Construction method and application of animal model for inducing heart failure by knocking out Per1 gene

Abstract

The invention discloses a construction method and application of an animal model for inducing heart failure by knocking out a Per1 gene, and belongs to the technical field of animal models. The construction method of the heart failure animal model comprises the step of knocking out animal myocardial cell Per1 genes. According to the invention, a heart failure animal model is constructed by specifically knocking out a myocardial cell Per1 gene, and the Per1 gene plays an important role in occurrence and development of heart failure. The mouse model obtained by the method can be used for treating heart failure and screening heart failure drugs, particularly provides a new thought for time therapeutics of heart failure, and further provides a new theoretical basis for application of circadian rhythm in drug development and clinical treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and particularly to a method for constructing and applying an animal model of heart failure induced by knocking out the Per1 gene. Background Art

[0002] Heart failure (HF), simply referred to as heart failure, is a severe manifestation or the mid - to - late stage of various cardiovascular diseases. It has a high incidence rate, with a 5 - year mortality rate of approximately 50%, and the survival rate is similar to that of malignant tumors. The prevalence of heart failure in the global adult population is 1% - 3%, and in the past 40 years, the death toll caused by heart failure has increased by 6 times. Due to the high mortality rate and poor prognosis of heart failure, it has become an important public health problem globally, but the molecular mechanism of heart failure has not been fully elucidated. Therefore, there is an urgent need to create a new animal model of heart failure to further study the molecular mechanism of heart failure and find new therapeutic targets for heart failure.

[0003] Studies have found that long - term shift work or irregular sleep can cause disruption of the biological clock (i.e., circadian rhythm) and metabolic disorders, leading to an increased incidence rate and earlier onset age of cardiovascular diseases (such as hypertension, coronary heart disease, dilated cardiomyopathy, acute myocardial infarction). Clinical studies have shown that the risk of any cardiovascular disease in shift workers is 17% higher than that in casual workers, and the incidence risk of coronary heart disease (CHD) is 26% higher. However, which circadian rhythm factors play a key role in heart failure and their regulatory mechanisms are not fully understood.

[0004] The circadian rhythm is a rhythm phenomenon with a 24-hour cycle that the body produces to adapt to the light / dark cycle changes on Earth, showing periodic changes in behavior and physiology, such as the sleep-wake cycle and rhythmic oscillations in neural, metabolic, endocrine, cardiovascular, and immune functions. The core circadian clock consists of transcription factors Bmal1 (brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1), Clock (circadian locomotor output cycles kaput), Per1 / 2 / 3 (period 1, 2, 3), and Cry1 / 2 (cryptochrome 1, 2). Clock and Bmal1 form a heterodimer to constitute a positive feedback loop, bind to the E-box DNA regulatory element, and activate the expression of target genes Per and Cry. Conversely, the overexpressed Per and Cry proteins are transported into the nucleus, thereby inhibiting the transcription mediated by Clock / Bmal1 and further inhibiting their own activation, completing the negative feedback loop. In mammals, the circadian clock consists of multiple highly interconnected transcription factors that activate or inhibit each other, jointly driving the 24-hour circadian rhythm at the molecular level and gradually being recognized as important regulators of physiology and diseases.

[0005] By knocking out the core circadian rhythm factors of the transcription-translation positive feedback loop through the Cre / Loxp recombinase system, a heart failure model induced by circadian rhythm disorder is constructed. For example, mice with cardiomyocyte-specific knockout of Bmal1 and mice with cardiomyocyte-specific knockout of Rev-erbα / β are prone to age-related dilated cardiomyopathy, manifested as decreased ejection fraction and fractional shortening of the short axis, left ventricular dilation, and ventricular wall thinning. However, no study has shown that specifically knocking out the core circadian rhythm factors of the transcription-translation negative feedback loop in the heart can be used to construct an animal model of heart failure. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for constructing a heart failure animal model by knocking out the Per1 gene and its application to solve the problems existing in the above-mentioned prior art. The present invention constructs a heart failure animal model by specifically knocking out the Per1 gene in cardiomyocytes and discovers that the Per1 gene plays an important role in the occurrence and development of heart failure. The mouse model obtained by this method can be used for the treatment of heart failure, the screening of heart failure drugs, especially providing new ideas for the chronotherapy of heart failure, and more importantly, providing a new theoretical basis for the application of circadian rhythm in drug development and clinical treatment.

[0007] To achieve the above purpose, the present invention provides the following solutions:

[0008] The present invention provides a method for constructing a heart failure animal model by knocking out the Per1 gene, including the step of knocking out the Per1 gene in animal cardiomyocytes.

