Use of stanniocalcin 2 in the preparation of a medicament for treating myocardial infarction

Abstract

The application relates to the field of biological medicine, and discloses application of stanniocalcin 2 in preparation of myocardial infarction treatment drugs. It is found for the first time that overexpression of stanniocalcin 2 can promote survival and proliferation of myocardial cells after heart infarction, and it is further found that STC2 drives survival and proliferation of myocardial cells after heart infarction by combining with PURA. Therefore, a new treatment target for heart repair after heart infarction is expected to be provided.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of calcitonin 2 in the preparation of drugs for the treatment of myocardial infarction. Background Technology

[0002] Cardiovascular disease is the leading cause of morbidity and mortality worldwide, with heart failure (heart failure) resulting from myocardial infarction (MI) being the primary cause of death from cardiovascular disease. Despite the efficacy of existing drugs and interventional procedures, the risk of death and disability from heart failure after MI remains high. Therefore, there is an urgent need to develop novel and complementary strategies to promote cardiac repair after MI. Unlike some regenerative organs, the adult human heart is almost unable to repair itself after MI, and the massive and irreversible death of functional cardiomyocytes is a major cause of adverse cardiac remodeling and the development of heart failure after MI. Therefore, timely rescue of dying cardiomyocytes and promotion of the proliferation and regeneration of surviving cardiomyocytes are key to treating MI. Elucidating the mechanisms regulating cardiomyocyte survival and proliferation after MI holds promise for opening up new therapies for MI and subsequent heart failure.

[0003] Small extracellular vesicles (sEVs) are lipid bilayer structures with a diameter of 50-150 nm released from cells. Under normal and pathological conditions, sEVs mediate functional intercellular communication by stably transporting active proteins, lipids, mRNA, and small non-coding RNA to target cells. Previous studies have shown that sEVs secreted by mesenchymal stem cells can exert cardioprotective effects, promote angiogenesis, reduce cardiac fibrosis, and regulate immunity by delivering key active molecules, thereby promoting cardiac injury repair. Therefore, utilizing sEVs to deliver active molecules that promote cardiomyocyte survival and proliferation holds promise as an important means of treating myocardial infarction.

[0004] Stanniocalcin 2 (STC2) is a disulfide-linked homodimer secreted glycoprotein widely expressed in various organs and tissues, including the mammary gland, muscle, heart, testes, and pancreas. As a paracrine / autocrine factor, STC2 participates in regulating various pathophysiological processes, such as cell metabolism, inflammation, oxidative stress, and calcium and phosphorus homeostasis. The STC2 gene is located on human chromosome 5, and the encoded protein contains 302 amino acids, unlike STC1, which is located on human chromosome 8 and encodes a protein containing 247 amino acids, and is currently the focus of cardiovascular research. STC2 and STC1 share only 34% amino acid sequence similarity. To date, there are no international reports on STC2 promoting myocardial tissue repair and regeneration after myocardial infarction, especially promoting cardiomyocyte survival and proliferation, and the molecular mechanism remains unclear.

[0005] PURA (purine-rich element-binding protein alpha) is a member of the PUR protein family involved in the regulation of DNA replication, transcription, and RNA translation. As a pluripotent transcription factor, PURA regulates the transcriptional levels of its target genes by directly binding to specific DNA or RNA sequences or by binding to other transcription factors through protein-protein interactions. Previous studies have shown that PURA plays a crucial role in brain development, neurological function, and neurological diseases. Patients with PURA gene mutations may exhibit symptoms such as neurodevelopmental delay, intellectual disability, hypotonia, and epilepsy. However, whether PURA regulates cardiac repair after myocardial infarction, particularly whether STC2 drives cardiomyocyte survival and proliferation after myocardial infarction by binding to PURA, remains unreported in the literature. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of calcitonin 2 in the preparation of drugs for the treatment of myocardial infarction. This invention is the first to discover that overexpression of calcitonin 2 can promote the survival and proliferation of cardiomyocytes after myocardial infarction, thus potentially providing a new therapeutic target for cardiac repair after myocardial infarction.

[0007] The specific technical solution of this invention is as follows: First, the present invention provides the use of calcitonin 2 or substances overexpressing calcitonin 2 in the preparation of drugs for the treatment of myocardial infarction.

[0008] This invention, through experimental findings, is the first to discover that human small extracellular vesicles overexpressing STC2 can promote the survival and proliferation of cardiomyocytes in mice after myocardial infarction, while knocking down STC2 weakens the ability of human small extracellular vesicles to promote cardiomyocyte survival and proliferation. Therefore, calcitonin 2 or substances overexpressing calcitonin 2 may hold promise as therapeutic agents for myocardial infarction.

[0009] Furthermore, the myocardial infarction is an acute myocardial infarction.

