Medical application of DNAJB6 in prevention or treatment of myocardial infarction

Abstract

The invention belongs to the field of biological medicines, and particularly discloses application of DNAJB6 in preparation of medicines for preventing or treating myocardial infarction (MI). In-vivo and in-vitro experiments find that the expression of DNAJB6 protein in an MI model is remarkably reduced, and the over-expression of DNAJB6 can improve the cardiac function of a mouse after MI, relieve myocardial fibrosis and inhibit hypoxia-induced myocardial apoptosis; and cell apoptosis is aggravated by low expression of DNAJB6. The overexpression of the DNAJB6 by using the myocardial specific adeno-associated virus (AAV) can be used as a new strategy for preventing and treating the MI, and an experimental basis is provided for developing a drug for targeting the DNAJB6.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the medical use of DNAJB6 in the prevention or treatment of myocardial infarction. Background Art

[0002] Myocardial infarction (MI), as the leading cause of death and disability from cardiovascular disease worldwide, has long been a focus of medical research. MI is usually caused by thrombosis in the coronary arteries, leading to complete or partial blockage of the arteries, resulting in a sudden decrease in myocardial blood flow, leading to heart failure and death. Although advanced methods such as thrombolysis, percutaneous coronary intervention, and adjuvant drug therapy have been widely used to treat MI, the prognosis of MI patients remains poor. The latest data on the diagnosis and treatment of heart failure in China show that the number of hospitalized patients with heart failure in 2023 increased by 38.9% compared with 2022, from 10.29 million cases to 14.29 million cases. Therefore, finding effective intervention and treatment targets has important theoretical and clinical significance for preventing the occurrence of myocardial infarction and improving prognosis.

[0003] MI is often accompanied by myocardial cell damage and death, a process closely related to myocardial cell apoptosis. Apoptosis is a highly regulated, programmed cell death form that plays a vital role in physiological processes such as the body's development, immune surveillance, and cell renewal. Studies have shown that apoptosis after MI mainly occurs in damaged myocardial cells, especially in the infarct edge zone. However, apoptosis after MI is not only limited to the infarct area but can also extend to non-infarct areas. In addition, an increase in the number of apoptotic myocardial cells is closely related to worsening cardiac function and heart failure. Apoptosis not only leads to a loss of myocardial cell number, but also affects the later repair and remodeling of myocardial tissue by promoting local inflammatory responses and changing the composition of the extracellular matrix.

[0004] DNAJB6 is a member of the Hsp40 family. The Hsp40 family can be divided into three major types (I, II, and III), all of which share a J domain of approximately 70 amino acids. DNAJB6 is involved in a variety of physiological processes, from transcription and cell signaling to cell adhesion. DNAJB6 knockout mice display embryonic lethality, in part due to placental abnormalities and neural tube defects. DNAJB6's inhibitory effect on Wnt-β-catenin signaling negatively regulates tumor growth and metastasis. Furthermore, DNAJB6 is also essential for cell growth, division, and migration. Studies have shown that DNAJB6 inhibits the transcriptional activity of nuclear factor of activated T cells by recruiting class II histone deacetylases, thereby reducing calcineurin-induced cardiomyocyte growth. This observation suggests that DNAJB6 plays a role in preventing cardiac hypertrophy. Recent studies on DNAJB6 have shown that DNAJB6 deficiency leads to mitochondrial defects, suggesting that DNAJB6 plays an important role in mitochondrial regulation. In summary, DNAJB6 plays a crucial role in biological growth and development, but its role in myocardial ischemia has not been reported. Therefore, the present invention aims to provide a method for inducing DNAJB6 expression or promoting its active fragment for the medical use in preventing or treating MI. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention aims to provide a medical use of DNAJB6 in the prevention or treatment of myocardial infarction, specifically a medical use of an adeno-associated virus overexpressing DNAJB6 for the prevention or treatment of MI.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The present invention discloses use of DNAJB6 protein or its active fragment in preparing medicine for preventing or treating myocardial infarction.

[0008] Furthermore, the drug achieves a therapeutic effect by upregulating the expression of DNAJB6 or enhancing its activity.

