Medical use of hspd1 acetylation modification in prevention or treatment of myocardial infarction

Abstract

The application relates to the technical field of biological medicine, and discloses medical uses of HSPD1 acetylation modification in prevention or treatment of myocardial infarction. The application first finds that the acetylation level of an HSPD1 protein K352 site is specifically up-regulated in the pathological process of myocardial infarction, and that the heart function after myocardial infarction can be significantly improved by up-regulating the acetylation level of the site, and myocardial fibrosis and myocardial cell hypertrophy can be inhibited; the acetylation level of the site is down-regulated, and myocardial ischemic injury is aggravated, and the heart function is deteriorated. The application confirms that the HSPD1 K352 site acetylation can be used as a new target point for prevention, treatment and auxiliary diagnosis of myocardial infarction, and provides a theoretical basis and experimental basis for research and development of myocardial infarction targeted drugs and gene therapy products.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the pharmaceutical use of HSPD1 acetylation modification in the prevention or treatment of myocardial infarction (MI). Background Technology

[0002] Acute myocardial infarction (AMI) is a major threat to human health and life due to its high incidence and mortality rates. According to statistics, approximately 17.9 million people died from cardiovascular disease globally in 2019, of which about 7 million died from AMI. AMI is a leading cause of morbidity and mortality worldwide. Although the incidence of AMI has declined in developed countries, it continues to rise in developing countries. The rising incidence of AMI in developing countries is partly due to the prevalence of certain lifestyles and dietary habits. In many low- and middle-income countries, insufficient medical resources contribute to the high mortality rate of AMI. In my country, AMI is an even more serious public health problem. According to the China Cardiovascular Epidemiological Survey, approximately 2.4 million people die from coronary heart disease each year, accounting for 61% of cardiovascular deaths and 25% of all-cause deaths. Furthermore, the mortality rate within 30 days of a first-time AMI patient is as high as 28%, and more than 10% of patients die from a subsequent AMI or heart failure within the following year. The incidence of AMI has risen sharply, from 8.86 cases per 100,000 people in 2007 to 12.79 cases per 100,000 people in 2018.

[0003] Myocardial infarction (MI) is often accompanied by cardiomyocyte damage and death, a process closely related to cardiomyocyte apoptosis. Apoptosis is a highly regulated form of programmed cell death that plays a crucial role in physiological processes such as development, immune surveillance, and cell renewal. Studies have shown that apoptosis after MI mainly occurs in damaged cardiomyocytes, especially in the infarct border zone. However, apoptosis after MI is not limited to the infarcted area but can extend to non-infarcted areas. Furthermore, an increase in the number of apoptotic cardiomyocytes is closely associated with deterioration of cardiac function and heart failure. Apoptosis not only leads to a loss of cardiomyocytes but also affects the later repair and remodeling of myocardial tissue by promoting local inflammatory responses and altering the composition of the extracellular matrix.

[0004] HSPD1 is a 60 kDa protein, also known as HSP60, encoded by the nuclear gene HSPD1 on human chromosome 2. It is transcribed in the nucleus, translated in the cytoplasm, and introduced into the mitochondria as a unfolded polypeptide. Highly conserved, HSPD1 is a large protein complex composed of 14 subunits, forming a cylindrical structure. Each subunit consists of three distinct domains. Each subunit of the HSPD1 protein contains a large domain, also known as the α / β domain. This domain has a typical α-helix and β-sheet structure and forms a globular shell around the complex. The large domain plays a crucial role in the stability and function of the HSPD1 protein complex. The large domain of the HSPD1 protein contains an ATP-binding site. Located at the center of the large domain, the ATP-binding site binds to ATP, thereby regulating the conformation and function of the complex.

[0005] In cells, 70% of HSPD1 is distributed in mitochondria, ensuring the correct folding of proteins transcribed from nuclear genes after they are introduced into the mitochondria. The remaining HSPD1 is distributed in the cytoplasm and is associated with cardiomyocyte apoptosis. However, under certain stress conditions, HSPD1 proteins released extracellularly or redistributed to the cell membrane can become self-antigens, inducing immune responses and promoting disease development. In the heart, intracellular HSPD1 has a protective effect on cardiomyocytes. In a study of myocardial-specific HSPD1 deficiency in mice, it was found that the absence of HSPD1 in adult mouse cardiomyocytes altered the activity of the mitochondrial complex, reduced mitochondrial membrane potential, and promoted ROS generation, ultimately leading to myocardial dysfunction. Under no other precipitating conditions, this can cause cardiac enlargement and heart failure, ultimately resulting in death. Therefore, HSPD1 plays an important role in maintaining normal heart morphology and function by regulating mitochondrial protein homeostasis and mitochondrial function. However, the role of HSPD1 acetylation modification in myocardial injury remains unclear. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides the pharmaceutical use of HSPD1 acetylation levels in the prevention or treatment of MI. Specifically, it provides the pharmaceutical use of adeno-associated viruses with HSPD1 acetylation overexpression for the prevention or treatment of MI.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0008] This invention discloses the use of reagents that upregulate the acetylation level of HSPD1 protein in the preparation of medicaments for the prevention and / or treatment of MI.

