CN111358951A - Method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes - Google Patents

Method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes Download PDF

Info

Publication number
CN111358951A
CN111358951A CN202010223638.7A CN202010223638A CN111358951A CN 111358951 A CN111358951 A CN 111358951A CN 202010223638 A CN202010223638 A CN 202010223638A CN 111358951 A CN111358951 A CN 111358951A
Authority
CN
China
Prior art keywords
cbp
cardiomyocytes
mature
right ventricular
flox
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010223638.7A
Other languages
Chinese (zh)
Other versions
CN111358951B (en
Inventor
叶霖财
洪海筏
刘锦纷
仇黎生
姜川
张海波
张�浩
肖颖颖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Childrens Medical Center Affiliated to Shanghai Jiaotong University School of Medicine
Original Assignee
Shanghai Childrens Medical Center Affiliated to Shanghai Jiaotong University School of Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Childrens Medical Center Affiliated to Shanghai Jiaotong University School of Medicine filed Critical Shanghai Childrens Medical Center Affiliated to Shanghai Jiaotong University School of Medicine
Priority to CN202010223638.7A priority Critical patent/CN111358951B/en
Publication of CN111358951A publication Critical patent/CN111358951A/en
Application granted granted Critical
Publication of CN111358951B publication Critical patent/CN111358951B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • 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
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01048—Histone acetyltransferase (2.3.1.48)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Abstract

The present invention relates to a method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes. The invention discovers that after histone acetyltransferase CBP/p300 of partial right ventricular cardiomyocytes of a mouse is knocked out specifically, the right ventricular cardiomyocytes are transdifferentiated into an immature state (the cells become small, the expression of a muscle node related gene is reduced, the expression of a T tube which is a specific mark of the mature cardiomyocytes is reduced or disappeared, and the calcium ion processing capability of the cardiomyocytes is reduced), and the cardiomyocytes form a plurality of proliferation clusters, which shows that the right ventricular mature cardiomyocytes can be transdifferentiated into the immature cardiomyocytes by inhibiting the CBP/p300, so that the cells have the proliferation and regeneration capability. The present invention provides an alternative method for the treatment of right heart failure.

