Application of S100A4 in preparation of medicine for promoting myocardial cell proliferation

Abstract

The invention provides application of S100A4 in preparation of a medicine for promoting myocardial cell proliferation. It is found that S100A4 not only can significantly induce proliferation of newborn mouse primary myocardial cells and hiPSC-CMs in vitro, but also can significantly improve the cardiac function after myocardial infarction and relieve ventricular fibrosis through AAV9-mediated cardiac targeting overexpression in vivo, and no obvious systemic toxic or side effect or tumorigenic signs are observed. Therefore, the S100A4 protein and the means of overexpressing the S100A4 can be used for promoting myocardial cell proliferation, so that the S100A4 protein can be used for improving or treating heart injury diseases such as ischemic heart disease and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of S100A4 in preparation of a drug for promoting proliferation of myocardial cells. BACKGROUND

[0002] Myocardial cells belong to highly terminal differentiated cells, and adult mammal myocardial cells basically have no effective regeneration capacity after injury. Therefore, irreversible loss of myocardial cell quantity is one of the fundamental reasons for continuous deterioration of heart function, ventricular remodeling and eventually heart failure after myocardial ischemia.

[0003] Based on the field consensus formed by a large number of lineage tracing and genetic marker studies, no cardiac stem cell population capable of stably differentiating and supplementing functional myocardial cells under physiological or pathological conditions has been found in adult hearts. In particular, the so-called c-kit positive cardiac stem cells that have been widely concerned and considered to have myocardial differentiation potential have been confirmed by a number of rigorous in vivo lineage tracing studies that they hardly participate in the generation of myocardial cells in adult hearts, nor do they have substantial myogenic differentiation capacity. Existing evidence consistently shows that, whether in the heart development stage, the repair process after injury, or the steady-state update of adult hearts, the increase in the number of myocardial cells mainly depends on the division and proliferation of existing myocardial cells, rather than differentiation and supplementation from exogenous or endogenous stem cells. Therefore, in recent years, the focus of cardiac regeneration medicine research has gradually focused on activating the re-proliferation capacity of endogenous myocardial cells to achieve the repair of myocardial tissue structure and function. Studies have attempted to induce myocardial cells to re-enter the cell cycle by miRNA regulation (such as miR-590, miR-199a), cell cycle factor expression (such as CDK1 / 2, CyclinA2), transcription factor transduction (such as Gata4, Mef2c, Tbx5) and other ways to achieve the purpose of promoting myocardial regeneration. Most of these methods are based on direct activation of the cell cycle core pathway, and although some of them have achieved certain proliferation effect in animal models, long-term expression will lead to arrhythmia and even sudden death. In addition, since these factors are usually closely related to tumor occurrence, and are mostly nuclear proteins or non-coding RNAs, the expression level is difficult to accurately control, and there is a certain potential off-target risk and long-term safety hazard, therefore, it still faces challenges in clinical transformation. SUMMARY

[0004] To solve the above problems, the inventors of the present application found that the endogenous secreted protein S100A4 has myocardial cell proliferation induction effect and good application potential.

[0005] Specifically, S100A4 is a calcium-binding protein with a molecular weight of about 11.5 kDa, which belongs to one of the members of the S100 protein family, and is first discovered in tumor metastasis research. Subsequent studies have shown that it is involved in regulating cell migration, apoptosis, fibrosis and immune response in various pathological conditions. However, its association with myocardial cell proliferation or heart regeneration is still blank.

[0006] The inventors of the present application found that S100A4 can not only significantly induce the proliferation of neonatal mouse primary myocardial cells and hiPSC-CMs in vitro, but also significantly improve cardiac function after myocardial infarction and reduce ventricular fibrosis in vivo through AAV9-mediated cardiac targeting overexpression, and no obvious systemic toxic side effects or tumorigenic signs have been observed. This provides a new path for protein-based mild regulation strategy, and also demonstrates the unique advantages of S100A4 as a potential regenerative therapeutic candidate factor.

[0007] Further research found that the mechanism of action of S100A4 is that S100A4, as a secreted protein, can bind to myocardial cells to activate downstream signaling pathways, thereby regulating the metabolic program in myocardial cells. Specifically, S100A4 can inhibit the expression of PPARa, a key transcription factor of the fatty acid oxidation pathway, resulting in a significant decrease in the transcription level of downstream fatty acid metabolism-related enzymes (such as CPT1, LCAD, etc.), thereby inhibiting the activity of the fatty acid oxidation process in myocardial cells; At the same time, the level of alpha-ketoglutarate, an intermediate product of the tricarboxylic acid cycle metabolism, in the cell is significantly increased, suggesting that metabolic flow reprogramming occurs. This metabolic remodeling is believed to help myocardial cells break through the metabolic homeostasis barrier and enter the mitotic cycle, thereby achieving the re-proliferation of myocardial cells.