[0009] Furthermore, the reagent that upregulates the acetylation level of HSPD1 protein is a reagent that specifically upregulates the acetylation level of the lysine residue at position 352 of the HSPD1 protein.

[0010] Preferably, the HSPD1 protein K352 site acetylation mimic mutant is the HSPD1-K352Q mutant, which is a mutant in which the wild-type HSPD1 protein has a lysine residue at position 352 mutated to glutamine.

[0011] Preferably, the nucleic acid molecule encoding the acetylation mimic mutant is delivered via an adeno-associated virus vector driven by a myocardial-specific promoter.

[0012] The present invention also discloses a pharmaceutical composition for the prevention and / or treatment of MI, characterized in that it comprises an effective amount of an HSPD1 protein K352 site acetylation upregulator and a pharmaceutically acceptable carrier.

[0013] Furthermore, the dosage form of the pharmaceutical composition is any one of injection, lyophilized powder for injection, or sustained-release formulation.

[0014] This invention also discloses the application of a reagent for detecting the acetylation level at the K352 site of the HSPD1 protein in the preparation of products for MI auxiliary diagnosis or prognostic assessment.

[0015] Furthermore, the reagent includes at least one of an antibody or its antigen-binding fragment that specifically recognizes the acetylation modification at the K352 site of the HSPD1 protein, a primer pair that specifically amplifies the HSPD1 gene, and a nucleic acid probe that specifically hybridizes to the nucleotide sequence of the HSPD1 gene.

[0016] Furthermore, the product is any one of the following: protein detection kit, real-time PCR detection kit, immunochromatographic test strip, and gene detection chip.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] This invention, for the first time, clarifies the core regulatory role of acetylation modification at the K352 site of HSPD1 protein in the occurrence and development of myocardial ischemia, filling a gap in existing technologies. Through in vitro and in vivo experiments, this invention demonstrates that under myocardial ischemia / hypoxia stress, the total expression of HSPD1 protein does not change significantly, and the core regulator of its cardioprotective function is acetylation modification at the K352 site. This clarifies the key regulatory mechanism of HSPD1 in myocardial ischemia injury, providing a novel direction for the study of the pathological mechanisms of MI.

[0019] This invention validates, through both positive and negative animal models, that upregulating the acetylation level at the HSPD1 K352 site has a clear preventive and therapeutic effect on myocardial infarction (MI). This invention constructs a mouse model overexpressing a myocardial-specific HSPD1-K352Q acetylation mutant, demonstrating that this mutant can significantly reverse the decrease in left ventricular ejection fraction (EF) and fractional shortening (FS) after MI, significantly improving cardiac function. Simultaneously, through a reverse validation using an HSPD1-K352R deacetylation mutant model, it is shown that downregulating the acetylation level at this site significantly exacerbates cardiac function damage after MI, and can directly induce myocardial fibrosis and cardiomyocyte hypertrophy without ischemic induction, providing sufficient and reliable experimental evidence for the pharmacological efficacy of this target.

[0020] This invention expands the clinical application scenarios of HSPD1 acetylation modification, and clarifies that the acetylation level of HSPD1 K352 site can serve as a novel molecular marker for the auxiliary diagnosis and disease monitoring of MI, providing a new detection target for the early screening and prognostic assessment of MI.

[0021] The targeted therapy based on HSPD1 K352 site acetylation provided by this invention uses an adeno-associated virus vector driven by a myocardial-specific promoter to achieve targeted regulation of the target. It has the advantages of strong targeting, clear efficacy and high myocardial specificity, and provides a mature technical solution for the development of gene therapy drugs for myocardial infarction (MI), with good clinical translational value. Attached Figure Description