[0010] Secondly, the present invention provides the application of calcitonin 2 or substances overexpressing calcitonin 2 in the preparation of reagents that promote the survival and proliferation of cardiomyocytes after myocardial ischemia, wherein the reagents are for non-therapeutic purposes.

[0011] Preferably, the substance overexpressing calcitonin 2 is a virus overexpressing calcitonin 2 or a small extracellular vesicle overexpressing calcitonin 2.

[0012] Preferably, the virus overexpressing calcitonin 2 is an adeno-associated virus, and its vector is GV571; the element sequence is cTNTp-MCS-3Flag-T2A-EGFP; wherein the sequence of the calcitonin 2 gene is the CDS sequence of the NM_003714.3 gene, as shown in SEQ ID NO.1.

[0013] Preferably, the small extracellular vesicles overexpressing calcitonin 2 are obtained by transfecting stem cells with a lentivirus overexpressing calcitonin 2; the vector of the lentivirus overexpressing calcitonin 2 is GV492; the element sequence is Ubc-MCS-3FLAG-CBh-gcGFP-IRES-puromycin; wherein the sequence of the calcitonin 2 gene is the CDS sequence of the NM_003714.3 gene, as shown in SEQ ID NO.1.

[0014] Third, the present invention provides the application of recombinant human calcitonin 2 protein in the preparation of drugs for the treatment of myocardial infarction, characterized in that: the amino acid sequence of the recombinant human calcitonin 2 protein is as shown in SEQ ID NO.2.

[0015] Fourth, the present invention provides the application of recombinant human calcitonin 2 protein in the preparation of a reagent that promotes the survival and proliferation of cardiomyocytes after myocardial ischemia, wherein the amino acid sequence of the recombinant human calcitonin 2 protein is shown in SEQ ID NO.2; the reagent is for non-therapeutic purposes.

[0016] Preferably, the drug is an injectable formulation.

[0017] Preferably, the drug further includes one or more pharmaceutically acceptable excipients, carriers, and excipients.

[0018] Fifth, the present invention provides a method for promoting the survival and proliferation of cardiomyocytes in vitro: under hypoxic and hyposeralemic conditions, recombinant human calcitonin 2 protein or small extracellular vesicles overexpressing calcitonin 2 are added to the culture medium of cardiomyocytes in vitro to promote the survival and proliferation of cardiomyocytes in vitro; the amino acid sequence of the recombinant human calcitonin 2 protein is shown in SEQ ID NO. 2.

[0019] Furthermore, the recombinant human calcitonin 2 protein or small extracellular vesicles overexpressing calcitonin 2 promote the survival and proliferation of in vitro cardiomyocytes by binding to the PURA protein in in vitro cardiomyocytes.

[0020] The present invention further discovered through experiments that STC2 drives the survival and proliferation of cardiomyocytes after myocardial infarction by binding to PURA.

[0021] Compared with the prior art, the beneficial effects of the present invention are: the present invention is the first to discover that overexpression of calcitonin 2 can promote the survival and proliferation of cardiomyocytes after myocardial infarction, thus potentially providing a new therapeutic target for cardiac repair after myocardial infarction. Attached Figure Description

[0022] Figure 1 After establishing an acute myocardial infarction model in mice, human small extracellular vesicles (sEVs) overexpressing STC2 were injected into the infarct area. OE STC2), control human small extracellular vesicles (sEVs) OENC (A) Western blot detection of sEVs and solvent control (Vehicle). OE STC2 (A) Overexpression of STC2 and corresponding statistics; (B) Dynamic changes in cardiac function in mice before myocardial infarction modeling (baseline) and 1, 7, 14 and 28 days after sEV injection; (C) Immunofluorescence staining of cardiac tissue apoptosis 3 days after sEV injection and statistical results of apoptotic cardiomyocytes in the peri-infarct area of ​​the three groups; green represents troponin, representing cardiomyocytes; red represents tunelin, representing apoptotic cells; blue represents Hoechst, representing cell nuclei; scale bar = 50 μm; (D) Immunofluorescence staining of cardiomyocyte proliferation 28 days after sEV injection and statistical results of proliferating cardiomyocytes in the peri-infarct area of ​​the three groups; green represents α-actinin, representing cardiomyocytes; red represents Ki67 and pH3, representing proliferating cells; blue represents Hoechst, representing cell nuclei; scale bar = 40 μm.

[0023] Figure 2 After establishing an acute myocardial infarction model in mice, human small extracellular vesicles (sEVs) overexpressing STC2 were injected into the infarct area. OE STC2 ), control human small extracellular vesicles (sEVs) OENC (A) Construction and experimental design of Myh6-MerCreMer; R26R-Confetti genetically traced mice; (B) Tracing diagram of proliferating cardiomyocytes in Confetti mice 28 days after sEV injection and statistical results of proliferating cardiomyocytes in the peri-infarct area of ​​three groups; white represents WGA, used to delineate single cardiomyocytes; red represents RFP-positive cardiomyocytes; green represents nGFP-positive cardiomyocytes; blue represents Hoechst, representing cell nuclei; scale bar = 50 μm.

