Application of IGFBPL1 and regulatory factor thereof in preparation of medicine for treating myocardial hypertrophy

Abstract

The invention provides an application of IGFBPL1 and a regulatory factor thereof in preparation of a medicine for treating myocardial hypertrophy. The invention discloses that MYLK4 in a phenylephrine-induced newborn mouse myocardial cell hypertrophy model can participate in regulation and control of expression of IGFBPL1, overexpression of MYLK4 or knock-down of IGFBPL1 alleviates phenylephrine-induced newborn mouse myocardial cell hypertrophy, and in a mouse myocardial hypertrophy model caused by pressure load, the MYLK4 can participate in regulation and control of expression of IGFBPL1. MYLK4 overexpression reduces the expression level of IGFBPL1 in the heart of a mouse with cardiac hypertrophy, the pathological change of cardiac hypertrophy induced by TAC operation is significantly relieved, and the pathological change of cardiac hypertrophy can be significantly aggravated by overexpression of IGFBPL1.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to the application of IGFBPL1 and its regulatory factors in the preparation of drugs for treating myocardial hypertrophy. Background Art

[0002] Cardiovascular diseases remain the leading cause of death globally. With the improvement of living standards and the continuous aggravation of population aging, the incidence of myocardial hypertrophy and the prevalence of heart failure have increased significantly. Myocardial hypertrophy is an adaptive response developed by the body under physiological or pathological stimulation conditions to meet the demand for cardiac output. Physiological myocardial hypertrophy occurs with the increase in heart size during growth, pregnancy, and exercise training, but it does not have a negative effect on systolic function and does not have adverse effects over time. Pathological myocardial hypertrophy can be seen in diseases such as hypertension, hypertrophic cardiomyopathy, or valvular dysfunction. Long-term excessive cardiac load, left ventricular outflow tract obstruction, or myocardial injury lead to cardiac systolic dysfunction, myocardial stiffness, and interstitial fibrosis. The stimulating factors and regulatory mechanisms required for the generation of these two types of hypertrophy are different, and in some cases, they are antagonistic to each other. The two work together to ultimately determine the nature of hypertrophy and exhibit different hypertrophic characteristics.

[0003] Physiological hypertrophy is manifested as a slight increase in heart mass, the growth of the length and width of individual cardiomyocytes, and almost no changes such as interstitial fibrosis. In physiological myocardial hypertrophy, the cells increase proportionally to maintain or increase cardiac output, and myocardial hypertrophy can be reversed when the demand for cardiac output normalizes. The manifestations of pathological hypertrophy are related to the disease generation mechanism. For example, the cardiac pathological remodeling caused by hyperglycemia, hyperinsulinemia, and insulin resistance in patients with diabetic cardiomyopathy is different from that in patients with hypertensive heart disease. Under the condition of excessive pressure load, myocardial hypertrophy begins to be compensatory. At this time, the heart shows an increase in wall thickness without significant enlargement of the chamber, and cardiac function is preserved. However, the further development of pathological myocardial hypertrophy ultimately leads to chamber dilation, thinning of the ventricular wall, and reduction of cardiac output. Excessive volume load is another common influencing factor, which usually leads to eccentric hypertrophy at the beginning. The generation of pathological myocardial hypertrophy is related to epigenetic changes. Therefore, the study of the mechanism of pathological myocardial hypertrophy helps to discover the molecular characteristics of reversing myocardial remodeling, develop new methods to promote the regression of pathological myocardial hypertrophy, and is of great significance for the prevention or treatment of heart failure.

[0004] Previous studies on pathological myocardial hypertrophy mainly focused on transcription factors that promote the expression of hypertrophy-related genes, inducing cell death, fibrosis, and mitochondrial dysfunction by activating downstream cell signal transduction pathways. However, more and more studies have now found that the regulation of gene expression plays an important role in myocardial hypertrophy. For example, RNA splicing regulators perform alternative splicing on mRNA to generate multiple mature transcripts, affecting the expression of a broad spectrum of cardiac genes and leading to corresponding cardiac pathological changes. RNA binding Fox-1 homolog 1 (RBFOX1) is necessary for the conserved splicing of the transcription factor myocyte enhancer factor 2 (MEF2) family members, and this process can generate different MEF2 subtypes, affecting the expression of genes related to the pathology of myocardial hypertrophy. MicroRNA (miR)-mediated gene silencing can regulate gene expression post-transcriptionally and affect the function of this gene at the protein level. miR-30d negatively regulates the protein expression of mitogen-activated protein 4 kinase 4 (MAP4K4) by binding to the 3′-untranslated region (3′-UTR) in cardiomyocytes, inhibiting the conduction of the calcineurin-NFAT signaling pathway. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides the use of IGFBPL1 and its regulatory factors in the preparation of drugs for the treatment of myocardial hypertrophy. The present invention reveals the roles of the MYLK4 and IGFBPL1 signals in pathological myocardial hypertrophy during the process of cardiac hypertrophy, providing a new idea for understanding the complex regulatory network during myocardial hypertrophy.

[0006] The technical solution adopted by the present invention to achieve the technical purpose is as follows:

[0007] The present invention provides the use of IGFBPL1 and its regulatory factors in the preparation of drugs for the treatment of myocardial hypertrophy.

[0008] Preferably, the increase in the expression of IGFBPL1 mRNA and protein in phenylephrine-induced hypertrophic cardiomyocytes can be reduced after overexpressing MYLK4.

[0009] Preferably, overexpressing MYLK4 or knocking down IGFBPL1 can alleviate phenylephrine-induced cardiomyocyte hypertrophy.

[0010] More preferably, overexpressing MYLK4 or knocking down IGFBPL1 can reduce the expression of hypertrophy markers β-MHC and BNP and reduce the surface area of cardiomyocytes.

[0011] Preferably, the elevated expression of IGFBPL1 in the heart with myocardial hypertrophy caused by pressure load can be reduced after overexpressing MYLK4.

[0012] Preferably, overexpressing MYLK4 or knocking down IGFBPL1 can significantly alleviate the pathological changes of myocardial hypertrophy caused by pressure load.

[0013] Furthermore, the pressure load includes pressure overload induced by TAC surgery.

[0014] Furthermore, the pathological changes of the myocardial hypertrophy include at least one of an increase in LVPWd and LVPWs, an increase in heart / body weight ratio, an enlarged heart, and an increase in the cross-sectional area of cardiomyocytes.