[0022] Figure 1 It was confirmed that HSPD1 protein can undergo acetylation under physiological conditions, with the acetylation site being K352. Figure A shows the acetylation of the synthesized protein in 293T cells transfected with the HSPD1-eGFP-HA plasmid, as detected by COIP. Figure B shows the expression level of HSPD1 protein in myocardial tissue at different time points after myocardial infarction, as detected by Western blot. Figure C shows the acetylation site of HSPD1 in mouse myocardial tissue, detected by proteometry. Figure D shows the construction of the HSPD1-K352R mutant plasmid based on mass spectrometry results. Figure E shows the reduced acetylation level of the synthesized protein by the HSPD1-K352R plasmid, verified by COIP. Figure F shows the increased acetylation level of HSPD1-K352 at the K352 site in hypoxic-treated primary cardiomyocytes, as detected by Western blot. (n=6)

[0023] Figure 2This study describes the construction of mice with elevated HSPD1-K352 acetylation expression specific to the myocardium. Figure A is a schematic diagram of the construction of AAV2-cTNT-HSPD1-K352R adeno-associated virus; Figures BC show the results and statistical graphs of HSPD1-K352 acetylation levels in myocardial tissue detected by Western blot; Figure D is a statistical graph of cardiac function in mice 28 days after the establishment of a myocardial infarction model using small animal ultrasound detection. (n=6)

[0024] Figure 3 The study aimed to construct mice with reduced HSPD1-K352 acetylation specific to the myocardium. Figure A is a schematic diagram of the mouse myocardial infarction model; Figures B and D are ultrasound images and cardiac function statistics of the mice 28 days after the establishment of the myocardial infarction model by overexpressing HSPD1-K352R (n=5~26).

[0025] Figure 4 Decreased HSPD1-K352 acetylation directly leads to myocardial fibrosis in mice. Figures AB show the construction of mice injected via tail vein with AAV2-cTNT-NC and AAV2-cTNT-HSPD1-K352R, and the viral transfection efficiency was detected using Western blot. Figures CE and CE show statistical graphs of cardiac function tests in mice at different time points, as well as a schematic diagram of ultrasound. Figure FH shows the H&E, Masson, and WGA staining results and statistical graphs at 8 weeks after tail vein injection. (n=6) Detailed Implementation

[0026] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products. All data in this invention were processed using the GraphPad Prism 8.0 software package, with p < 0.05 considered statistically significant.

[0027] Example 1: Changes in HSPD1 expression in ischemic myocardial tissue and hypoxic cardiomyocytes in mice after myocardial infarction.

[0028] 1. Laboratory animals and their care.

[0029] Experimental animal species, sex, age, and origin: C57BL / 6J mice, male, 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 temperature of (22±2)℃ and a humidity of 45%~70%, with a 12-hour light cycle and free access to food and water.

[0030] 2. Establishment of the MI model in C57BL / 6J mice.

[0031] 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 shaved and skin-prepared, then fixed in a supine position on the operating table. A cannula was inserted through the mouse's mouth, and a ventilator was connected. The success of the cannulation was determined by the mouse's chest respiratory movement and the ventilator frequency. The skin was disinfected with iodine, and an incision was made along the left axilla towards the xiphoid process of the sternum. The pectoralis major and pectoralis minor muscles were bluntly dissected. An incision of approximately 0.5 cm was made along the left third and fourth intercostal spaces at the point where the heartbeat was most pronounced. The pericardium and associated connective tissue were carefully torn open, revealing the large blood vessels on the surface of the heart. The left anterior descending coronary artery was ligated using 6-0 surgical sutures. If the ligation was successful, the anterior ventricular wall would gradually turn pale. After expelling air from the pleural cavity, the chest wall and wound skin were sutured with 4-0 sutures. A small animal electrocardiograph was used to connect the mice post-surgery; ST segment elevation exceeding 50% indicated successful modeling. The expression levels of HSPD1 in the MI and sham groups were measured.

[0032] 3. Establishment of primary rat cardiomyocyte and H9C2 cardiomyocyte hypoxia models.

[0033] Primary rat cardiomyocytes and H9C2 cardiomyocytes were treated with sugar-free DMEM medium and cultured in a cell culture incubator at 37°C, 5% CO2, and 1% oxygen concentration. After 24 hours of further culture, they could be used for experiments.

[0034] 4. Western blot method was used to detect the expression of HSPD1 in mouse heart tissue and cardiomyocytes.