[0009] Preferably, the method for knocking out the Per1 gene in animal cardiomyocytes is the method of knocking out using the Cre / Loxp recombinase system or the method of using the CRISPR / Cas9 gene editing technology.

[0010] Preferably, the method of knocking out using the Cre / Loxp recombinase system includes the following steps:

[0011] (1) Target two loxP alleles to exon 2 of Per1 to construct an animal with the genotype Per1 fl / fl ;

[0012] (2) Target the Cre recombinase to the promoter site of the cardiomyocyte-specific α-myosin heavy chain gene to construct an α-MHC-Cre transgenic animal that spontaneously expresses the Cre recombinase;

[0013] (3) Mate the animal with the genotype Per1 fl / fl with the α-MHC-Cre transgenic animal to obtain the next generation, which is the heart failure animal model.

[0014] The present invention also provides an application of the heart failure animal model constructed by the above construction method in screening heart failure drugs for treating myocardial fibrosis.

[0015] The present invention also provides an application of the heart failure animal model constructed by the above construction method in screening heart failure drugs for treating myocardial hypertrophy.

[0016] The present invention also provides an application of the heart failure animal model constructed by the above construction method in screening drugs for treating heart failure caused by circadian rhythm disorders.

[0017] The present invention also provides an application of the heart failure animal model constructed by the above construction method in screening the administration time of heart failure drugs.

[0018] The present invention also provides an application of the heart failure animal model constructed by the above construction method in studying the pharmacokinetic properties of heart failure drugs.

[0019] The present invention discloses the following technical effects:

[0020] Period 1 (Per1) is a core regulator in the circadian transcriptional-translational negative feedback loop. Its expression is highly specific to the myocardium and is closely related to the rhythmic expression of the key factor in the intracellular calcium cycle of cardiomyocytes, namely, the calcium-transporting ATPase. This function helps maintain the circadian fluctuations of blood pressure and heart rate and is crucial for maintaining the normal rhythm and function of the heart. In this invention, a heart failure animal model was constructed by specifically knocking out the Per1 gene in cardiomyocytes, which showed symptoms such as myocardial hypertrophy, fibrosis, and activation of signaling pathways related to oxidative stress, demonstrating the important role of the Per1 gene in the occurrence and development of heart failure. Since the Per1 gene is a circadian regulatory factor, the heart failure mouse model obtained by this method belongs to heart failure caused by circadian rhythm disorders, providing new ideas for the treatment of heart failure, the screening of heart failure drugs, especially for screening the optimal administration time of heart failure drugs, studying pharmacokinetic properties, improving the efficacy of heart failure drugs, etc., and providing a new theoretical basis for the application of circadian rhythm in drug development and clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 It shows the detection results of Per1 protein expression levels and representative heart pictures of mice in the experimental group and the control group. Among them, (A) shows the Per1 protein expression level and relative quantitative analysis results in the myocardial tissue of 2.5-month-old mice; (B) shows the overall picture of the heart of 6.5-month-old mice.

[0023] Figure 2 It shows the echocardiogram detection results of mice in the experimental group and the control group. Among them, (A) shows the M-mode echocardiograms of mice at 2.5, 4.5, and 6.5 months old; (B) shows the ejection fraction (EF%) and fractional shortening rate (FS%) of the heart of mice at 2.5, 4.5, and 6.5 months old.

[0024] Figure 3 It shows the pathological staining results of the heart tissues of mice in the experimental group and the control group. Among them, (A) shows the detection of the cross-sectional area of cardiomyocytes by WGA staining and the quantitative analysis results; (B) shows the detection of the degree of myocardial tissue fibrosis by Masson staining and the quantitative analysis results; (C) shows the detection of the distribution of α-SMA by immunohistochemical staining and the quantitative analysis results; (D) shows the detection of the level of reactive oxygen species (ROS) by DHE staining and the quantitative results.

[0025] Figure 4Real-time quantitative PCR results of mice in the experimental group and the control group; (A) qPCR was used to detect the mRNA expression levels of atrial natriuretic peptide (NPPA) and B-type natriuretic peptide (NPPB) in myocardial tissue (n = 6); (B) qPCR was used to detect the mRNA expression levels of type I collagen (COL1a1) and type III collagen (COL3a1) in myocardial tissue (n = 6); (C) qPCR was used to detect the mRNA expression levels of NADPH oxidase 2 (CYBB) and NADPH oxidase 4 (NOX4) in myocardial tissue (n = 6).