[0009] Furthermore, the drug comprises a nucleic acid sequence encoding DNAJB6.

[0010] Furthermore, the nucleic acid sequence is delivered via an adeno-associated virus (AAV) vector.

[0011] The present invention also discloses a pharmaceutical composition for preventing or treating myocardial infarction, comprising an effective amount of DNAJB6 protein or an active fragment thereof, and a pharmaceutically acceptable carrier.

[0012] Furthermore, the dosage form of the pharmaceutical composition includes a pharmaceutically acceptable dosage form.

[0013] The present invention also discloses the use of a detection reagent for the expression level of the DNAJB6 gene or its encoded protein in the preparation of an auxiliary diagnosis product for myocardial infarction.

[0014] Furthermore, the product contains specific primers for amplifying the DNAJB6 gene and a probe for hybridizing with the nucleotide sequence of the DNAJB6 gene.

[0015] Furthermore, the specific primer sequences for amplifying the DNAJB6 gene are shown in SEQ ID NOs. 1 to 4.

[0016] Compared with the prior art, the present invention has the following beneficial effects.

[0017] Through a large number of experiments, the present invention found that DNAJB6 protein expression was significantly decreased in the MI model. A mouse myocardial-specific DNAJB6 overexpression or underexpression model was constructed by tail vein injection of AAV adeno-associated virus, and it was found that overexpression of DNAJB6 in mice significantly improved cardiac function after MI. In in vitro cytology, when H9C2 / NRCMs were given hypoxia stimulation, DNAJB6 protein expression was significantly decreased. Low expression of DNAJB6 promoted hypoxia-induced cardiomyocyte apoptosis; conversely, overexpression of DNAJB6 could improve hypoxia-induced cardiomyocyte apoptosis. The above results indicate that DNAJB6 plays an important regulatory role in the occurrence and development of MI and may be a new target for the prevention or treatment of MI. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 4 shows changes in DNAJB6 expression in ischemic myocardial tissue and hypoxic cardiomyocytes in mice after myocardial infarction. Figures AB show the results of western blot analysis of DNAJB6 protein expression in myocardial tissue and their statistical graphs; Figure C shows the results of RT-PCR analysis of DNAJB6 mRNA expression in myocardial tissue; Figures DE show the results of western blot analysis of DNAJB6 protein expression in cardiomyocytes and their statistical graphs. Figure F shows the results of RT-PCR analysis of DNAJB6 mRNA expression in cardiomyocytes; (*** p <0.001; n = 3).

[0019] Figure 2 This study constructs a mouse model of myocardial-specific DNAJB6 overexpression. Figure A is a schematic diagram of constructing the AAV2-cTNT-DNAJB6 adeno-associated virus; Figures BC are the results of western blot analysis of DNAJB6 protein expression in myocardial tissue and their statistical graphs; Figure D is RT-PCR analysis of DNAJB6 mRNA expression in myocardial tissue; (*** p <0.001; n = 3).

[0020] Figure 3 DNAJB6 overexpression improves cardiac function in mice after myocardial infarction. Figure A is a schematic diagram of the mouse myocardial infarction model; Figure B shows ultrasound images of the heart, cardiac function, and heart-to-body weight ratio of mice overexpressing DNAJB6 28 days after establishing a myocardial infarction model (***P < 0.001 vs. AAV2-cTNT-NC group; ##P < 0.01 vs. AAV2-cTNT-NC+MI group; n = 5-18).

[0021] Figure 4 DNAJB6 overexpression ameliorates myocardial fibrosis after myocardial infarction. Panel A shows H&E, Masson's, and WGA staining results of AAV2-cTNT-NC and AAV2-cTNT-GNG2 mice 28 days after myocardial infarction (MI). Panel B shows the statistical results of MI area in Panel A. Panel C shows the statistical results of myocardial fibrosis area in Panel A. Panel D shows the statistical results of WGA staining in Panel A. (***P < 0.001 vs. AAV2-cTNT-NC group; ###P < 0.001 vs. AAV2-cTNT-NC + MI group; n = 3).