[0015] The beneficial effects of the present invention are as follows:

[0016] The present invention discloses that MYLK4 can participate in regulating the expression of IGFBPL1 in a phenylephrine-induced neonatal mouse cardiomyocyte hypertrophy model. Overexpressing MYLK4 or knocking down IGFBPL1 alleviates phenylephrine-induced neonatal mouse cardiomyocyte hypertrophy, including increased expression of myocardial hypertrophy molecular markers and increased cross-sectional area of cardiomyocytes. In a mouse myocardial hypertrophy model caused by pressure load, the expression of MYLK4 is decreased. Lentivirus (LV)-mediated overexpression of MYLK4 reduces the expression level of IGFBPL1 in the hearts of myocardial hypertrophy mice and significantly alleviates the pathological changes of TAC surgery-induced myocardial hypertrophy. Overexpressing IGFBPL1 can significantly exacerbate the pathological changes of myocardial hypertrophy. The conclusions of the present invention may also be applicable to other disease states causing myocardial hypertrophy, such as angiotensin II-induced myocardial hypertrophy. Description of the Drawings

[0017] Figure 1 Expression of MYLK4 after cardiomyocyte hypertrophy. (A) qPCR was used to detect the mRNA expression of cardiomyocyte hypertrophy markers after PE treatment. (B-D) WB was used to detect the protein expression of cardiomyocyte hypertrophy markers. (E) mRNA expression of MYLK4 in hypertrophied cardiomyocytes. (F-G) WB was used to detect the protein expression of MYLK4 in hypertrophied cardiomyocytes. (n = 3, *p < 0.05)

[0018] Figure 2 MYLK4 affects the expression of IGFBPL1. (A) qPCR was used to detect the mRNA level of IGFBPL1 when MYLK4 was overexpressed. (B-D) WB was used to detect the protein expression of MYLK4 and IGFBPL1 after overexpressing MYLK4. (n = 3, ns indicates no statistical significance, *p < 0.05)

[0019] Figure 3 Overexpression of MYLK4 or knockdown of IGFBPL1 alleviates cardiomyocyte hypertrophy. (A-D) Protein expressions of the hypertrophy markers β-MHC and BNP were detected after overexpression of MYLK4. (E-F) Immunofluorescence staining of the nucleus with DAPI (blue) and the cell outline with α-actinin (red) was used to observe the change in cardiomyocyte area (scale bar = 20 μm). (G) qPCR was used to verify the knockdown effect of siIGFBPL1. (H-K) Protein expressions of IGFBPL1 and the hypertrophy markers β-MHC and BNP were detected by WB after knockdown of IGFBPL1. (L-M) Immunofluorescence staining of the nucleus with DAPI (blue) and the cell outline with α-actinin (red) was used to observe the change in cardiomyocyte area (scale bar = 20 μm). (n = 3, *p < 0.05)

[0020] Figure 4 Expressions of MYLK4 and IGFBPL1 in the myocardium of mice in different groups. (A-C) Expressions of MYLK4 and IGFBPL1 in the myocardial tissues of mice were detected by WB according to the experimental grouping. (n = 3, ns indicates no statistical significance, *p < 0.05)

[0021] Figure 5 MYLK4 affects myocardial hypertrophy through IGFBPL1. (A-D) Mice were subjected to echocardiography 4 weeks after TAC to compare LVPWd, LVPWs, LVEF, and FS among groups of mice. (E) Heart / body weight ratio among groups of mice. (F-G) HE showed the pathological changes in the heart structure among groups of mice (scale bar = 1000 μm), and WGA staining was used to quantitatively measure the size of cardiomyocytes in mice (scale bar = 50 μm). (n = 3, ns indicates no statistical significance, *p < 0.05) Detailed implementation manners

[0022] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Without special instructions, they can all be obtained from commercial channels. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The following experiments such as mouse aortic arch constriction, neonatal mouse cardiomyocyte extraction, cardiomyocyte hypertrophy induction, qRT-PCR (qPCR), western blot (WB), immunofluorescence staining, WGA (Wheatgerm agglutinin) staining, HE (Hematoxylin-Eosin) staining, and siRNA technology are all conventional experimental operations, and the methods are true and reliable.

[0023] Example

[0024] I. Experimental method

[0025] 1. Extraction of primary neonatal rat cardiomyocytes

[0026] 1.1 Preparation of materials and instruments

[0027] Place the surgical instruments to be used in an aluminum lunch box for autoclaving. Prepare three clean small plastic boxes, 75% ethanol disinfectant solution, DMEM high-glucose medium, fetal bovine serum (FBS), penicillin-streptomycin solution (100×), trypsin cell digestive solution (0.25% trypsin), sterile phosphate buffer solution (PBS, pH 7.2 - 7.4, 0.01M), 5-bromo-2-deoxyuridine (5-BrdU), 6 cm sterile culture dish, T25 sterile cell culture flask, 1.5 mL sterile EP tube, 15 mL sterile centrifuge tube, 50 mL sterile centrifuge tube, 100 μm sterile cell sieve, 3 mL sterile Pasteur pipette. Wipe and dry the small plastic boxes with 75% ethanol disinfectant solution, place them in the biosafety cabinet and sterilize them with ultraviolet light for 30 minutes. Pour an appropriate volume of high-glucose medium into three 6 cm culture dishes for standby. Prepare a cell culture medium containing 10% FBS in a 50 mL centrifuge tube.

[0028] 1.2 Extraction of neonatal rat hearts

[0029] Place C57BL / 6J neonatal rats in the first plastic small box, and pour 75% ethanol disinfectant solution into the second plastic box for mouse disinfection. Use a straight ophthalmic scissors in the right hand to transect the neonatal rats at the neck, and put the removed heads into the third plastic box; cut open the upper sternum at the position of the anterior midline, and gently squeeze the chest area with the left thumb. At this time, the beating heart can be seen at the cut. Replace the scissors in the right hand with a sharp-pointed curved forceps, clamp and remove the heart during the cardiac contraction period, and place it in a 6 cm sterile culture dish containing high-glucose medium. The mouse carcasses are also placed in the third plastic box. After all the mouse hearts are removed by the above method, withdraw the three plastic boxes from the biosafety cabinet, put the mouse carcasses into a yellow plastic bag and place them in a -20°C refrigerator.

[0030] 1.3 Cleaning and trimming of neonatal rat hearts

[0031] Gently squeeze the heart placed in the petri dish with a sharp curved forceps to remove the residual blood in the heart cavity. Use a curved ophthalmic scissors to remove the redundant blood vessels and atria at the base of the heart, carefully peel off the tissues such as fascia covering the surface of the heart, and place it into a second 6-cm sterile petri dish containing high-glucose medium. After treating all the hearts according to the above method, pick up the heart with a sharp curved forceps, and use an ophthalmic scissors to divide each heart into 2 - 3 tissue blocks of similar size, and place them into a third 6-cm sterile petri dish containing high-glucose medium.