[0026] Figure 5 Results of cardiac proteomic analysis of mice in the experimental group and the control group; (A-C) Results of enrichment analysis of GO, KEGG, and phenotypes of differentially expressed proteins in myocardial tissues of 6.5-month-old control mice and Per1-cKO mice.

[0027] Figure 6 Results of detection of the expression levels of related proteins in the cardiac tissues of mice in the experimental group and the control group; among them, (A) Protein levels and relative quantitative analysis results of NOX2 and NOX4 in myocardial tissues of 6.5-month-old mice in the experimental group and the control group; (B) Protein levels and relative quantitative analysis results of p-AKT and AKT in myocardial tissues of 6.5-month-old mice in the experimental group and the control group; (C) Protein levels and relative quantitative analysis results of TGF-β in myocardial tissues of 6.5-month-old mice in the experimental group and the control group. Detailed implementation manners

[0028] The various exemplary implementation manners of the present invention will be described in detail below. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention has been described only with reference to preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0032] Regarding the use of "comprising", "including", "having", "containing", etc. in this text, they are all open-ended terms, meaning including but not limited to.

[0033] Example 1

[0034] I. Animal Model

[0035] All animal experimental protocols were approved by the Animal Protection and Use Committee of Dalian Medical University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (publication number: 85-23, 1996). The temperature in the animal room was 22 - 25 °C, with daylight lighting, a light-dark cycle of 12 h / 12 h, and standardized supply of breeding feed and drinking water, with free access to food and water. All the mice had been backcrossed for 10 generations with mice of C57BL / 6 background.

[0036] One pair each of Per1 fl / + heterozygous mice and α-MHC-Cre mice were introduced from Jackson Laboratory (Bar Harbor, ME, USA). Per1 fl / + heterozygous female mice were mated with male mice to obtain offspring mice with the genotype of Per1 fl / fl . Mice with the genotype of Per1 fl / fl have two loxP sites at both ends of the Per1 gene sequence in cardiomyocytes, targeting exon 2 of Per1. The α-MHC-Cre mice were obtained by targeting the Cre recombinase at the specific α-MHC promoter site in mouse cardiomyocytes and can spontaneously express Cre-recombinase.

[0037] Mice with the genotype of Per1 fl / fl were crossed with α-MHC-Cre mice to obtain cardiomyocyte-specific Per1 knockout mice (Per1fl / fl,Cre Mice), abbreviated as Per1-cKO mice. After birth, the Per1 gene sequence in Per1-cKO mice was spontaneously knocked out by Cre recombinase driven by cardiac-specific α-MHC, thereby constructing an animal model with specific knockout of Per1 in cardiomyocytes.

[0038] II. Detection methods for animal models

[0039] Using mice with the genotype Per1 fl / fl as the control group (abbreviated as Ctrl), and Per1-cKO mice as the experimental group, they were uniformly raised in the animal house, and the following indicators were detected:

[0040] 1. Echocardiogram

[0041] At 2.5 months, 4.5 months, and 6.5 months of age in mice, ultrasound was performed using a high-resolution ultrasound imaging system (probe frequency 30-MHz). Before ultrasound, depilatory cream was evenly applied to the left side of the chest of all mice to remove the chest hair, fully exposing the location of the heart, fixed on a thermostatic plate, ultrasound coupling agent was applied to the precordial area, and the ultrasound probe was adjusted to collect M-mode echocardiogram in the long-axis section of the heart beside the sternum, and at least 3 independent cardiac cycle averages were taken to evaluate left ventricular function. The main indicators included ejection fraction (EF) and fractional shortening (FS).

[0042] 2. Immunoblotting

[0043] RIPA lysis buffer (P0013K, Beyotime) and PMSF (ST506, Beyotime) were configured into a protein lysis buffer at a ratio of 100:1. Myocardial tissue and cardiomyocyte proteins were lysed on ice, and the protein concentration of the samples was determined using the BCA protein quantification kit (P0010S, Beyotime). According to the molecular weight of the target protein, acrylamide gels with corresponding concentrations were prepared for electrophoresis, transfer, blocking, incubation with primary and secondary antibodies to detect the expression levels of related proteins.

[0044] 3. Pathological staining

[0045] Heart tissues fixed with 4% paraformaldehyde were stored at room temperature. After 48 hours of fixation, they were routinely dehydrated, paraffin-embedded, and made into 4-μm tissue sections. Paraffin sections of heart tissues with complete structures were selected, and after dewaxing, various pathological stainings such as wheat germ agglutinin (WGA) staining, Masson staining, immunohistochemical staining, and dihydroethidium (DHE) staining were performed.