[0008] Based on the above findings, the present application provides an application of S100A4 in the preparation of a drug for promoting myocardial cell proliferation, characterized in that the drug is used to promote S100A4 overexpression.

[0009] Preferably, in the application of S100A4 in the preparation of a drug for promoting myocardial cell proliferation provided by the present application, the drug is a delivery system containing S100A4 overexpression genes, and the delivery system includes any one or a combination of several of viral vectors, mRNA delivery, plasmid transfection, liposome encapsulation, and nanoparticle delivery.

[0010] The present application also provides another application of S100A4 in the preparation of a drug for promoting myocardial cell proliferation, characterized in that: wherein the drug is a small molecule drug that can achieve S100A4 upregulation or activity enhancement.

[0011] In addition, the application further provides another application of S100A4 in the preparation of a drug for promoting the proliferation of myocardial cells, and the drug comprises any one or a combination of several of S100A4 protein, a mutant of S100A4 protein and a modified variant of S100A4 protein.

[0012] In the application of S100A4 in the preparation of a drug for promoting the proliferation of myocardial cells according to any one of the above, the drug can be used for treating or improving a heart injury disease. Further, the heart injury disease can be ischemic heart disease. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a result graph of the proliferation effect of the recombinant S100A4 protein of embodiment 1 of the application on neonatal mouse primary myocardial cells.

[0014] Figure 2 is a result graph of the proliferation effect of the recombinant S100A4 protein of embodiment 1 of the application on hiPSC-CMs.

[0015] Figure 3 is a schematic diagram of the experimental process of embodiment 2 of the application.

[0016] Figure 4 is a result graph of the effect of S100A4 gene delivery on the degree of myocardial fibrosis and collagen deposition area of mice in embodiment 2 of the application.

[0017] Figure 5 is a M-mode echocardiogram of embodiment 2 of the application.

[0018] Figure 6 is a result graph of the measurement of heart function in embodiment 2 of the application.

[0019] Figure 7 is a result graph of myocardial cell immunofluorescence staining in embodiment 3 of the application.

[0020] Figure 8 is a result graph of myocardial cell immunofluorescence staining in embodiment 3 of the application.

[0021] Figure 9 is a result graph of single-cell transcriptome data of B cells in embodiment 4 of the application.

[0022] Figure 10 is a result graph of parallel quantitative analysis of protein concentrations of S100A4 and S100A6 in the primary culture of neonatal mouse spleen B lymphocytes in embodiment 4 of the application.

[0023] Figure 11 is a result graph of the expression distribution of S100A4 gene in the immune microenvironment of an injured heart in embodiment 4 of the application.

[0024] Figure 12 is a schematic diagram of the process of the genetically edited mouse of embodiment 4 of the present application.

[0025] Figure 13 is a fluorescence intensity result graph of S100A4 in B cells of embodiment 4 of the present application.

[0026] Figure 14 is a result graph of immunofluorescence staining of myocardial cells treated by supernatant of B lymphocytes in embodiment 4 of the present application.

[0027] Figure 15 is a result graph of immunofluorescence analysis of co-culture of B lymphocytes and myocardial cells in embodiment 4 of the present application. DETAILED DESCRIPTION

[0028] The specific embodiments of the present application are described below in conjunction with the accompanying drawings and examples.

[0029] Example 1

[0030] This embodiment is a study on the proliferative effect of recombinant S100A4 protein on neonatal mouse primary myocardial cells and human induced pluripotent stem cell-derived myocardial cells (hiPSC-CMs).

[0031] 1. Proliferative effect of recombinant S100A4 protein on neonatal mouse primary myocardial cells

[0032] The specific process is as follows:

[0033] Primary myocardial cells of C57BL / 6J wild-type mice at 1 day after birth (animals purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.) were isolated and cultured, and were treated with 100 nM concentration of recombinant S100A4 protein (purchased from R&D Systems, dissolved with PBS), and the control group used the same volume of PBS. After 48 hours of culture, immunofluorescence co-staining was performed using myocardial cell marker a-actinin and cell proliferation marker pH3 (both purchased from Abeam Co., Ltd.).