[0035] To clarify the expression changes of HSPD1 in ischemic and hypoxic myocardium, Western blot was used to detect the expression of HSPD1 in cardiac tissue and cardiomyocytes. Samples were added to an appropriate amount of protein lysis buffer and lysed on ice for 30 min, inverting and mixing every 5 min. Centrifugation was performed at 12000g for 20 min at 4℃, 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 electrophoresis was started. The following voltage and time were used: 100V for 30 min, 120V for 60 min, and the power was turned off after bromophenol blue electrophoresis reached the bottom of the glass plate. Samples were transferred to a PVDF membrane at 90V for 2 h. The PVDF membrane was then placed in 5% milk blocking buffer for 1 h, followed by incubation with primary antibody at 4℃ overnight. HSPD1 (Wuhan Sanying Co., Ltd.) and βactin (Wuhan Sanying Co., Ltd.) were used as primary antibodies, and horseradish peroxidase-labeled goat anti-mouse (or anti-rabbit) antibody (CellSignaling Technology) was used as secondary antibody. ECL imaging was performed using an Amersham kit. ImageJ 1.51 software was used to measure the grayscale values ​​of the bands and perform statistical analysis.

[0036] The results showed that, compared with the sham group, there was no change in HSPD1 protein expression in the heart tissue of mice in the MI group; compared with the Control group, there was no change in HSPD1 protein expression in cardiomyocytes of the hypoxia group. Figure 1 (AB, DE). All of the above results suggest that HSPD1 may participate in the development and progression of MI through post-translational modification.

[0037] Example 2: Myocardial-specific HSPD1-K352Q mice were successfully constructed, and cardiac function was tested.

[0038] 1. Construct and identify HSPD1-K352Q mice.

[0039] The adeno-associated virus (AAV2-cTNT-HSPD1-K352Q) and its negative control AAV2-cTnT-NC, specifically for myocardial HSPD1-K352Q, were constructed at Heyuan Biotechnology Co., Ltd. Myocardial-specific HSPD1-K352Q mice were constructed in vivo via tail vein injection.

[0040] 2. Establishment of the HSPD1-K352Q mouse MI model.

[0041] Four weeks after intravenous injection of AAV2-cTNT-HSPD1-K352Q and its negative control virus, transfection with HSPD1-K352Q was successful. Figure 2The MI model was constructed, and the specific experimental method was the same as in Example 1.

[0042] 3. Detection of cardiac function by ultrasound imaging in small animals.

[0043] Before ultrasound examination, hair was removed from the chest of mice using depilatory cream. Mice were then lightly anesthetized with isoflurane gas inhalation, maintaining a heart rate of 450–550 beats / min. Using the Vevo 2100 small animal ultrasound system, coupling gel was applied to the chest and upper abdomen of the mice, and M-mode echocardiograms were obtained over 10 cardiac cycles under the guidance of a two-dimensional probe. Two-dimensional images of the left ventricle were acquired in the short-axis view of the left ventricle, and the images were recorded to measure various cardiac function parameters.

[0044] 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; while compared with the AAV2-cTNT-NC+MI group, the cardiac function indicators of the AAV2-cTNT-HSPD1-K352Q+MI group were significantly improved. Figure 2 ).

[0045] Example 3: Myocardial-specific HSPD1-K352R mice were successfully constructed and their cardiac function was tested.

[0046] 1. Construct and identify HSPD1-K352R mice.

[0047] The adeno-associated virus (AAV2-cTNT-HSPD1-K352R) for constructing myocardial-specific HSPD1-K352R and its negative control AAV2-cTnT-NC were both constructed by Heyuan Biotechnology Co., Ltd. Myocardial-specific HSPD1-K352R mice were constructed in vivo via tail vein injection.

[0048] 2. Establishment of the HSPD1-K352R mouse MI model.

[0049] Four weeks after intravenous injection of AAV2-cTNT-HSPD1-K352R and its negative control virus, transfection with HSPD1-K352R was successfully detected. Figure 2 The MI model was constructed, and the specific experimental method was the same as in Example 1.

[0050] 3. Detection of cardiac function by ultrasound imaging in small animals.

[0051] Before ultrasound examination, hair was removed from the chest of mice using depilatory cream. Mice were then lightly anesthetized with isoflurane gas inhalation, maintaining a heart rate of 450–550 beats / min. Using the Vevo 2100 small animal ultrasound system, coupling gel was applied to the chest and upper abdomen of the mice, and M-mode echocardiograms were obtained over 10 cardiac cycles under the guidance of a two-dimensional probe. Two-dimensional images of the left ventricle were acquired in the short-axis view of the left ventricle, and the images were recorded to measure various cardiac function parameters.

[0052] 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 compared with the AAV2-cTNT-NC+MI group, the cardiac function indicators of the AAV2-cTNT-HSPD1-K352R+MI group were significantly decreased. Figure 2 ) Example 4: Myocardial-specific HSPD1-K352R mice were successfully constructed, and their heart function was tested at different time points.

[0053] 1. Construct and identify HSPD1-K352R mice.