[0046] 4. Detection of mRNA expression by real-time fluorescence quantitative polymerase chain reaction (qPCR)

[0047] Extract and quantify mRNA from myocardial tissue by the Trizol phenol-chloroform extraction method. Use a reverse transcription kit (11141ES60, Yeasen) to reverse transcribe mRNA into cDNA. Detect the expression levels of mRNA of myocardial hypertrophy markers atrial natriuretic peptide (NPPA) and B-type natriuretic peptide (NPPB), fibrosis markers type I collagen (COL1a1) and type III collagen (COL3a1), and NADPH oxidase 2 (CYBB) and NADPH oxidase 4 (NOX4) using a Green fluorescence PCR kit (RR820A, Takara). The primer sequences are shown in Table 1.

[0048] Table 1 Primer sequences

[0049]

[0050] 5. Liquid chromatography-tandem mass spectrometry analysis

[0051] Select 4 Per1-cKO mice and 4 control mice at 6.5 months of age. After anesthesia, fix them on a sampling board, cut out the heart, drain the blood, and freeze it in liquid nitrogen. Send it to the company at low temperature for subsequent LC-MS / MS analysis. The obtained MS / MS data is processed using the Maxquant search engine (version 1.5.2.8). Then, identify proteins by searching the mass spectrometry and MS / MS data and comparing them with the decoy version of the complete proteome in the UniProt mouse database. When screening and identifying peptides, set the screening conditions as q value < 0.01, false discovery rate (FDR) < 1%, and minimum score ≥ 40 points. Select genes with differential expression between the two groups (P < 0.05, fold change ≥ 1.1-fold) and perform enrichment analysis of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and phenotypes using the WebGestalt data analysis website.

[0052] 6. Statistical methods

[0053] Use GraphPad Prism 8.0 software for data analysis. The results are expressed as mean ± standard deviation. Student's t-test is used to compare the differences between groups of normally distributed samples; when the sample data does not meet the normal distribution, the Mann-Whitney U test is used to compare the differences between groups of non-normally distributed data. p < 0.05 is considered statistically significant.

[0054] III. Detection results

[0055] 1. The level of Per1 protein in the heart tissue of Per1-cKO mice was significantly reduced

[0056] The knockout efficiency of Per1 in Per1-cKO mice at 2.5 months of age was detected by Western blotting. The results showed that the protein level of Per1 in the myocardial tissue of Per1-cKO mice was significantly lower than that in the control group (see Figure 1 A), and the knockout effect was obvious.

[0057] 2. The heart volume of Per1-cKO mice was significantly increased

[0058] At 6.5 months of age, compared with the control group, the heart volume of Per1-cKO mice was significantly increased, and the ratios of heart weight to body weight (HW / BW) and heart weight to tibia length (HW / TL) were both significantly increased (see Figure 1 B).

[0059] 3. Echocardiography showed that Per1-cKO mice progressed from myocardial hypertrophy to heart failure

[0060] Echocardiography (see Figure 2 A-B) results showed that compared with the control group, the cardiac systolic function of Per1-cKO mice was normal at 2.5 months of age; at 4.5 months of age, Per1-cKO mice showed myocardial hypertrophy and abnormal cardiac systolic function, manifested as significant increases in ejection fraction (EF%) and fractional shortening (FS%), while at 6.5 months of age, EF% and FS% decreased significantly, eventually leading to heart failure.

[0061] 4. Myocardial-specific knockout of Per1 induced myocardial hypertrophy, fibrosis and oxidative stress in mice

[0062] Using wheat germ agglutinin (WGA) staining, Masson staining, immunohistochemical staining and dihydroethidium (DHE) staining, it was found that at 6.5 months of age, the cross-sectional area of myocardial cells in Per1-cKO mice was significantly higher than that in the control group (see Figure 3 A), the degree of myocardial fibrosis in Per1-cKO mice was significantly higher than that in the control group (see Figure 3 B-C), and the ROS level was also significantly higher than that in the control group (see Figure 3 D). Then, the qPCR results showed that the mRNA levels of myocardial hypertrophy markers atrial natriuretic peptide (NPPA) and B-type natriuretic peptide (NPPB), fibrosis markers collagen type I (COL1a1) and collagen type III (COL3a1), and NADPH oxidase 2 (CYBB) and NADPH oxidase 4 (NOX4) were all significantly increased (see Figure 4 A-C). These data indicate that myocardial-specific knockout of the Per1 gene can lead to increased myocardial hypertrophy, fibrosis and oxidative stress in mice.