[0034] Figure 1 is a result graph of the proliferative effect of recombinant S100A4 protein on neonatal mouse primary myocardial cells in embodiment 1 of the present application.

[0035] As shown in Figure 1 , compared with the PBS control group, the number of myocardial cells positive for pH3 significantly increased in the S100A4 treatment group, indicating that S100A4 significantly induced cells to enter the mitotic phase and had a proliferative effect on myocardial cells.

[0036] 2. Proliferative effect of recombinant S100A4 protein on human myocardial cells

[0037] The specific process is as follows:

[0038] Using the above processing procedure of neonatal mouse primary cardiomyocytes, the mouse primary cardiomyocytes therein are replaced with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provided by Nanjing Kasmo Biotechnology Co., Ltd. After treatment (i.e. after 48 hours of culture), α-actinin and Ki67 antibodies are used for co-staining.

[0039] Figure 2 is a result graph of the proliferation effect of the recombinant S100A4 protein of embodiment 1 on hiPSC-CMs.

[0040] As shown in Figure 2 , compared with the PBS control group, the proportion of Ki67 positive cells in the hiPSC-CMs of the S100A4 group is significantly increased, indicating that the S100A4 protein also has the effect of promoting the proliferation of cardiomyocytes in the cross-species background.

[0041] Embodiment 2

[0042] This embodiment is a study on the effect of S100A4 gene delivery targeting the heart on ventricular adverse remodeling and left ventricular systolic function after myocardial ischemia.

[0043] Figure 3 is a schematic diagram of the experimental procedure of embodiment 2 of the present application.

[0044] As shown in Figure 3 , the specific process of this embodiment is as follows:

[0045] In the in vivo experiment, AAV9-S100A4 overexpression virus was constructed, and AAV9-Ctrl was used as a control. A left coronary artery ligation-induced myocardial infarction model was established in 8-10 week-old adult C57BL / 6J mice, and AAV9-S100A4 or AAV9-Ctrl control virus was injected, with a total virus particle number of 1×10¹² vg (virus genome copy number) per mouse, a total amount of 15 μL per point, and a 34G extremely fine needle used to inject the virus into the cardiac gap to avoid tissue damage as much as possible. On day 28 after the operation, Masson staining was performed on the heart to show the degree of myocardial fibrosis and collagen deposition area.

[0046] Figure 4 is a result graph of the effect of S100A4 gene delivery on the degree of myocardial fibrosis and collagen deposition area of mice in embodiment 2 of the present application.

[0047] As shown in Figure 4 , compared with the control group, the degree of myocardial fibrosis of the S100A4 group of mice is significantly reduced, and the collagen deposition area is reduced.

[0048] Further, the heart function was tracked and evaluated using a Vevo-2100 small animal ultrasound system at 0, 7, 14, and 28 days after the operation to measure the left ventricular ejection fraction (EF), fractional shortening (FS), and end-systolic internal diameter (LVIDs).

[0049] Figure 5 is an M-mode echocardiogram of Example 2 of the present application, Figure 6 is a heart function measurement result graph of Example 2 of the present application.

[0050] As shown in Figures 5-6 , the heart function indicators of the S100A4 overexpression group at multiple time points are all better than those of the control group, and in particular, the EF and FS significantly increase at 28 days, and the LVIDs decreases, indicating that it has a significant improvement effect on the ventricular systolic function.

[0051] Example 3

[0052] This example is an experiment on the myocardial cell proliferation effect of S100A4 gene delivery targeting the heart after myocardial ischemia, and the details are as follows:

[0053] The same steps as in Example 2 were used to construct AAV9-S100A4 overexpression and AAV9-Ctrl control adult C57BL / 6J mice, and the heart tissue was collected at 7 days after the operation, and the myocardial cell marker cTnT and the cell nucleus proliferation marker Ki67 were used for immunostaining.

[0054] Figure 7 is a myocardial cell immunostaining result graph of Example 3 of the present application.

[0055] As shown in Figure 7 , compared with the control group, the proportion of myocardial cells positive for Ki67 in the AAV9-S100A4 overexpression group significantly increased, indicating that S100A4 overexpression can make more myocardial cells re-enter the cell cycle.

[0056] Further, to verify whether the myocardial cells truly divided, the cytoplasm division marker AuroraB was used for immunostaining of the S100A4 overexpression group.

[0057] Figure 8 is a myocardial cell immunostaining result graph of Example 3 of the present application.