[0022] Figure 5 DNAJB6 overexpression inhibits cardiomyocyte apoptosis under hypoxia. Panel A shows the effect of DNAJB6 knockdown on the expression levels of cleaved caspase-3 and Bcl-2, markers of hypoxia-induced cardiomyocyte apoptosis, as assessed by Western blotting. Panels BC show the quantitative grayscale analysis of the Western blotting bands in panel A. Panels DE show the effect of DNAJB6 overexpression on hypoxia-induced cardiomyocyte apoptosis as assessed by TUNEL staining (***P < 0.01 vs. adnc group; #P < 0.05, ##P < 0.01 vs. adnc + Cocl-2 group; n = 3).

[0023] Figure 6 DNAJB6 knockdown exacerbates cardiomyocyte apoptosis under hypoxia. Panel A shows the effect of DNAJB6 knockdown on the expression levels of cleaved caspase-3 and Bcl-2, markers of hypoxia-induced cardiomyocyte apoptosis, as assessed by Western blotting. Panels B and D show the quantitative grayscale analysis of the Western blotting bands in panel A. Panels E and F show the effect of DNAJB6 overexpression on hypoxia-induced cardiomyocyte apoptosis as assessed by TUNEL staining (*P < 0.05, **P < 0.01, ***P < 0.001 vs. sinc group; #P < 0.05, ##P < 0.01 vs. sinc + CoCl-2 group; n = 3). DETAILED DESCRIPTION

[0024] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the present invention. Where specific conditions are not specified in the examples, the results were performed under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments used is not specified, they are all commercially available conventional products. The present invention was processed using the GraphPad Prism 8.0 software package, and statistical significance was considered to be p < 0.05.

[0025] Example 1: Changes in DNAJB6 expression in ischemic myocardial tissue and hypoxic myocardial cells in mice after myocardial infarction.

[0026] 1. Experimental animals and their husbandry.

[0027] Animal species, sex, age, and source: Male C57BL / 6J mice, 8 weeks old. C57BL / 6J mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Mice were housed in a specific pathogen-free (SPF) animal room at a room temperature of (22 ± 2)°C, a humidity range of 45%–70%, and a 12-h light cycle with free access to food and water.

[0028] 2. Establishment of MI model in C57BL / 6J mice.

[0029] C57BL / 6J mice were randomly divided into a control group and an experimental group, with 8 mice in each group. After anesthesia, the mice were depilated and skin prepared, then secured in the supine position on an operating table. A cannula was placed through the mouth of the mice and connected to a ventilator. Successful intubation was assessed by chest movement and ventilator frequency. After disinfection with iodine, the skin was incised along the left axilla to the xiphoid process of the sternum, and the pectoralis major and minor muscles were bluntly dissected. A 0.5 cm incision was made along the left third and fourth intercostal spaces at the site of the most pronounced cardiac pulsation. The pericardium and associated connective tissue were carefully dissected to reveal the major vessels on the cardiac surface. The left anterior descending coronary artery was ligated with 6-0 surgical sutures. Successful ligation was observed by gradual pallor of the anterior ventricular wall. After pleural effusion was expelled by squeezing, the chest wall and skin at the wound site were sutured with 4-0 sutures. The mice were connected to a small animal electrocardiograph. Successful modeling was confirmed by ST-segment elevation exceeding 50%. DNAJB6 expression levels were measured in the MI and sham groups.

[0030] 3. Establishment of rat primary cardiomyocyte and H9C2 cardiomyocyte hypoxia models.

[0031] Primary rat cardiomyocytes and H9C2 cardiomyocytes were cultured in a 37°C, 5% CO2 cell culture incubator and subjected to hypoxia or the addition of 600µM CoCl2. The cells were cultured for 24 hours before use in the experiment.

[0032] 4. Fluorescence real-time quantitative PCR (Real-time PCR) method was used to detect the expression of DNAJB6 in cardiac tissues and cells of MI model.

[0033] 1) RNA extraction from cardiac tissue.