[0063] 5. Proteomic analysis of the hearts of Per1-cKO mice

[0064] Proteomic analysis was performed on Per1-cKO mice and littermate control mice using liquid chromatography and tandem mass spectrometry (LC-MS / MS). The results of GO analysis showed that at the level of biological process (BP), the main enrichment was in the redox process; at the level of cellular component (CC), the enriched terms were mainly in mitochondria and the envelope; at the level of molecular function (MF), the main enrichment was in RNA binding and oxidoreductase activity ( Figure 5 of A). KEGG pathway enrichment analysis showed that the signaling pathways involved by the differentially expressed genes were mainly the biosynthesis of ubiquinone and other terpenoids - ubiquinone ( Figure 5 of B). The results of phenotypic enrichment analysis showed that the activities of a variety of antioxidant enzymes related to the redox process were decreased ( Figure 5 of C). These results suggest that myocardial-specific knockout of Per1 may be closely related to the imbalance of the redox process and the production of reactive oxygen species (ROS).

[0065] 6. Myocardial-specific knockout of Per1 induces the activation of signaling pathways related to myocardial hypertrophy, fibrosis and oxidative stress

[0066] To further clarify the molecular mechanism of myocardial-specific knockout of Per1-induced heart failure, Western blotting was used to detect the expression of key proteins in the signaling pathways of hypertrophy, fibrosis and oxidative stress in mouse myocardial tissues. The results showed that compared with the control group mice, the related proteins NOX2 and NOX4 of nicotinamide adenine dinucleotide phosphate oxidase (NOX) in the myocardial tissues of Per1-cKO mice were significantly increased (see Figure 6 of A), and the expression levels of the myocardial hypertrophy-related protein p-AKT / AKT and the fibrosis-related protein TGF-β were also significantly increased ( Figure 6 of B-C). These results indicate that myocardial-specific knockout of Per1 induces the activation of signaling pathways related to myocardial hypertrophy, fibrosis and oxidative stress in mice, thereby leading to the occurrence of heart failure.

[0067] In summary, the present invention found that myocardial-specific knockout of Per1 mice developed myocardial hypertrophy at 4.5 months, heart dysfunction and heart failure at 6.5 months, and the signaling pathways related to myocardial hypertrophy, fibrosis and oxidative stress in myocardial-specific knockout of Per1 mice were activated, thereby leading to heart failure. It was first confirmed that Per1 is a new molecule for the prevention and treatment of heart failure; maintaining the normal rhythmic expression of Per1 can be used as a new strategy for intervening in heart failure; the heart failure model with circadian rhythm disorder provides a new tool for the treatment of heart failure from the perspective of chronotherapy.

[0068] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a heart failure animal model by knocking out the Per1 gene, characterized in that, It includes the step of knocking out the Per1 gene in cardiomyocytes of an animal.

2. The construction method according to claim 1, wherein The method for knocking out the Per1 gene in cardiomyocytes of an animal is the method of knocking out by using the Cre / Loxp recombinase system or the method of using the CRISPR / Cas9 gene editing technology.

3. The construction method according to claim 2, wherein The method of knocking out by using the Cre / Loxp recombinase system includes the following steps: (1) Target two loxP alleles to exon 2 of Per1 to construct animals with the genotype Per1 fl / fl ; (2) Target the Cre recombinase at the promoter site of the cardiomyocyte-specific α-myosin heavy chain gene to construct an α-MHC-Cre transgenic animal that spontaneously expresses the Cre recombinase. (3) Cross the animal with the genotype Per1 fl / fl with the ɑ-MHC-Cre transgenic animal to obtain the next generation, which is the heart failure animal model.

4. Application of a heart failure animal model constructed by the construction method according to any one of claims 1 to 3 in screening drugs for treating heart failure with myocardial fibrosis.

5. Application of a heart failure animal model constructed by the construction method according to any one of claims 1 to 3 in screening drugs for treating heart failure with myocardial hypertrophy.

6. Application of a heart failure animal model constructed by the construction method according to any one of claims 1 to 3 in screening drugs for treating heart failure caused by circadian rhythm disorders.

7. Application of a heart failure animal model constructed by the construction method according to any one of claims 1 to 3 in screening the administration time of heart failure drugs.

8. Application of a heart failure animal model constructed by the construction method according to any one of claims 1 to 3 in studying the pharmacokinetic properties of heart failure drugs.

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