Description

Method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes
Technical Field
The invention relates to the field of biotechnology, in particular to a method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes.
Background
Congenital heart disease (abbreviated as congenital heart disease) refers to a disease caused by abnormal cardiac anatomy due to the formation of heart and large blood vessels or abnormal development during embryonic development, or by the failure of an automatically closed channel to close after birth (in the fetal genus). China is one of countries with high incidence of congenital heart disease in the world, about 30 more than ten thousand congenital heart disease children are born each year, and are the first causes of birth defects, wherein about 80% of the children need surgical treatment and are one of the leading causes of death of children under 5 years old.
Congenital heart diseases in China mainly take the right heart system as the main factor, and right heart failure is one of important complications affecting the long-term survival quality of children with the heart disease in China. The essence of right heart failure is that there is a massive decrease in cardiomyocytes with normal contractile function, and mature cardiomyocytes are terminally differentiated cells and cannot proliferate and regenerate. No method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes is currently available.
CBP/P300 is an important macromolecular protein in Histone Acetyltransferase (HAT), is considered to have the same function due to the high homology of the structure, can regulate a plurality of transcription factors, is absorbed to a promoter position through interaction with a specific sequence activator so as to mediate the activation of transcription, CBP/P300 is involved in the activation of hundreds of transcription factors and is involved in a transduction pathway of numerous signals, the dysfunction of CBP/P300 is also involved in the pathogenesis of various human diseases such as tumors, diabetes, inflammation, heart diseases and the like, wherein the research in tumor diseases is more, for example, documents (the expression of human breast cancer tissues β, CBP/P300 protein and the clinical significance thereof, the fourth national tumor diagnosis and treatment new progress and new technology science conference, 2009, 10 months 1) disclose that CBP/P300 protein in human breast cancer is related to the formation of tumors, the expression of human breast cancer tissue β, CBP/P300 protein in human breast cancer is likely to inhibit the metastasis of breast cancer, the expression of breast cancer metastasis of lymph node, the acute tumor metastasis of breast cancer cell lines is found to be inhibited in the colon cancer cell line expression of CBP receptor, the cervical cancer metastasis of CBP receptor protein, the receptor of CBP/P receptor gene expression of CBP 300, the receptor gene expression of CBP, the receptor protein of pancreatic cancer cell line expression of CBP/P protein, the receptor protein of CBP, the receptor protein of the receptor gene of the receptor of lung cancer cell line, the receptor of the receptor of.
However, no technical scheme for knocking out or down-regulating CBP/p300 to promote the conversion of mature right ventricular cardiomyocytes to immature cardiomyocytes and further treat heart failure is reported at present.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for preventing and treating right heart failure by inhibiting CBP/p 300.
In a first aspect, the invention provides an application of CBP/p300 in preparing a medicament for preventing and treating right heart failure.
In a second aspect, the invention provides an application of an inhibitor of CBP/p300 in preparing a medicament for preventing and treating right heart failure.
As a preferable example, the inhibitor of CBP/p300 is a substance for reducing the expression level of CBP/p 300.
As another preferred example, the inhibitor of CBP/p300 is selected from small molecule compounds or biological macromolecules.
More preferably, the biological macromolecule is small interfering RNA, dsRNA, shRNA, microRNA or antisense nucleic acid which takes CBP/p300 protein or transcript thereof as a target sequence and can inhibit CBP/p300 protein expression or gene transcription; or a construct capable of expressing or forming said small interfering RNA, dsRNA, microRNA, antisense nucleic acid.
In a third aspect, the present invention provides a method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes for non-therapeutic purposes, comprising the step of knocking down CBP/p300 in said mature right ventricular cardiomyocytes.
As a preferred example, the mature right ventricular cardiomyocytes are mature right ventricular cardiomyocytes cultured in vitro, or mature right ventricular cardiomyocytes in a non-human animal.
As another preferred example, the method comprises the steps of: AAV-TnT-Cre was injected subcutaneously into CBPflox /flox；p300flox/flox；Rosa26-Trap/trapThe right ventricle of the mouse.
Herein, the term "right heart failure" is also called right heart failure and refers to the clinical syndrome that the systolic or diastolic dysfunction of the right ventricle is insufficient to provide the desired cardiac output for the body due to any cause. Generally, the basic etiologies that cause right heart failure include:
1. myocardial lesions
(1) Primary myocardial damage: myocardial damage caused by myocardial ischemia (myocardial infarction, coronary atherosclerotic heart disease), inflammation, immune reaction (myocarditis, dilated cardiomyopathy), genetic diseases (hypertrophic cardiomyopathy, myocardial insufficiency), etc.
(2) Secondary myocardial damage: myocardial damage caused by metabolic diseases (diabetes, hyperthyroidism), systemic infiltrative diseases (myocardial amyloidosis), connective tissue diseases, myocardial toxic drug damage, and the like.
2. Cardiac overload
Mainly refers to diseases that can cause heart overload, and these diseases can make heart and cardiac muscle meet the demand of heart pumping blood by changing their own structure, but it is not a long-term measure, the compensatory ability of heart is limited, and after a certain limit, the structure and function of cardiac muscle can lose compensatory ability, and then the heart failure occurs. Further comprising:
(1) excessive pressure load: it is seen in hypertension, pulmonary hypertension, aortic stenosis, etc.
(2) Excessive capacity load: it is seen in cardiac valve insufficiency, congenital cardiovascular diseases, chronic anemia, hyperthyroidism, etc.
(3) Insufficient ventricular preload: it is seen in mitral stenosis, cardiac tamponade, restrictive pericarditis, constrictive pericarditis, etc.
Right heart failure can be classified according to the occurrence of acute or chronic heart failure, and therefore right heart failure according to the present invention also includes acute right heart failure and chronic right heart failure.
The invention has the advantages that:
the invention discovers that after histone acetyltransferase CBP/p300 of partial right ventricular cardiomyocytes of a mouse is knocked out specifically, the right ventricular cardiomyocytes are transdifferentiated into an immature state (the cells become small, the expression of genes related to muscle joint is reduced, the expression of a T tube which is a specific mark of the mature cardiomyocytes is reduced or disappeared, the calcium ion processing capacity of the cardiomyocytes is reduced), and the cardiomyocytes form a plurality of proliferation clusters, so that the invention provides a method for converting the mature cardiomyocytes of the right ventricle into the immature cardiomyocytes in vivo. Mature myocardial cells cannot proliferate, and immature myocardial cells have strong proliferation capacity. The essence of right heart failure is that there is a massive decrease in cardiomyocytes with normal contractile function, since mature cardiomyocytes are terminally differentiated cells that cannot proliferate and regenerate. The invention can transdifferentiate the mature cardiac muscle cell of the right ventricle into the immature cardiac muscle cell, so that the immature cardiac muscle cell has the capability of proliferation and regeneration, and an alternative method is provided for treating the right heart failure.
Drawings
FIG. 1 shows that the expression of sarcomeric genes is down-regulated and the cells become smaller in CBP/p300 knocked-out cardiomyocytes (green) compared with surrounding cardiomyocytes.
FIG. 2 is a drawing: WES protein semi-quantitative technical detection shows that the expression of the myocardial cell sarcomere related gene of knocking CBP/p300 out is reduced.
FIG. 3: the CBP/p300 knocked-out myocardial cell T tube disappears, and the cell becomes small. LE: a T-tube long axis element; TE: t-tube minor axis element. MM 4-64: t-tube dye.
FIG. 4 is a drawing: the calcium ion processing capacity of the CBP/p300 knocked-out myocardial cells is reduced, the contraction amplitude of the cells is reduced, and the time of reaching a peak value is prolonged. A: the dynamic change of calcium ions of the cardiac muscle cells under the stimulation of 20Hz frequency and 15 mV; b: graph a is a peak graph after image J processing.
FIG. 5: CBP/p300 knock-out cardiomyocytes formed 3 proliferative clusters.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
Example 1
1. Experimental Material
CBPflox/flox；p300flox/flox；Rosa26-Trap/trapMice were given by professor William Pu of harvard medical school. Also available from Jackson lab: CBPfloxMouse, cat number: STOCK NO: 025178/CBP flox; p300floxMouse, cat number: STOCK NO: 025526/p300 flox; CBPflox/+CBP can be obtained by mouse self matingflox/flox；p300+/+A mouse; p300flox/+P300 can be obtained by self mating of miceflox/flox；CBP+/+A mouse; subjecting CBP toflox/flox；p300+/+Mice and p300flox/flox；CBP+/+CBP can be obtained by mating miceflox/+；p300flox/+A mouse; then CBP is addedflox/+；p300flox/+The CBP can be obtained by self mating of miceflox/flox；p300flox/floxA mouse.
AAV-TnT-Cre was purchased from Addgene, cat #: # 69916.
2. Experimental methods
We will dose low (2.0 × 10) in mice in adulthood (postnatal day 28)10VP/g AAV-TnT-Cre was injected subcutaneously into CBPflox/flox；p300flox/flox；Rosa26-Trap/trapMouse, the histone acetyltransferase CB of the partial right ventricle cardiac muscle cell of the mouse is knocked out specificallyP/P300. A control group was also set: control mice were wild-type: CBP+/+；p300+/+；Rosa26-Trap/trap(ii) a The administration mode is the same as that of the experimental group.
3. Results of the experiment
FIG. 1 shows that the expression level of right mouse ventricular cardiomyocyte CBP/p300 is obviously reduced.
Further examination revealed that right ventricular cardiomyocytes knocked out of CBP/p300 transdifferentiated to an immature state (cells became small, expression of sarcomere-associated genes was down-regulated, expression of T-tubes, a marker specific to mature cardiomyocytes, was reduced or disappeared, ability of cardiomyocytes to handle calcium ions was down-regulated) (fig. 1-4), and that cardiomyocytes formed multiple proliferative clusters (fig. 5).
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims (8)