[0054] Adeno-associated virus (AAV2-cTNT-HSPD1-K352R) overexpressing myocardial-specific HSPD1-K352R and its negative control AAV2-cTnT-NC were both constructed at Heyuan Biotechnology Co., Ltd. Myocardial-specific HSPD1-K352R mice were constructed in vivo via tail vein injection.

[0055] 2. Detection of cardiac function by ultrasound imaging in small animals.

[0056] Before ultrasound examination, hair was removed from the chest of mice using depilatory cream. Mice were then lightly anesthetized with isoflurane gas inhalation, maintaining a heart rate of 450–550 beats / min. Using the Vevo 2100 small animal ultrasound system, coupling gel was applied to the chest and upper abdomen of the mice, and M-mode echocardiograms were obtained over 10 cardiac cycles under the guidance of a two-dimensional probe. Two-dimensional images of the left ventricle were acquired in the short-axis view of the left ventricle, and the images were recorded to measure various cardiac function parameters.

[0057] The results showed that at 4 weeks after tail vein injection, the cardiac function of mice was significantly reduced and gradually decreased over time. At 8 weeks, compared with the 6-week-old mice, there was no significant reduction in cardiac function, and this was taken as the time endpoint.

[0058] 3. H&E and MASSON staining were used to examine the gross morphology of the heart and myocardial fibrosis.

[0059] Heart tissues were collected from the AAV2-cTnT-NC and AAV2-cTnT-HSPD1-K352R groups 8 weeks after tail vein injection and subjected to H&E staining and MASSON staining.

[0060] 5. WGA staining to detect cardiomyocyte size.

[0061] Heart tissues from the AAV2-cTnT-NC and AAV2-cTnT-HSPD1-K352R groups were collected 8 weeks after tail vein injection and stained with WGA.

[0062] The results showed that compared with the AAV2-cTNT-NC group, the AAV2-cTnT-HSPD1-K352R group mice had significantly larger hearts and more severe myocardial fibrosis; at the same time, WGA staining showed that the cross-sectional area of ​​cardiomyocytes in the AAV2-cTnT-HSPD1-K352R group mice was increased ( Figure 4 The above results suggest that HSPD1-K352R has a significant damaging effect on myocardial fibrosis.

[0063] This invention, through extensive experiments, revealed that while HSPD1 protein expression did not significantly change in MI models, HSPD1 acetylation levels were significantly elevated. Mouse myocardial-specific HSPD1 acetylation overexpression or underexpression models were constructed using tail vein injection of AAV (adeno-associated virus). The results showed that excessively low HSPD1 acetylation levels significantly impaired cardiac function in mice. In in vitro cell culture, H9C2 / NRCMs subjected to hypoxic stimulation showed no significant change in HSPD1 protein expression, but HSPD1 acetylation levels significantly increased. These results indicate that HSPD1 acetylation levels play a crucial regulatory role in the development and progression of MI, and may represent a novel target for MI prevention or treatment.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of reagents that upregulate HSPD1 protein acetylation levels in the preparation of drugs for the prevention and / or treatment of myocardial infarction.

2. The application according to claim 1, characterized in that, The reagent that upregulates the acetylation level of HSPD1 protein is a reagent that specifically upregulates the acetylation level of the lysine residue at position 352 of the HSPD1 protein.

3. The application according to claim 2, characterized in that, The HSPD1 protein K352 site acetylation mimic mutant is the HSPD1-K352Q mutant, which is a mutant in which the wild-type HSPD1 protein has a lysine residue at position 352 mutated to glutamine.

4. The application according to claim 2, characterized in that, The nucleic acid molecule encoding the acetylation mimic mutant is delivered via an adeno-associated virus vector driven by a myocardial-specific promoter.

5. A pharmaceutical composition for the prevention and / or treatment of myocardial infarction, characterized in that, It contains an effective amount of an upregulator of HSPD1 protein K352 acetylation levels, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is any one of injection, lyophilized powder for injection, or sustained-release formulation.

7. Application of reagents for detecting acetylation levels at the K352 site of HSPD1 protein in the preparation of products for auxiliary diagnosis or prognostic assessment of myocardial infarction.

8. The application according to claim 7, characterized in that, The reagent includes at least one of an antibody or its antigen-binding fragment that specifically recognizes the acetylation modification of the K352 site of the HSPD1 protein, a primer pair that specifically amplifies the HSPD1 gene, and a nucleic acid probe that specifically hybridizes to the nucleotide sequence of the HSPD1 gene.

9. The application according to claim 7, characterized in that, The product is any one of the following: protein detection kit, real-time PCR detection kit, immunochromatographic test strip, and gene detection chip.

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