[0032] 1.4 Digestion of neonatal rat hearts and preparation of single-cell suspension

[0033] Use a sterile Pasteur pipette to aspirate the minced heart tissue blocks in the petri dish into a 15-mL sterile centrifuge tube, and remove the medium aspirated together with the tissue blocks. Add an appropriate amount of trypsin cell digestion solution and shake the centrifuge tube. After rinsing the residual medium in the centrifuge tube, remove the trypsin cell digestion solution. Add 2 times the volume of trypsin cell digestion solution according to the volume of cardiomyocytes in the centrifuge tube, shake the centrifuge tube at 37 °C for 2 - 5 minutes. When the clear cell digestion solution becomes turbid, use a Pasteur pipette to transfer the digestion solution in the tube into a centrifuge tube containing serum-containing cell medium to terminate the digestion. Repeat the above steps until all the myocardial tissues in the centrifuge tube are completely digested. Take another 50-mL sterile centrifuge tube, place the cell sieve on the tube opening, and use a Pasteur pipette to filter the medium containing neonatal rat primary cardiomyocytes through the cell sieve to obtain a single-cell suspension containing cardiomyocytes.

[0034] 1.5 Differential attachment to separate cardiomyocytes and cardiac fibroblasts

[0035] Centrifuge the cell suspension at 1500 r / min for 7 minutes. After centrifugation, discard the supernatant, add an appropriate amount of cell medium containing 10% FBS, and gently blow it evenly with a Pasteur pipette or a sterile pipette tip. According to the number of neonatal rats used, inoculate the cell suspension into a 6-cm sterile petri dish or a T25 sterile cell culture flask. The seeding density is preferably no more than 30 neonatal rats in a T25 cell culture flask. Since the attachment rates of cardiomyocytes and cardiac fibroblasts are different, place the above culture flask or petri dish in a 37 °C cell culture incubator containing 5% CO2 and incubate for about 1.5 hours. After differential attachment is completed, use a Pasteur pipette or a sterile pipette tip to aspirate the medium in the culture flask or petri dish. This medium is the cardiomyocyte suspension. The cardiac fibroblasts in the culture flask or petri dish can be cultured further by adding cell medium containing 10% FBS or discarded.

[0036] 1.6 Adherent culture of cardiomyocytes

[0037] Resuspend the above cardiomyocyte suspension with 0.1 mmol / L 5-BrdU again to inhibit the proliferation of cardiac fibroblasts. Evenly seed the cardiomyocytes in a cell culture dish and change the medium after culturing for 24 h. When changing the medium, first discard the medium in the dish, wash it three times with phosphate buffer to remove the residual tissue fragments in the culture dish, and then add the cardiomyocyte medium containing 0.1 mmol / L 5-BrdU. Observe the morphology and beating of the cardiomyocytes. If the cardiomyocytes are in good condition, they can be used for subsequent experiments.

[0038] 2. Treatment and grouping of cardiomyocytes

[0039] According to different cell models required for the experiment, the cells are treated accordingly:

[0040] a. Treatment of cardiomyocytes with phenylephrine

[0041] To construct a cardiomyocyte hypertrophy model, phenylephrine (PE) is added to the cardiomyocyte medium, and the concentration of PE in the medium is 200 μmol / L to induce cardiomyocyte hypertrophy.

[0042] Day 1 Day 2 Day 3 Day 4 Day 5 Control group Medium change Medium change Medium change plus phosphate buffered saline Medium change Cell harvest Hypertrophy group Medium change Medium change Medium change plus PE solution Medium change Cell harvest

[0043] b. Gene overexpression in cardiomyocytes by lentiviral transfection

[0044] Entrust Suzhou GenePharma Co., Ltd. to construct a mouse MYLK4 lentivirus (LV) overexpression vector (LV-MYLK4) and a negative control lentivirus (LV-NC). The sequences are shown in the following table:

[0045]

[0046]

[0047] The virus is stored in a -80 °C ultra-low temperature refrigerator for later use. Before transfection, take out the virus from the -80 °C refrigerator and melt it on ice. According to the results of the preliminary experiment, aspirate an appropriate volume of the virus stock solution, mix the virus stock solution and the cardiomyocyte medium, and add Polybrene at a final concentration of 5 μg / mL. Mix again and then aspirate the medium in the cell culture well and replace it with the medium containing the virus and Polybrene. After transfection for 24 hours, change it to fresh medium and continue to culture for a period of time. During this period, change the medium or perform treatment as needed.

[0048] Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 LV-NC group Medium change Medium change Medium change plus LV-NC Medium change plus phosphate buffered saline Medium change Cell harvest LV-NC+PE group Medium change Medium change Medium change plus LV-NC Medium change plus phenylephrine Medium change Cell harvest LV-MYLK4 group Medium change Medium change Medium change plus LV-MYLK4 Medium change plus phosphate buffered saline Medium change Cell harvest LV-MYLK4+PE group Medium change Medium change Medium change plus LV-MYLK4 Medium change plus phenylephrine Medium change Cell harvest

[0049] c. Gene silencing in cardiomyocytes by siRNA transfection

[0050] Inhibit the expression of certain genes in cells by siRNA transfection. The IGFBPL1-specific siRNA oligonucleotides were purchased from Suzhou GenePharma Co., Ltd. The target sequences of the siRNAs used are as follows:

[0051]

[0052] Before transfection, the growth status of the cells needs to be closely observed. Generally, transfection is carried out when the cell density is about 60%. Now, taking one well of a 6-well plate as an example, the system used for transfection is outlined. Add 200 μL of serum-reduced medium Opti-MEM into two sterile 1.5 mL EP tubes respectively. Add 150 pmol of small interfering RNA (siRNA) into one tube and 6 μL of liposomal transfection reagent Lipofectamine 3000 into the other tube. Gently mix with a pipette and let it stand at room temperature for five minutes. Mix the liquids in the two tubes and mix again with a pipette. After mixing, let it stand for 20 minutes and immediately add it into the well plate that has been replaced with fresh medium. Incubate in a 37 °C incubator containing 5% CO2 for 36 h and then detect the mRNA expression. The protein expression can be detected 48 h later.

[0053] Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Control group Medium change Medium change Medium change plus siNC transfection Medium change plus phosphate buffered saline Medium change Cell harvest Hypertrophy group Medium change Medium change Medium change plus siNC transfection Medium change plus phenylephrine Medium change Cell harvest siRNA transfection group Medium change Medium change Medium change plus siRNA transfection Medium change plus phosphate buffered saline Medium change Cell harvest siRNA transfection plus hypertrophy group Medium change Medium change Medium change plus siRNA transfection Medium change plus phenylephrine Medium change Cell harvest

[0054] 3. Construction of a mouse model of myocardial hypertrophy

[0055] 3.1 Grouping of experimental animals

[0056] Purchase SPF-grade 6-week-old male C57BL / 6J mice and make good marks. After 2 weeks of adapting to the environment, randomly group them, and the specific treatments are as follows:

[0057] Group Treatment method Con group After performing sham operation, the animals were raised in a SPF-level environment for 28 days TAC group After TAC surgery, the animals were raised in a SPF-level environment for 28 days TAC+NC group One day after TAC surgery, NC virus was injected into the tail vein, and the animals were raised in a SPF-level environment for 28 days TAC+MYLK4 group One day after TAC surgery, MYLK4 virus was injected into the tail vein, and the animals were raised in a SPF-level environment for 28 days TAC+shIGFBPL1 group One day after TAC surgery, shIGFBPL1 virus was injected into the tail vein, and the animals were raised in a SPF-level environment for 28 days TAC+MYLK4+IGFBPL1 group One day after TAC surgery, MYLK4+IGFBPL1 virus was injected into the tail vein, and the animals were raised in a SPF-level environment for 28 days

[0058] Echocardiography was performed 4 weeks after TAC. The mouse heart tissues were collected, and the expressions of MYLK4 and IGFBPL1 in the mouse myocardial tissues were detected by WB. The heart / body weight ratio of mice in different groups was measured. The heart size was observed by HE staining, and the cross-sectional area of cardiomyocytes was detected by WGA staining.

[0059] 3.2 Construction of a mouse model of myocardial hypertrophy using TAC

[0060] 3.2.1 Preparation before surgery

[0061] Place the stereomicroscope on the experimental operation table and adjust the cold light source to ensure a clear and bright surgical field of view. Prepare anesthetic (1% sodium pentobarbital solution), furosemide injection, depilatory cream, medical tape, medical cotton swabs, sterile cotton balls, sterile gauze, 0.9% sodium chloride injection, iodophor skin disinfectant, 75% ethanol disinfectant, medical 6-0 and 4-0 suture threads, 6-cm culture dish (sterile), 27G blunt needle (bent to about 90°), self-made thread-passing hook (grind the bent part of an 8-gauge stainless steel mouse curved gavage needle with a fine file until a notch is formed between the ground part and the hollow pipe of the gavage needle, and the notch can accommodate the passage of the suture thread), and a chest expander made of a paper clip (separate the small inner ring and the large outer ring of the paper clip and bend them into an S shape. The slightly upturned parts on both sides of the U shape of the small ring can form a hook). After the surgical instruments are sterilized by high-pressure steam, wipe and disinfect them again with 75% ethanol disinfectant before the operation. After turning on the constant temperature heating pad, set the temperature to 42 °C for the mouse to wake up after the operation. Pour an appropriate amount of normal saline into the culture dish, cut the 6-0 suture thread into small sections about 3 cm long and soak them in the normal saline for later use.

[0062] 3.2.2 Weighing, Anesthetizing, and Depilation

[0063] Weigh the mouse (about 25 g) with a weighing scale and anesthetize it by intraperitoneal injection of sodium pentobarbital solution at a dose of 40 mg / kg. After completely anesthetizing the mouse, evenly apply the depilatory cream from the mouse's neck to the costal margin, reaching the midaxillary line on both sides. After about 5 - 10 minutes, wipe the applied area with a cotton swab to remove the shed hair and the depilatory cream. Soak a sterile cotton ball in iodophor skin disinfectant, disinfect the surgical area from the middle to the outside and repeat 3 times. After the disinfectant dries naturally, proceed to the next step.

[0064] 3.2.3 Mouse Fixation

[0065] Turn the mouse's head towards the operator and place it in a supine position. Fix the four limbs to the base of the stereomicroscope with medical tape, and stick the tail with adhesive tape after straightening it. To keep the airway unobstructed and fully expose the mouse's neck for subsequent surgical operations, the incisors of the mouse also need to be fixed with a thin thread by stretching. Adjust the positions of the cold light sources on both sides of the microscope again, and expose the mouse's neck and chest to the center of the light source.

[0066] 3.2.4 Aortic Constriction

[0067] Lift the skin of the mouse's neck with forceps, and make a small opening along the anterior midline from the mouse's neck to the chest with a straight ophthalmic scissors. The opening extends from the neck to the end of the sternum, about 1.5 cm long. Use a curved forceps to bluntly separate the connective tissue at the suprasternal fossa, including the muscles on both sides of the tracheal midline and part of the chest wall muscles, and be gentle to avoid damaging the small subcutaneous blood vessels and affecting the surgical field of view. Slightly force the curved forceps (with the head facing up) into the suprasternal fossa, pick up the sternum from the suprasternal fossa, and use a straight ophthalmic scissors to cut the sternum once from the suprasternal fossa towards the xiphoid process, with a length reaching about the second rib level. Pull the hooked ends of the self-made chest expander to the left and right sides to open the cut sternum, fully exposing the surgical field of view. At this time, white thymus tissue and superficial mediastinal fat can be observed. Gently tear the thymus in the middle with micro forceps and push it to both sides, readjust the position of the chest expander to fix the thymus tissue on both sides, carefully clean the surrounding adipose tissue. When the brachiocephalic trunk and the left common carotid artery are distinguished, the intersection of the two on the aortic arch can be found along their running directions, and this is the ligation site. This step is very likely to damage small blood vessels and cause bleeding. Try to be gentle. If there is little bleeding, use a sterile cotton ball dipped in physiological saline to clean the surgical field of view. Excessive bleeding will cause the death of the mouse. Pick up the soaked 6-0 suture in the culture dish with forceps, insert it from the notch of the self-made suture-passing hook and pass through the needle tip. Slowly pass the suture-bearing hook under the aortic arch, hold one end of the thread with forceps and slowly withdraw the hook, so that both ends of the suture straddle both sides of the aortic arch. Place a 27G blunt needle beside the aortic arch, place the suture at the ligation site and ligate the aortic arch and the 27G blunt needle together with forceps, and at the same time gently and quickly withdraw the 27G blunt needle to achieve transverse narrowing of the aortic arch. Finally, a single knot can be added to prevent the thread from loosening and slipping. Remove the chest expanders on both sides, suture the chest wall muscles on both sides with 6-0 suture, and suture the skin with 4-0 suture. After suture, disinfect the wound with iodophor disinfectant, and at the same time inject 0.1 ml of furosemide intraperitoneally to prevent the mouse from dying due to acute left heart failure after TAC. Place the mouse in the prone position on a thermostatic heating pad, closely observe its vital signs such as breathing, heart rate and body temperature, and place it in a mouse cage with clean bedding for separate cages after it wakes up. The sham operation group does not ligate the aortic arch. After the operation, both groups of mice were cultured in the same SPF-level environment for 28 days.

[0068] 3.3 Intravenous injection of virus into the mouse tail

[0069] Use the NC virus, MYLK4 overexpression virus, IGFBPL1 knockdown virus (shIGFBPL1), and MYLK4 + IGFBPL1 overexpression virus constructed by Shanghai Genechem Co., Ltd. Knockdown or overexpression of genes was achieved by intravenous injection of the virus into the mouse tail 1 day after TAC, and the total injection volume was 2.5×10 11 vg / mouse, and the detailed information is shown in the following table.

[0070]

[0071] 4. Verification of the myocardial hypertrophy model by echocardiography in mice

[0072] All mice in each group survived smoothly until 28 days. The echocardiography examination of the mice was entrusted to Kesi'ai Biotechnology (Suzhou) Co., Ltd. The model of the ultrasound instrument used by this company is Feiyno VINNO 6. The echocardiography examination was performed by the same person to avoid measurement errors. Both the examination process and data processing adopted the double-blind method. For echocardiography examination, the hair of the mice should be removed at least one day in advance. The steps were similar to those when constructing the TAC model. The hair removal area included the left chest and the prethoracic area. The mice were placed in the induction box of the anesthesia machine. After quickly inducing anesthesia in the mice with high-concentration isoflurane, their limbs were fixed on the detection plate in a supine position. Anesthesia was maintained using low-concentration isoflurane through the anesthesia machine pipeline. The coupling agent was applied to the limbs of the mice and an electrocardiogram was connected. The anesthesia dose was appropriately adjusted to control the mouse heart rate at 300 - 500 beats / min. After applying the coupling agent to the prethoracic area of the mice, the probe was placed on the left side of the sternum, and the corresponding echocardiogram data were obtained in B-mode and M-mode respectively. Subsequently, the left ventricular posterior wall thickness in diastole (LVPWd) and the left ventricular posterior wall thickness in systole (LVPWs) of the mice were measured successively in 3 cardiac cycles and the average values were calculated.

[0073] 5. Sampling of mouse heart tissue

[0074] The tissue sampling of the mice was carried out after the echocardiography examination. First, the body weight of the mice was weighed on an electronic scale and recorded. The mice were euthanized by intraperitoneal injection of a sufficient amount of sodium pentobarbital solution. The limbs of the mice were fixed, and the skin of the mice was cut open with straight ophthalmic scissors. The thoracic cage of the mice was cut open from the xiphoid process and folded to both sides to expose the heart. The right auricle of the mice was cut off to form a perfusion fluid outlet. A 50 mL syringe barrel was used to aspirate 40 mL of 0.9% sodium chloride injection. After connecting the intravenous infusion needle to the syringe barrel, it was inserted 3 - 4 mm into the cardiac apex, and normal saline was injected into the left ventricle of the mice to wash the residual blood in the heart and tissues. The heart was removed along the aortic root with scissors and placed in ice-cold normal saline at 4°C for rinsing. The excess normal saline on the surface of the heart was wiped off with sterile gauze, weighed and recorded, and then placed in a cryopreservation tube, or handed over to Wuhan Sevier Biotechnology Co., Ltd. for subsequent experiments. The mouse number, group and date were recorded. The cryopreservation tube could be stored in a liquid nitrogen tank. The mouse carcasses were put into yellow plastic bags and placed in a -20°C refrigerator, which was uniformly processed by the laboratory.

[0075] 6. Section staining of heart tissue

[0076] After the mouse tissue sampling was completed, it was handed over to Wuhan Saiweier Biotechnology Co., Ltd. for sectioning and staining. The staining items included WGA staining and HE staining. ImageJ was used for the statistical analysis of cardiomyocyte size among samples.

[0077] 7. qPCR verification of gene mRNA level expression

[0078] 7.1 Extraction of RNA from myocardial tissue and cardiomyocytes

[0079] Put the mouse tissue samples into a 1.5 mL enzyme-free EP tube, make marks, rinse three times with phosphate buffer solution. Prepare a pestle and mortar that have been treated with RNaseZap solution. After carefully adding liquid nitrogen for pre-cooling, add an appropriate amount of liquid nitrogen to ensure that the mouse heart tissue can be completely immersed in liquid nitrogen. Quickly transfer the heart tissue to liquid nitrogen, use the pestle to crush the tissue into small pieces and grind it into powder. Immediately add 1 mL of TRIzol and mix well. Transfer the lysate to a new 1.5 mL enzyme-free EP tube, make marks, and let it stand at room temperature for 5 minutes.

[0080] Aspirate the cell culture medium in the 6-well plate, wash twice with sterile phosphate buffer solution, remove the buffer solution used for washing. Add 1 mL of TRIzol to each well, pipette after blowing, and transfer the TRIzol lysate to a centrifuge tube, make marks, and let it stand at room temperature for 5 minutes.

[0081] Add 1 / 5 volume (200 μL) of chloroform to the cell or tissue lysate, invert and mix 10 times, and let it stand for 5 min. Centrifuge at 12000 r / min for 15 minutes in a 4℃ centrifuge. After taking out the centrifuged EP tube, it can be observed that the sample is divided into three layers. Carefully pipette 400 μL of the colorless upper aqueous phase with a 200 μL pipette into a new centrifuge tube and make marks with a marker pen. Add 400 μL of isopropanol and mix well. Let it stand for 10 min and then centrifuge for 10 minutes again. Discard the supernatant. Add 1 mL of 75% ethanol pre-cooled on ice (prepared with DEPC water) into the tube, suspend the RNA precipitate on the tube wall with an enzyme-free pipette tip and place it in a 4℃ centrifuge. After centrifuging at 7500 r / min for 5 minutes, take it out. Discard the supernatant, repeat the above steps once, and place the EP tube in the fume hood to dry for 10 minutes. Dissolve the RNA in 20 - 50 μL of DEPC water according to the precipitation amount, and fully pipette with an enzyme-free pipette tip to dissolve the RNA. The extracted RNA can be immediately used for reverse transcription or stored at -80℃ for short-term use.

[0082] 7.2 Quality detection of RNA and reverse transcription reaction

[0083] The total RNA extracted from each sample was detected using DeNovix DS-11. When the measured OD260 / OD280 was in the range of 1.8 - 2.0, it indicated that the purity of the extracted RNA met the requirements and could be used for subsequent reverse transcription experiments. In this experiment, the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit from Novoprotein was selected for reverse transcription. The required RNA amount was uniformly set to 1 μg and the volume of the used RNA (V 模板 ) was calculated. The experimental procedure was as follows:

[0084] a. Removal of genomic DNA

[0085] Prepare the mixture in an enzyme-free centrifuge tube according to the following table, with a total volume of 16 μL. After preparation, gently pipette and mix well, then centrifuge briefly in a tabletop centrifuge and incubate at 42 °C for 2 minutes.

[0086]

[0087]

[0088] b. Preparation of the reverse transcription reaction system and reverse transcription

[0089] Directly add 4 μL of 5×HiScriptⅢ qRT SuperMix to the reaction tube in the first step, with a total volume of 20 μL. After mixing, centrifuge briefly and proceed to the next step:

[0090] 37℃ 15 minutes 85℃ 5 seconds

[0091] The product can be directly used for qPCR reactions or stored at -20 °C and used within half a year. For long-term storage, it can be aliquoted and stored at -70 °C to avoid repeated freezing and thawing.

[0092] 7.3 Real-time fluorescence quantitative polymerase chain reaction

[0093] Add an appropriate amount of DEPC water to dilute the cDNA product stock solution obtained from reverse transcription (the added DEPC water should not be less than 80 μL) and mix well. According to the usage procedure of the Novoprotein Taq Pro Universal SYBR qPCR Master Mix, prepare the qPCR reaction system in a 96-well reaction plate. The preparation process requires protection from light and should be carried out on ice.

[0094] a. Preparation of the qPCR reaction system

[0095] Component Volume 2×Taq Pro Universal SYBR qPCR Master Mix 5μL Forward primer 0.5μL Reverse primer 0.5μL Diluted cDNA 4μL Total 10μL

[0096] b. After loading the samples, centrifuge the 96-well reaction plate briefly, set the reaction program on the instrument according to the loading order, and perform the qPCR reaction:

[0097]

[0098] According to the Ct value results of each well, statistical analysis was performed using 2 -ΔΔCt methods. The melting curve can be used to determine whether the qPCR product is single.

[0099] Primer sequence:

[0100]

[0101]

[0102] 8. Western blot

[0103] 8.1 Extraction and concentration determination of total protein

[0104] To prepare the protein lysate, RIPA lysis buffer and PMSF protease inhibitor need to be mixed in a certain ratio before use. Add 1 μL of PMSF to every 100 μL of RIPA lysis buffer. After preparation, it can be placed on ice for later use. Both RIPA lysis buffer and PMSF are stored at -20 °C and are easily degraded in water, so they need to be prepared freshly before use. For the cells to be used for protein extraction (taking a 6-well plate as an example), aspirate the culture medium, wash the cells once with phosphate-buffered saline and then discard it. Add approximately 30 μL of the prepared lysis buffer, and lyse the cells on ice for 10 minutes. Use a cell scraper to press against the bottom of the well plate and scrape the cells at the bottom of the dish as cleanly as possible in one direction. Use a pipette to transfer the scraped cell suspension to a 1.5 mL EP tube, and pipette up and down repeatedly to promote sufficient lysis of the cells. Insert the EP tube containing the protein sample on ice. Replace the cell scraper before scraping cells from each well, and the whole process needs to be carried out on ice to avoid protein degradation. After all the cells in the well plate are collected, continue to lyse on ice for 30 minutes, then place it in a centrifuge at 4 °C and centrifuge at 12,000 r / min for 15 - 20 minutes. Aspirate the supernatant into a new EP tube to obtain the protein sample. Note the volume of the aspirated supernatant, make a good mark and place it on ice for later use. To ensure that the protein amount in each loading well is the same during SDS-polyacrylamide gel electrophoresis, it is necessary to measure the protein concentration in each sample after protein extraction. Prepare a 25 mg / mL protein standard solution with the BCA protein concentration assay kit, and dilute it to 0.5 mg / mL with phosphate-buffered saline for later use. Both can be stored at -20 °C for a long time. Add 1 part of BCA reagent B to 50 parts of BCA reagent A, and mix well to obtain the BCA working solution. Add the 0.5 mg / mL protein standard solution to a 96-well plate in gradients, and make up each well to 10 μL with phosphate-buffered saline, which is equivalent to diluting the standard solution in each well to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL. Add 1 μL of the protein sample to the 96-well plate, make up to 10 μL with phosphate-buffered saline, add 200 μL of BCA working solution to each well, incubate at 37 °C for 30 minutes, then measure the absorbance value and calculate the protein concentration of the sample. According to the protein sample concentration, add protein lysis buffer to make the concentrations of all samples the same. Add an appropriate volume of SDS-PAGE loading buffer (5X), heat at 100 °C for 5 minutes (the heating time can be appropriately extended according to the sample concentration). After the sample cools to room temperature, it can be directly used for loading, or stored in a -20 °C refrigerator for later use.

[0105] 8.2 Preparation and Electrophoresis of SDS-Polyacrylamide Gel (SDS-PAGE)

[0106] After the glass plate is fixed on the rubber rack, it is leak-tested with deionized water. After the leak test is completed, the deionized water is poured out and the residual liquid is blotted dry with filter paper. The lower-layer rubber solution and the lower-layer rubber buffer are mixed evenly at a ratio of 1:1, and a modified coagulant is added. Then it is injected into the glass plate for making the gel. After sealing the edge with anhydrous ethanol, it is left standing. When an obvious fold line can be seen between the separating gel and the anhydrous ethanol, it indicates that the separating gel is completely solidified. Then the anhydrous ethanol is poured out and blotted dry with filter paper. The upper-layer rubber solution and the lower-layer rubber buffer are prepared at a ratio of 1:1 and a coagulant is added. After mixing evenly, it is injected into the glass plate and a gel comb is immediately inserted. It is left standing at room temperature for 20 minutes. The Tris-glycine-SDS electrophoresis buffer (10×) is diluted with deionized water to 1× and then mixed evenly for standby. The prepared gel is placed in the electrophoresis tank and the diluted electrophoresis buffer is added. After pulling out the gel comb, 20 μg of protein sample and pre-stained protein Marker (4 μL) are added to each well. The power switch is turned on, and the voltage is set to 80 V for electrophoresis. After the Marker is separated, the voltage is adjusted to 150 V. When the blue dye in the loading buffer electrophoreses to near the bottom of the lower layer of the gel (about 75 minutes), the electrophoresis can be stopped and the power is turned off.

[0107] 8.3 Transfer membrane and blocking

[0108] Prepare 1× transfer buffer the next day: Dilute 100 mL of transfer buffer (10×) with 700 mL of deionized water, add 200 mL of methanol, mix well, place in a 4°C refrigerator, and pre-cool an ice box in a -20°C refrigerator overnight. Cut the PVDF membrane into a size of 8.5×6.5 cm, soak it in methanol for a few seconds, and place it in the prepared 1× transfer buffer for equilibration after it becomes transparent. Pour an appropriate amount of pre-cooled 1× transfer buffer into an enamel tray, transfer the equilibrated PVDF membrane to the tray with forceps, and place two filter paper-free transfer sponge pads in the transfer buffer to equilibrate for about 2 minutes. Place the black side of the transfer cassette at the bottom of the tray and place the first transfer sponge pad on it. Take out the gel from the electrophoresis tank, pry open the short glass plate with a gel lifter, cut off the colored upper gel part of the gel, lift the lower gel from the long glass plate with a gel lifter, and carefully transfer it horizontally to the first transfer sponge pad. Place the PVDF membrane on the transferred gel with forceps, use a roller to expel the air bubbles between the PVDF membrane and the gel, cover the second transfer sponge pad on the membrane, fold the white side of the transfer cassette over the sponge pad and clamp the top fixing clip. Align the black side of the prepared transfer cassette with the cathode of the transfer electrode and place the transfer electrode in the transfer tank. Take out the ice box from the -20°C refrigerator and place it in the transfer tank. Pour an appropriate amount of transfer solution and then transfer the entire transfer tank to a foam box filled with ice. Cover the lid and turn on the power supply. Set the constant current to 350 mA and transfer for 90 minutes. Dilute the 10× TBST concentrate with deionized water to 1× TBST buffer. After the transfer is completed, take out the PVDF membrane and quickly place it in the diluted TBST buffer, and rinse it on a shaker at medium speed for 1 - 2 minutes. Replace the TBST with 5% skim milk or 5% BSA, shake it at low speed at room temperature, and block for 1.5 hours. After the blocking is completed, recover the skim milk or BSA blocking solution, adjust the shaker to medium speed and rinse the PVDF membrane with TBST buffer 3 times, 10 minutes each time.

[0109] 8.4 Incubation with primary antibody and secondary antibody

[0110] Name of reagent and consumable Brand and catalog number GAPDH Monoclonal antibody Proteintech 60004-1-Ig MYLK4 Polyclonal antibody Proteintech 24309-1-AP IGFBPL1 antibody (C-5) Santa Cruz Biotechnology sc-398875 Anti-BNP antibody Abcam ab19645 MYH7 / β-MHC Rabbit pAb ABclonal A7564 Goat anti-rabbit secondary antibody-HRP Linker Biologics GAR007 Goat anti-mouse secondary antibody-HRP Linker Biologics GAM007

[0111] Dilute the primary antibody according to the ratio indicated in the antibody instruction manual with antibody diluent and place it on ice for later use. Immerse the PVDF membrane in TBST buffer, and cut the PVDF membrane into strips of appropriate width according to the position indicated by the Marker and the molecular weight of the target protein. If there are many strips, different marks can be made in the upper left corner of the membrane for distinction. Add the diluted primary antibody to the strips and incubate overnight at 4°C on a shaker. The next day, dilute the secondary antibody with antibody diluent according to the ratio indicated in the instruction manual and place it on ice for later use. After recovering the primary antibody diluent, immerse the strips in the antibody incubation box with TBST buffer and rinse them 3 times at medium speed on a shaker at room temperature for 10 minutes each time. After rinsing, add the diluted secondary antibody and incubate slowly at room temperature on a shaker for 1.5 hours. After incubation, recover the secondary antibody and rinse the strips 3 times at medium speed with TBST buffer on a shaker at room temperature for 10 minutes each time.

[0112] 8.5 Chemiluminescence Detection of Strips

[0113] In a dark room, mix the peroxide solution and luminol reagent in the chemiluminescence HRP substrate kit in equal amounts, and immerse the strips completely in the developer and incubate appropriately at room temperature. Turn on the imager and complete the calibration. Place the strips with the protein adsorption side down, spread them evenly on the imager screen, ensuring a tight fit between the membrane and the screen without any air bubbles remaining. Gently close the lid, select an appropriate exposure time, complete the image acquisition, and save the experimental results.

[0114] 8.6 Gray Value Statistics and Result Analysis

[0115] Use Adobe Photoshop software to obtain the gray values of the target protein and the internal reference for calculating the relative expression level of the target protein.

[0116] 9. Immunofluorescence Staining

[0117] The cardiomyocytes extracted in step 1 are placed in a confocal dish for culture, and the cells are processed accordingly according to step 2. Use a pipette to remove the cardiomyocyte medium in the dish, and wash the culture dish with phosphate buffer to avoid residue. Add 1 mL of 4% paraformaldehyde to each dish and fix at room temperature for 20 minutes, then wash the culture dish 2 times with phosphate buffer. Add 1 mL of immunostaining permeabilization solution (Triton X-100) to each confocal dish and incubate at room temperature for 10 minutes. After that, wash the culture dish 2 times with phosphate buffer. Prepare a 2% BSA solution with phosphate buffer (0.2 g of BSA powder is fully dissolved in phosphate buffer and made up to 10 mL) and incubate at room temperature for 1 hour for blocking. After blocking, wash the culture dish 2 times with phosphate buffer. Prepare a 1% BSA solution (0.1 g of BSA powder is fully dissolved in phosphate buffer and made up to 10 mL) to dilute the primary antibody (Anti-sarcomericalpha Actinin antibody, Abcam ab137346). If the cells only need to be labeled with a single antibody except for DAPI, dilute it with 1% BSA solution according to the dilution ratio recommended in the primary antibody instruction manual and incubate overnight at 4°C. The next day, first wash the culture dish 1 time with phosphate buffer, then wash the culture dish 3 times with 0.05% Triton X-100, and finally wash the culture dish 1 time with phosphate buffer. Select a suitable secondary antibody (Goat Anti-Rabbit IgG H&L(Alexa 647), Abcam ab150079) according to the species of the primary antibody. Dilute it with phosphate buffer at the concentration shown in the instruction manual and incubate in the dark at room temperature for 30 minutes. Dissolve the DAPI powder with deionized water, the stock solution concentration is 5 mg / mL, and store it in aliquots at -20°C in the dark. When preparing the DAPI working solution, thaw the frozen stock solution at room temperature until fully melted and warmed up, dilute it to 5 μg / mL with phosphate buffer at a ratio of 1:1000, mix well and keep it on ice in the dark for later use. After the secondary antibody incubation is completed, wash the culture dish 1 time with phosphate buffer, add the DAPI working solution to the culture dish, stain in the dark at room temperature for 5 minutes, and wash the culture dish 2 times with phosphate buffer. The culture dish can be stored in the dark in a 4°C refrigerator for short-term storage, and then observed and photographed with a laser confocal microscope, and the cell sizes between samples are calculated using ImageJ.

[0118] 10. Statistical analysis

[0119] All data were analyzed using GraphPad Prism 9.5.0, and the results are expressed as mean ± standard deviation. The t-test was used for data comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison among multiple groups of data. A p value < 0.05 was considered statistically significant.

[0120] II. Results and Analysis

[0121] The experimental results in the examples are as follows:

[0122] 1. The expression of MYLK4 decreases during myocardial hypertrophy

[0123] PE is a commonly used drug to induce a myocardial hypertrophy cell model. After treating cardiomyocytes with 200 μmol / L of PE, the expression of cardiomyocyte hypertrophy markers ANP, BNP, and β-MHC was verified by qPCR ( Figure 1 A). Under the induction of 200 μmol / L of PE, we also verified the protein expression of cardiomyocyte hypertrophy markers BNP and β-MHC by WB ( Figure 1 B, C, D). The qPCR results showed that the expression of MYLK4 decreased in cardiomyocytes ( Figure 1 E). Then we detected the protein level of MYLK4 in hypertrophic cardiomyocytes and found that the protein expression of MYLK4 was also decreased in hypertrophic cardiomyocytes ( Figure 1 F, G).

[0124] 2. MYLK4 can participate in regulating the expression of IGFBPL1

[0125] The cardiomyocytes were divided into 4 groups: LV-NC group, LV-NC + PE group, LV-MYLK4 group, and LV-MYLK4 + PE group. By qPCR and WB, it was found that the mRNA and protein expression of IGFBPL1 increased during PE induction, and the increased gene expression could be reduced after overexpressing MYLK4 ( Figure 2 A, B, C, D).

[0126] 3. Overexpressing MYLK4 or knocking down IGFBPL1 alleviates phenylephrine-induced neonatal mouse cardiomyocyte hypertrophy

[0127] The cardiomyocytes were divided into 4 groups: LV-NC group, LV-MYLK4 group, LV-NC + PE group, and LV-MYLK4 + PE group. Western blot analysis found that overexpressing MYLK4 could reduce the expression of hypertrophy markers β-MHC and BNP ( Figure 3 A, B, C, D), and immunofluorescence staining further confirmed that overexpressing MYLK4 could significantly reduce the surface area of cardiomyocytes ( Figure 3 E, F). To further prove the effect of IGFBPL1 on myocardial hypertrophy, the cardiomyocytes were divided into four groups: siNC group, siNC + PE group, siIGFBPL1 group, and siIGFBPL1 + PE group. The inhibitory effect of the siRNA sequence on IGFBPL1 was detected by qPCR ( Figure 3G). Western blot analysis demonstrated that knockdown of IGFBPL1 decreased the expression of the hypertrophy markers β-MHC and BNP ( Figure 3 H, I, J, K), and immunofluorescence staining further confirmed that knockdown of IGFBPL1 significantly reduced the surface area of cardiomyocytes ( Figure 3 L, M).

[0128] 4. In a mouse model of pressure overload-induced myocardial hypertrophy, MYLK4 expression was decreased while IGFBPL1 expression was increased. Overexpression of MYLK4 reduced the expression level of IGFBPL1 in the hearts of mice with myocardial hypertrophy.

[0129] Consistent with the results in the cell model, the expression of MYLK4 in the heart tissues of mice in the TAC group and the TAC+NC group was significantly lower than that in the control group, while the expression of IGFBPL1 was significantly increased, and there was no significant difference in the expression of these two genes between the TAC and TAC+NC groups, excluding the influence of the virus itself on MYLK4 and IGFBPL1. After injection of the MYLK4 overexpression virus, the expression of MYLK4 in the myocardial tissues of mice was significantly increased, and the expression of IGFBPL1 was inhibited, but this inhibitory effect could be relieved after overexpression of IGFBPL1. The above results further confirmed the regulatory effect of MYLK4 on IGFBPL1. In addition, tail vein injection of the IGFBPL1 knockdown virus significantly reduced the increase in IGFBPL1 protein level induced by TAC ( Figure 4 A, B, C).

[0130] 5. In a mouse model of pressure overload-induced myocardial hypertrophy, overexpression of MYLK4 or knockdown of IGFBPL1 significantly alleviated the pathological changes of myocardial hypertrophy induced by TAC surgery. Re-overexpression of IGFBPL1 after overexpression of MYLK4 significantly aggravated the pathological changes of myocardial hypertrophy.

[0131] Echocardiography results showed that LVPWd and LVPWs in mice in the TAC group and the TAC+NC group were significantly increased, while they were significantly decreased after overexpression of MYLK4 or knockdown of IGFBPL1, which confirmed the regulatory effects of MYLK4 and IGFBPL1 on myocardial hypertrophy. At the same time, the two indexes in the TAC+MYLK4+IGFBPL1 group were significantly increased compared with those in the TAC+MYLK4 group ( Figure 5 A, B). There were no significant differences in LVEF and FS among the mice in each group, indicating normal cardiac function of the mice ( Figure 5 C, D). In addition to the echocardiography results, the heart / body weight ratio of mice in the TAC group and the TAC+NC group was significantly increased, while it returned to normal after overexpression of MYLK4 or knockdown of IGFBPL1, and the heart / body weight ratio of mice increased again after overexpression of IGFBPL1 following overexpression of MYLK4 ( Figure 5E). These results indicate that MYLK4 exerts its regulatory function through IGFBPL1.

[0132] Meanwhile, HE staining confirmed that the size of the mouse heart increased after TAC surgery. When MYLK4 was overexpressed or IGFBPL1 was knocked down after TAC surgery, the heart size returned to normal. When MYLK4 was overexpressed and IGFBPL1 was overexpressed simultaneously, the heart size increased again ( Figure 5 F). WGA staining showed that the cross-sectional area of mouse cardiomyocytes increased after TAC surgery. The cross-sectional area decreased when MYLK4 was overexpressed or IGFBPL1 was knocked down, and increased again in the TAC + MYLK4 + IGFBPL1 group ( Figure 5 F, G). The above results further confirmed that MYLK4 can affect myocardial hypertrophy in mice through IGFBPL1.

[0133] Obviously, the above-mentioned embodiments of the present invention are merely examples for more clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation methods here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. Use of IGFBPL1 and its regulatory factors in the preparation of drugs for treating myocardial hypertrophy.

2. The application according to claim 1, characterized in that The increase in the expression of IGFBPL1 mRNA and protein in phenylephrine-induced hypertrophic cardiomyocytes can be reduced after overexpression of MYLK4.

3. The application according to claim 1, characterized in that, Overexpression of MYLK4 or knockdown of IGFBPL1 can alleviate phenylephrine-induced cardiomyocyte hypertrophy.

4. The application according to claim 3, wherein Overexpression of MYLK4 or knockdown of IGFBPL1 can reduce the expression of hypertrophy markers β-MHC and BNP and decrease the surface area of cardiomyocytes.

5. The application according to claim 1, wherein The increase in the expression of IGFBPL1 in the hearts with myocardial hypertrophy induced by pressure load can be reduced after overexpression of MYLK4.

6. The application according to claim 1, characterized in that, Overexpression of MYLK4 or knockdown of IGFBPL1 can significantly alleviate the pathological changes of myocardial hypertrophy induced by pressure load.

7. The application according to claim 5 or 6, characterized in that, The pressure load includes pressure overload induced by TAC surgery.

8. The application according to claim 6, characterized in that, The pathological changes of myocardial hypertrophy include at least one of the increase in LVPWd and LVPWs, the increase in heart / body weight ratio, heart enlargement, and the increase in the cross-sectional area of cardiomyocytes.

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