[0034] A. Mouse heart tissue and centrifuged cardiomyocytes were placed in an RNase-free EP tube. 1 mL of Trizol was added, and three RNase-free grinding beads were added. The tube was then ground in a tissue grinder homogenizer for 120 s at 70 Hz.

[0035] B. Place at room temperature for 5 minutes, add 1 / 5 of chloroform, then mix it upside down and let it stand for 15 minutes.

[0036] C. Centrifuge at 10,000 rpm, 4°C for 10 min.

[0037] D. Aspirate the supernatant, add an equal volume of isopropanol, invert and mix thoroughly, and let stand at room temperature for 10 minutes.

[0038] E. Centrifuge at 10,000 rpm, 4°C for 10 min and discard the supernatant.

[0039] F. Add 1 mL of 75% ethanol and gently shake the tube to resuspend the precipitate.

[0040] G. Centrifuge at 10,000 rpm, 4°C for 10 min and discard the supernatant.

[0041] H. Allow to dry at room temperature until transparent.

[0042] I. Add 25 μL of enzyme-free water to dissolve RNA.

[0043] 2) Reverse transcription of RNA to cDNA: Use the Takara reverse transcription kit and perform the experiment according to the kit instructions.

[0044] 3) Primer sequence .

[0045] 4) Fluorescence quantitative PCR: Follow the instructions.

[0046] The results showed that compared with the sham group, Dnajb6 Compared with the Control group, the transcription level of myocardial cells in the hypoxia group was increased. Dnajb6 Increased transcription levels ( Figure 1 C, F).

[0047] 4.Western blot method was used to detect the expression of DNAJB6 in mouse heart tissue and cardiomyocytes.

[0048] To clarify the role of DNAJB6 in ischemic and hypoxic myocardium, we used Western blot to examine DNAJB6 expression in cardiac tissue and cardiomyocytes. Samples were added to an appropriate amount of protein lysis buffer and lysed on ice for 30 minutes, mixing by inversion every 5 minutes. The cells were centrifuged at 12,000 g for 20 minutes at 4°C, and the supernatant was collected as total protein. Protein concentration was determined using a BCA colorimetric assay kit. 20 μg of protein sample was added to each well, and the power supply was turned on and electrophoresis was initiated. The voltage and time were as follows: 100 V for 30 minutes, 120 V for 60 minutes, and the power supply was turned off after bromophenol blue reached the bottom of the glass plate. The samples were then transferred to a PVDF membrane at 90 V for 2 hours. The membrane was placed in 5% milk blocking solution for 1 hour, followed by incubation with the primary antibody overnight at 4°C. DNAJB6 (Wuhan Tri-Tac Co., Ltd.) and GAPDH (Wuhan Tri-Tac Co., Ltd.) were used as primary antibodies, respectively, and horseradish peroxidase-conjugated goat anti-mouse (or anti-rabbit) antibodies (Cell Signaling Technology) were used as secondary antibodies. ECL kit (Amersham) was used for luminescence imaging. Grayscale values of the bands were measured and statistically analyzed using ImageJ 1.51 software.

[0049] The results showed that compared with the sham group, the expression of DNAJB6 protein in the heart tissue of the MI group mice was significantly decreased; compared with the control group, the expression of DNAJB6 protein in the myocardial cells of the hypoxia group was significantly decreased ( Figure 1 AB, DE).

[0050] The above results suggest that DNAJB6 may be involved in the occurrence and development of MI.

[0051] Example 2: Myocardial-specific DNAJB6 overexpression mice were successfully constructed and cardiac function was detected.

[0052] 1. Construct and characterize DNAJB6-overexpressing mice.

[0053] Adeno-associated virus (AAV2-cTNT-DNAJB6) expressing cardiomyocyte-specific overexpression of DNAJB6 and its negative control AAV2-cTnT-NC were constructed by Heyuan Biotechnology Co., Ltd. Cardiomyocyte-specific overexpression of DNAJB6 was established in vivo by tail vein injection.

[0054] 2. Establishment of MI model in mice overexpressing DNAJB6.

[0055] Three weeks after tail vein injection of AAV2-cTNT-DNAJB6 and its negative control virus, DNAJB6 overexpression was successfully detected by western blot and RT-PCR ( Figure 2 ). A myocardial infarction model was constructed, and the specific experimental method was the same as in Example 1.

[0056] 3.Ultrasound imaging of small animals to detect cardiac function.

[0057] Before ultrasound examination, mice were depilatory and lightly anesthetized with isoflurane gas inhalation, maintaining a heart rate of 450–550 beats / min. Using a Vevo 2100 small animal ultrasound system, coupling gel was applied to the chest and upper abdomen of the mice. M-mode echocardiographic recordings were obtained over 10 cardiac cycles using a two-dimensional probe. Two-dimensional images of the left ventricle were acquired in the left ventricular short-axis view, and images were retained for measurement of various cardiac function indicators.

[0058] The results showed that compared with the AAV2-cTNT-NC group, the EF (%) and FS (%) values of the AAV2-cTNT-NC+MI group were significantly decreased, and the LVIDd and LVIDs values were increased. Compared with the AAV2-cTNT-NC+MI group, the cardiac function indicators of the AAV2-cTNT-DNAJB6+MI group were significantly improved. In addition, compared with the AAV2-cTNT-NC+MI group, the HW / BW value of the AAV2-cTNT-DNAJB6+MI group showed a decrease. Figure 3 ).

[0059] 4. H&E and MASSON staining were used to detect the gross morphology and myocardial fibrosis of the heart.

[0060] Heart tissues were collected from AAV2-cTnT-NC, AAV2-cTnT-NC+MI, AAV2-cTnT-DNAJB6, and AAV2-cTnT-DNAJB6+MI groups 28 days after the establishment of the myocardial infarction model, and subjected to H&E staining and MASSON staining.

[0061] 5. WGA staining was used to detect the size of myocardial cells.

[0062] Heart tissues were collected from AAV2-cTnT-NC, AAV2-cTnT-NC+MI, AAV2-cTnT-DNAJB6, and AAV2-cTnT-DNAJB6+MI groups 28 days after the establishment of the myocardial infarction model and subjected to WGA staining.

[0063] The results showed that compared with the AAV2-cTNT-NC group, the hearts of mice in the AAV2-cTNT-NC+MI group were significantly larger and myocardial fibrosis was significantly aggravated; while the heart size and degree of fibrosis in the AAV2-cTNT-DNAJB6+MI group were significantly smaller than those in the AAV2-cTNT-NC+MI group. At the same time, WGA staining showed that overexpression of DNAJB6 significantly improved the increase in the cross-sectional area of ischemic myocardial cells induced by MI ( Figure 4 These results suggest that DNAJB6 overexpression can significantly improve MI-induced ischemic myocardial fibrosis.

[0064] Example 3: Overexpression of DNAJB6 inhibits cardiomyocyte apoptosis after hypoxia.

[0065] 1. Overexpression of DNAJB6 in H9C2 cardiomyocytes and establishment of hypoxia model.

[0066] To overexpress GNG2 in H9C2 cardiomyocytes, cells were plated in six-well plates. When the cells reached 70%–80% confluence, plasmids containing the DNAJB6 overexpression vector and a control vector were added to the H9C2 cells. The cells were cultured in a 37°C, 5% CO2 incubator for 6–8 hours. Afterwards, 600 µM CoCl2 was added and the cells were cultured for an additional 24 hours before use in experiments. Plasmid construction was provided by Heyuan Biotechnology (Shanghai) Co., Ltd.

[0067] 2. Western blot was used to detect the effect of DNAJB6 overexpression on cardiomyocyte apoptosis after hypoxia. The apoptosis marker proteins were cleaved caspase 3 (Abcam), Bax (Proteintech), and Bcl2 (Abcam). The specific method was the same as in Example 1.

[0068] The results showed that compared with the control group, the apoptosis level of cardiomyocytes in the adnc+CoCl2 group was significantly increased, while compared with the adnc+CoCl2 group, the adDNAJB6+CoCl2 group significantly improved the hypoxia-induced apoptosis of cardiomyocytes, mainly manifested by a decrease in cleaved caspase3 and an increase in Bcl2 ( Figure 5 AC).

[0069] 3. TUNEL staining was used to detect the effect of GNG2 overexpression on apoptosis in H9C2 cardiomyocytes. The experiment was performed according to the instructions of the apoptosis kit.

[0070] The results showed that the TUNEL staining results were consistent with the Western blotting results, both demonstrating that DNAJB6 overexpression improved hypoxia-induced cardiomyocyte apoptosis ( Figure 5 DE).

[0071] Example 4: DNAJB6 knockdown promotes cardiomyocyte apoptosis after hypoxia.

[0072] 1. Establishment of low expression of DNAJB6 and hypoxia model in H9C2 cardiomyocytes.

[0073] To establish cardiomyocytes with low DNAJB6 expression, cells were plated in six-well plates and transfected with siRNA when the cells reached 70%-80% confluence. 0.5 mL of Opti-MEM medium was added to each well, and siNC and siDNAJB6 were transfected into rat H9C2 cardiomyocytes using lipofectamine 2000 transfection reagent. The cells were cultured in a 37°C, 5% CO2 incubator for 6-8 hours. The Opti-MEM medium in the six-well plates was then replaced with DMEM supplemented with 10% fetal bovine serum and 1% antibiotics, and 600 µM CoCl2 was added. The cells were cultured for an additional 24 hours before use in experiments.

[0074] 2. Western blot was used to detect the effect of low expression of DNAJB6 on myocardial cell apoptosis indicators after hypoxia.

[0075] The results showed that compared with the sinc group, the apoptosis level of cardiomyocytes in the sinc+CoCl2 group was increased, and compared with the sinc+CoCl2 group, the apoptosis level in the siDNAJB6+CoCl2 group was more significantly upregulated, mainly manifested by increased Cleavedcaspase3, increased Bax, and decreased Bcl2 ( Figure 6 AD).

[0076] 3. TUNEL staining was used to detect the effect of low expression of DNAJB6 on myocardial cell apoptosis.

[0077] The results showed that the TUNEL staining results were consistent with the Western blotting results, both demonstrating that DNAJB6 knockdown aggravated hypoxia-induced cardiomyocyte apoptosis ( Figure 6 EF).

[0078] In summary, experiments have demonstrated a significant decrease in DNAJB6 expression in the MI model. A cardiomyocyte-specific overexpression mouse model, constructed using an adeno-associated virus (AAV2-cTNT-DNAJB6), has been shown to improve cardiac function and reduce myocardial fibrosis and apoptosis after MI. In vitro experiments further demonstrated that DNAJB6 overexpression inhibited hypoxia-induced cardiomyocyte apoptosis (e.g., decreased cleaved caspase-3 and increased Bcl-2), whereas knockdown of DNAJB6 exacerbated apoptosis. The present invention provides the use of DNAJB6 or its active fragments in the preparation of MI therapeutics, particularly AAV-based gene therapy.

[0079] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of DNAJB6 protein or its active fragment in the preparation of a drug for preventing or treating myocardial infarction.

2. The use according to claim 1, characterized in that The drug achieves therapeutic effects by upregulating the expression of DNAJB6 or enhancing its activity.

3. The use according to claim 1, characterized in that The drug comprises a nucleic acid sequence encoding DNAJB6.

4. The use according to claim 3, characterized in that The nucleic acid sequence is delivered via an adeno-associated virus (AAV) vector.

5. A pharmaceutical composition for preventing or treating myocardial infarction, comprising an effective amount of DNAJB6 protein or an active fragment thereof, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, wherein The dosage form of the pharmaceutical composition includes pharmaceutically acceptable dosage forms.

7. Use of a detection reagent for the expression level of the DNAJB6 gene or its encoded protein in the preparation of an auxiliary diagnosis product for myocardial infarction.

8. The use according to claim 7, characterized in that The product contains specific primers for amplifying DNAJB6 gene and probes for hybridizing with the nucleotide sequence of DNAJB6 gene.

9. The use according to claim 8, characterized in that The specific primer sequences for amplifying the DNAJB6 gene are shown in SEQ ID NOs. 1 to 4.

Images