[0058] As shown in Figure 8 , the number of myocardial cells positive for AuroraB in the S100A4 overexpression group significantly increased, indicating that S100A4 overexpression can make myocardial cells divide.

[0059] In combination with Figure 7 , Figure 8The results show that S100A4 can effectively induce cardiomyocytes to re-enter the proliferation cycle from the resting state, start the mitotic process, and may be one of the key mechanisms for reducing myocardial ischemic injury and promoting tissue repair after myocardial infarction.

[0060] Example 4

[0061] This example is an analysis experiment of the correlation between S100A4 of B cell origin and myocardial regeneration process.

[0062] 1. Single cell transcriptome data analysis

[0063] Figure 9 is a single cell transcriptome data result graph of B cells in Example 4 of the application.

[0064] As Figure 9 shown, a myocardial injury model was established using neonatal C57BL / 6J mice (1 day after birth), and the damaged heart tissue was taken 1 week after injury, and single cell suspension was prepared by mixed enzyme digestion. Then, CD45 magnetic beads were used to sort immune cells, and single cell transcriptome sequencing was carried out. At the same time, the single cell transcriptome data of immune cells after myocardial injury in adult mice was integrated and analyzed, and after cell type annotation, the single cell transcriptome data of B cells as shown in Figure 9 was obtained.

[0065] Combined with the research results published by the applicant earlier (NPJ Regenerative Medicine, 2023, 8(1):7, doi:10.1038 / s41536-023-00282-7), it can be known that neonatal mouse B cells play a key role in myocardial regeneration, while adult mouse B cells aggravate myocardial damage after myocardial ischemia. Further in-depth analysis of the single cell transcriptome data of B cells found that there is a specific B lymphocyte subpopulation in the heart of neonatal mice that highly expresses S100a4 and S100a6. Among them, S100a4 and S100a6 are genes encoding secreted proteins, and are significantly highly expressed in this subpopulation. Based on the above results, it can be speculated that S100A4 or S100A6 may act as a key secreted effector factor of this B cell subpopulation, thereby mediating its biological role in promoting myocardial regeneration.

[0066] 2. In vitro functional protein secretion identification

[0067] To further confirm the dominant position of S100A4 or S100A6 in B cells, this example carries out in vitro identification of functional protein secretion in B cells, the specific process is as follows:

[0068] The spleen B lymphocytes of newborn C57BL6 / J mice (one day after birth) were separated for primary culture, the conditioned medium was collected, and the protein concentrations of S100A4 and S100A6 were quantitatively analyzed in parallel by enzyme-linked immunosorbent assay.

[0069] Figure 10 Figure is a parallel quantitative analysis result diagram of the protein concentrations of S100A4 and S100A6 in the primary culture of the spleen B lymphocytes of newborn mice in Embodiment 4 of the present application.

[0070] As shown in Figure Figure 10 , the concentration of S100A4 protein in the culture supernatant was significantly higher than that of S100A6. This comparative data shows that S100A4 may dominate compared to S100A6 in the potential secretome.

[0071] 3, Deep mining of single-cell transcriptome data

[0072] Figure 11 Figure is an expression distribution result diagram of the S100A4 gene in the damaged heart immune microenvironment in Embodiment 4 of the present application.

[0073] As shown in Figure Figure 11 , the obtained single-cell transcriptome data was deeply mined, and the expression distribution of the S100A4 gene in the damaged heart immune microenvironment was analyzed. It was found that after myocardial injury, the expression of S100A4 was not limited to traditional mononuclear cells, but also significantly up-regulated in the lymphocyte population.

[0074] 4, Expression level of gene editing mouse with B cell conditional knockout of S100A4

[0075] Figure 12 Figure is a process schematic diagram of the gene editing mouse in Embodiment 4 of the present application.

[0076] As shown in Figure Figure 12 , to determine whether S100A4 from B cells is directly involved in regulating the proliferation process of cardiomyocytes, a gene editing mouse model with B cell conditional knockout of S100A4 was constructed in this embodiment. Specifically, Cd19-Cre tool mice (specifically expressing Cre recombinase in B cells) and S100a4 flox / flox mice (loxP sites were inserted on both sides of the 2nd and 3rd exons of the S100a4 gene) were systematically mated and genotyped to obtain conditional knockout mice (cKO) with S100A4 deleted only in B cells. The genotype of this group of mice is Cd19-Cre⁺;S100a4 flox / flox ; the control group is littermate Cd19-Cre⁻;S100a4 flox / flox mice, denoted as wild type (WT).

[0077] To verify the validity of the model, the spleens of the above two groups of mice were separated, single cell suspensions were prepared, and the expression level of S100A4 protein in B cells was detected by flow cytometry.

[0078] Figure 13 is a fluorescence intensity result graph of S100A4 in B cells of Example 4 of the present application.

[0079] As shown in Figure 13 , the mean fluorescence intensity (MFI) of S100A4 in B cells of the cKO group was significantly lower than that of the WT group, confirming that the model achieved efficient B cell-specific knockout at the protein level.

[0080] 5, B cell supernatant treatment of cardiomyocytes

[0081] B lymphocytes of newborn mice were isolated, and their culture supernatant was collected. The collected supernatant was used to treat primary cultured cardiomyocytes.

[0082] Figure 14 is an immunofluorescence staining result graph of B lymphocyte supernatant treatment of cardiomyocytes in Example 4 of the present application.

[0083] As shown in Figure 14 , compared with the control group, the proportion of pH3 positive cells in cardiomyocytes treated with B cell supernatant was significantly increased, suggesting that B cells can promote cardiomyocyte proliferation by secreting certain factors.

[0084] 6, B lymphocyte and cardiomyocyte co-culture

[0085] B lymphocytes and cardiomyocytes were co-cultured in a Transwell chamber system. Specifically, primary cardiomyocytes were co-cultured with B lymphocytes from WT and cKO mice, respectively, and immunofluorescence analysis was performed after 1 day of culture.

[0086] Figure 15 is an immunofluorescence analysis result graph of B lymphocyte and cardiomyocyte co-culture of Example 4 of the present application.

[0087] As shown in Figure 15 , compared with the PBS control group, the presence of WT B lymphocytes can significantly enhance the proliferation ability of cardiomyocytes; while using cKO B lymphocytes lacking S100A4, the pro-proliferation effect is significantly weakened, indicating that S100A4 plays an important role in the regulation of B lymphocyte-mediated cardiomyocyte proliferation.

[0088] From the results of the above embodiments, it can be known that S100A4 can not only significantly induce the proliferation of neonatal mouse primary myocardial cells and hiPSC-CMs in vitro, but also significantly improve cardiac function and reduce ventricular fibrosis after myocardial infarction in vivo through AAV9-mediated cardiac targeting overexpression. Moreover, no obvious systemic toxic side effects or tumorigenicity were observed in the process. Therefore, the S100A4 protein itself and its mutants or modified variants with equivalent activity can be directly used to prepare drugs for promoting myocardial regeneration, and the means for overexpressing S100A4 can also be used to promote myocardial regeneration, such as using a delivery system containing an overexpression gene of S100A4 (viral vector, mRNA delivery, plasmid transfection, liposome encapsulation, nanoparticle delivery, etc.). In addition, means that can achieve up-regulation or activity enhancement of S100A4 can also be used to promote myocardial regeneration, such as small molecule drugs that can achieve up-regulation or activity enhancement of S100A4. These means for promoting myocardial regeneration can be used to treat or improve cardiac injury diseases, especially myocardial ischemic injury caused by ischemic heart disease.

Claims

1. The application of S100A4 in the preparation of drugs that promote cardiomyocyte proliferation, characterized in that, The drug is used to promote S100A4 overexpression.

2. The application of S100A4 according to claim 1 in the preparation of drugs that promote cardiomyocyte proliferation, characterized in that: in, The drug is a delivery system containing the S100A4 overexpression gene. The delivery system includes any one or a combination of several of the following: viral vector, mRNA delivery, plasmid transfection, liposome encapsulation, and nanoparticle delivery.

3. The application of S100A4 in the preparation of drugs that promote cardiomyocyte proliferation, characterized in that: in, The drug is a small molecule drug that can upregulate or enhance the activity of S100A4.

4. The application of S100A4 in the preparation of drugs that promote cardiomyocyte proliferation, characterized in that: in, The drug comprises any one or a combination of several of the following: S100A4 protein, mutants of S100A4 protein, and modified variants of S100A4 protein.

5. The use of S100A4 according to any one of claims 1-4 in the preparation of a drug for promoting cardiomyocyte proliferation, characterized in that: in, The drug is used to treat or improve heart damage.

6. The application of S100A4 according to claim 5 in the preparation of drugs that promote cardiomyocyte proliferation, characterized in that: in, The heart injury is ischemic heart disease.

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