  1. Application of CBP/p300 in preparing medicine for preventing and treating right heart failure.
  2. Application of CBP/p300 inhibitor in preparing medicine for preventing and treating right heart failure.
  3. 3. The use according to claim 2, wherein the inhibitor of CBP/p300 is a substance that reduces the expression level of CBP/p 300.
  4. 4. Use according to claim 2, wherein the inhibitor of CBP/p300 is selected from a small molecule compound or a biological macromolecule.
  5. 5. The use of claim 4, wherein the biomacromolecule is a small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid targeting the CBP/p300 protein or its transcript and capable of inhibiting the expression of the CBP/p300 protein or the transcription of a gene; or a construct capable of expressing or forming said small interfering RNA, dsRNA, microRNA, antisense nucleic acid.
  6. 6. A method for non-therapeutic purposes of converting mature right ventricular cardiomyocytes into immature cardiomyocytes, comprising the step of knocking down CBP/p300 in said mature right ventricular cardiomyocytes.
  7. 7. The use of claim 6, wherein the mature right ventricular cardiomyocytes are mature right ventricular cardiomyocytes cultured in vitro, or mature right ventricular cardiomyocytes in a non-human animal.
  8. 8. Use according to claim 6, characterized in that the method comprises the following steps: AAV-TnT-Cre was injected subcutaneously into CBPflox/flox；p300flox/flox；Rosa26-Trap/trapThe right ventricle of the mouse.
CN202010223638.7A 2020-03-26 2020-03-26 Method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes Active CN111358951B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010223638.7A CN111358951B (en) 2020-03-26 2020-03-26 Method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010223638.7A CN111358951B (en) 2020-03-26 2020-03-26 Method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes

Publications (2)

Publication Number Publication Date
CN111358951A true CN111358951A (en) 2020-07-03
CN111358951B CN111358951B (en) 2022-06-24

Family

ID=71200028

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010223638.7A Active CN111358951B (en) 2020-03-26 2020-03-26 Method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes

Country Status (1)

Country Link
CN (1) CN111358951B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116904469A (en) * 2023-09-12 2023-10-20 首都儿科研究所 Inhibitor for p300 protein expression, preparation method and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019161162A1 (en) * 2018-02-16 2019-08-22 Constellation Pharmaceuticals, Inc. P300/cbp hat inhibitors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019161162A1 (en) * 2018-02-16 2019-08-22 Constellation Pharmaceuticals, Inc. P300/cbp hat inhibitors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TETSUHIKO YANAZUME等: "Cardiac p300 Is Involved in Myocyte Growth with Decompensated Heart Failure", 《M OLECULAR AND C ELLULAR B IOLOGY》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116904469A (en) * 2023-09-12 2023-10-20 首都儿科研究所 Inhibitor for p300 protein expression, preparation method and application thereof
CN116904469B (en) * 2023-09-12 2024-01-23 首都儿科研究所 Inhibitor for p300 protein expression, preparation method and application thereof

Also Published As

Publication number Publication date
CN111358951B (en) 2022-06-24

Similar Documents

Publication Publication Date Title
Lorente-Cebrián et al. MicroRNAs and other non-coding RNAs in adipose tissue and obesity: emerging roles as biomarkers and therapeutic targets
Mia et al. Loss of Yap/Taz in cardiac fibroblasts attenuates adverse remodelling and improves cardiac function
El Azzouzi et al. The hypoxia-inducible microRNA cluster miR-199a∼ 214 targets myocardial PPARδ and impairs mitochondrial fatty acid oxidation
Masamune et al. NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells
Sano et al. The Foxc2 transcription factor regulates tumor angiogenesis
Huang et al. Overexpression of MicroRNA-1 improves the efficacy of mesenchymal stem cell transplantation after myocardial infarction
Drummond et al. Na/K-ATPase signaling regulates collagen synthesis through microRNA-29b-3p in cardiac fibroblasts
WO2016145974A1 (en) Use of gene engineering bacteria vpn 20009-m in preparation of medicaments for preventing and treating cancer metastasis
Valkov et al. MicroRNA-1-mediated inhibition of cardiac fibroblast proliferation through targeting cyclin D2 and CDK6
Liu et al. Ubiquitin-specific protease 14 regulates cardiac hypertrophy progression by increasing GSK-3β phosphorylation
CN104548134A (en) Application of miR-144 and inhibitor thereof
Chen et al. MicroRNA-27b enhances the hepatic regenerative properties of adipose-derived mesenchymal stem cells
WO2022088692A1 (en) Ezh2 alternative spliceosome and application thereof
Meng et al. Adiponectin modified BMSCs alleviate heart fibrosis via inhibition TGF-beta1/Smad in diabetic rats
CN111358951B (en) Method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes
Zhao et al. Hdac8 inhibitor alleviates transverse aortic constriction-induced heart failure in mice by downregulating Ace1
Usui et al. Endogenous muscle atrophy F-box is involved in the development of cardiac rupture after myocardial infarction
Chen et al. MiR-1908 improves cardiac fibrosis after myocardial infarction by targeting TGF-β1.
Tang et al. Significance of MR/OPN/HMGB1 axis in NAFLD-associated hepatic fibrogenesis
Pan et al. Single-cell transcriptome reveals effects of semaglutide on non-cardiomyocytes of obese mice
Yang et al. Cyclin-dependent kinase inhibitor 2b controls fibrosis and functional changes in ischemia-induced heart failure via the BMI1-p15-Rb signalling pathway
Nian et al. Gut microbiota metabolite TMAO promoted lipid deposition and fibrosis process via KRT17 in fatty liver cells in vitro
CN109223818A (en) Application of the miR-3158-3p in preparation diagnosis, prevention and/or treatment atopic dermatitis product
Hu et al. Peroxiredoxin-5 knockdown accelerates pressure overload-induced cardiac hypertrophy in mice
Torrado et al. Targeted gene-silencing reveals the functional significance of myocardin signaling in the failing heart

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant