 medium and cut into 1-2mm3 pieces. Transfer to serum bottles with rotors, aspirate DMEM/F12, add pancreatin digest. The rotation speed is 120r/min, digestion is carried out for 15min, the reaction is stopped for a few seconds, and the supernatant is discarded.
(3) Adding pancreatin digestive juice, and digesting for 15min at a rotating speed of 120 r/min. After standing for several seconds, the supernatant was aspirated, and digestion was stopped with 20% calf serum in DMEM/F12 medium and stored in a refrigerator at 4 ℃. This step is repeated, and cycled several times. The supernatant should be removed as much as possible, and digestion is terminated when the tissue mass becomes white and significantly smaller.
(4) The collected cardiomyocyte suspension was centrifuged at 1500rpm for 8min and the supernatant was discarded. An appropriate amount of culture medium was added to the centrifuge tube, the resuspended cells were gently blown, concentrated into 150 mL centrifuge tube, and the cell suspension was filtered through a 40 μm filter screen.
(5) Cells were seeded in 100mm dishes and adherent for 90min at time difference, and the unadhered cell suspension was aspirated and filtered. Brdu (final concentration 0.1 mM) was added according to the total amount of cell suspension, and after mixing, added to a vessel coated with 0.1% gelatin.
(6) The cells were gently dispersed and not vortexed. 37 ℃ and 5% CO 2 Incubate 48 hours, wash 1 with PBS, change media.
Effect of CHCHD3 overexpression (Ad-CHCHD 3) on hypoxia-stimulated primary cardiomyocyte mitochondrial morphology and function
Ad-CTR (adenovirus with no load, used as control) and Ad-CHCHD3 (adenovirus with CHCHD3 overexpression vector, specific structure is shown in FIG. 8A) 10MOIs were infected with primary cardiomyocytes cultured for 3 days, respectively, in a hypoxic environment (1.0% O after 12 hours 2 Three-gas incubator) or normal oxygen cultureAfter 48 hours of incubation, mitochondrial fluorescent probe detection and seahorse cell mitochondrial respiratory function detection were performed.
2.1 mitochondrial fluorescent probe detection
(1) Adding a small amount of 1mM Mito-Tracker Red storage solution into the myocardial cell culture solution according to the proportion of 1:5000, enabling the final concentration to be 200nM, and uniformly mixing to prepare Mito-Tracker Red working solution, and preheating the Mito-Tracker Red working solution at 37 ℃; (2) Removing the cell culture solution, adding the prepared Mito-Tracker Red staining working solution which is preincubated at 37 ℃, and incubating for 30 minutes with myocardial cells at 37 ℃; (3) Removing Mito-Tracker Red staining working solution, adding fresh cell culture solution preincubated at 37 ℃ for 2 times; (4) Mitochondrial morphology was observed with a laser confocal microscope and Image J software quantified mitochondrial length.
2.2 mitochondrial function detection
Mitochondrial oxidative respiration rate (OCR) in neonatal rat cardiomyocytes was measured using a Seahorse XFe 24 cell energy analyzer. The working concentrations of mitochondrial stress test drugs (oligomycin-a and FCCP) required for primary cardiomyocytes were determined by dose titration. Culturing each well in V-7 hippocampal plate at 10 or more 5 Primary cardiomyocytes. Before measurement, primary cardiomyocytes were free of CO at 37 ℃ 2 Is incubated in an incubator (1 mM pyruvate+glucose free seahorse assay medium) for 1 hour, a syringe is loaded to add 1.0. Mu.M oligomycin, 1.0. Mu.M FCCP and 2.0. Mu.M antimycin A, and mitochondrial oxidative respiration test parameters such as basal respiration, ATP production, maximum oxygen consumption, respiration potential are recorded as mitochondrial Oxygen Consumption Rate (OCR). The results indicate that the mitochondrial respiratory function of cardiomyocytes after Ad-CHCHD3 adenovirus infection is significantly enhanced compared with the Ad-CTR control group, i.e. CHCHD3 overexpression improves the mitochondrial function of cardiomyocytes (FIGS. 9C-D).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

  1. Use of CHCHD3 or chd3 expression promoter in the manufacture of a medicament for the prevention, alleviation and/or treatment of myocardial infarction.
  2. 2. The use according to claim 1, characterized in that: the medicine for preventing, relieving and/or treating myocardial infarction can relieve mitochondrial injury of myocardial tissues and improve mitochondrial oxidative phosphorylation function.
  3. 3. The use according to claim 1, characterized in that: CHCHD3 expression promoters include one or more of proteins, proteolytically targeted chimeras, polynucleotides, small molecule compounds.
  4. 4. The use according to claim 1, characterized in that: the CHCHD3 or chd3 expression promoter allows entry into the cell by one or more of the following: direct naked DNA injection method, liposome coated DNA direct injection method, gold coated DNA gene gun bombardment method, propagation defect bacteria carrying plasmid DNA method, replication defect adenovirus carrying target DNA method, PEG modified protein drug injection method, liposome coated protein intravenous injection method, protein microsphere preparation subcutaneous injection method.
  5. 5. The use according to claim 1, characterized in that: the CHCHD3 expression promoter is a recombinant plasmid which contains the CHD3 gene and can normally express the CHD3 protein.
  6. 6. The use according to claim 1, characterized in that: the CHCHD3 expression promoter is adenovirus Ad-CHCHD3 carrying a CHCHD3 sequence, and the Ad-CHCHD3 promotes the expression of CHCHD3 protein in myocardial tissues.
  7. 7. A medicament for preventing, alleviating and/or treating myocardial infarction, characterized by: the medicine is a preparation prepared by taking CHCHD3 expression promoter as an active substance and adding pharmaceutically acceptable auxiliary materials or auxiliary components.
  8. 8. The medicament for preventing, alleviating and/or treating myocardial infarction according to claim 7, characterized in that: the CHCHD3 expression promoter is a recombinant plasmid which contains the CHD3 gene and can normally express the CHD3 protein.
  9. 9. The medicament for preventing, alleviating and/or treating myocardial infarction according to claim 7, characterized in that: the CHCHD3 expression promoter is adeno-associated virus Ad-CHCHD3 carrying CHCHD3 sequence, and the Ad-CHCHD3 promotes the expression of CHCHD3 protein in myocardial tissue.
  10. 10. A pharmaceutical composition for preventing, alleviating and/or treating myocardial infarction, characterized in that: comprises 2 or more than 2 medicines for preventing, relieving and/or treating myocardial infarction, wherein one medicine takes CHD3 expression promoter as active substance.
CN202311623303.4A 2023-11-30 2023-11-30 Application of CHCHD3 expression promoter in preparation of myocardial infarction drugs Pending CN117599190A (en)

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