[0024] Figure 3 After establishing an acute myocardial infarction model in mice, human small extracellular vesicles (sEVs) with STC2 knockout were injected into the infarct area of ​​the mice. siSTC2 ), control human small extracellular vesicles (sEVs) siNC (A) Western blot detection of sEVs and solvent control (Vehicle). siSTC2(A) Knockdown of STC2 and corresponding statistics; (B) Dynamic changes in cardiac function in mice before myocardial infarction modeling (baseline) and 1, 7, 14 and 28 days after sEV injection; (C) Immunofluorescence staining of cardiac tissue apoptosis 3 days after sEV injection and statistical results of apoptotic cardiomyocytes in the peri-infarct area of ​​the three groups. Green represents troponin, representing cardiomyocytes; red represents tunel, representing apoptotic cells; blue represents Hoechst, representing cell nuclei; scale bar = 50 μm; (D) Immunofluorescence staining of cardiomyocyte proliferation 28 days after sEV injection and statistical results of proliferating cardiomyocytes in the peri-infarct area of ​​the three groups; green represents α-actinin, representing cardiomyocytes; red represents Ki67 and pH3, representing proliferating cells; blue represents Hoechst, representing cell nuclei; scale bar = 40 μm.

[0025] Figure 4 Tracer images of proliferating cardiomyocytes in Confetti mice 28 days after sEV injection and statistical results of proliferating cardiomyocytes in the peri-infarct area of ​​three groups; white represents WGA, used to delineate single cardiomyocytes; red represents RFP-positive cardiomyocytes; green represents nGFP-positive cardiomyocytes; blue represents Hoechst, representing cell nuclei; scale bar = 50 μm.

[0026] Figure 5 Immunofluorescence staining images and corresponding statistical results of cardiomyocytes (hiPSC-vCM) derived from human induced pluripotent stem cells after normal in vitro culture and after treatment with human recombinant STC2 protein or solvent control (PBS) for 24 hours under simulated ischemic conditions; green represents α-actinin, representing cardiomyocytes; red represents Tunel, Ki67, and pH3, where Tunel represents apoptotic cells, and Ki67 and pH3 represent proliferating cells; blue represents Hoechst, representing the cell nucleus; scale bar = 40 μm.

[0027] Figure 6 STC2 in human small extracellular vesicles binds to PURA in cardiomyocytes. (A) Schematic diagram of mass spectrometry screening for STC2-binding proteins. The Venn diagram shows the overlapping proteins between the three samples as candidate proteins, and the table shows the top 5 transcriptionally related proteins among the candidate proteins; (B) Immunoprecipitation and Western blot were used to detect the expression of STC2 and PURA in the pull-down protein to evaluate the interaction between STC2 and PURA; (C) AlphaFold 3 software was used to predict the direct binding sites of human STC2 protein and mouse PURA protein; (D) Western blot was used to detect the interaction between STC2 and PURA in neonatal rat cardiomyocytes under normoxic and ischemic stimulation. OE NC and sEVOE STC2 The cytoplasmic and nuclear localization of STC2 and PURA.

[0028] Figure 7 SEVs were added to neonatal rat cardiomyocytes and PURA knockdown neonatal rat cardiomyocytes under normoxic and in vitro ischemic conditions, respectively. OE STC2 sEV OE NC Immunofluorescence staining images of vehicles after 24 hours of culture and statistical results of five groups of apoptotic and proliferating cardiomyocytes; green represents α-actinin, representing cardiomyocytes; red represents Tunel, Ki67 and pH3, where Tunel represents apoptotic cells, and Ki67 and pH3 represent proliferating cells; blue represents Hoechst, representing cell nuclei; scale bar = 20 μm. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments.

[0030] Example 1: Intramyocardial injection of human small cell extracellular vesicles overexpressing STC2 significantly improved cardiac function and promoted cardiomyocyte survival and proliferation in mice after acute myocardial infarction. (1) Preparation of human STC2 overexpression lentivirus: The vector name of the STC2 overexpression lentivirus used in this embodiment is GV492; the element sequence is Ubc-MCS-3FLAG-CBh-gcGFP-IRES-puromycin; it was constructed by Shanghai Jikai Gene Technology Co., Ltd. The sequence of the calcitonin 2 gene is the CDS sequence of the NM_003714.3 gene, as shown in SEQ ID NO.1.

[0031] (2) Preparation of human small extracellular vesicles overexpressing STC2: After adhering to mesenchymal stem cells derived from human embryonic stem cells for 24 hours, they were transfected with STC2 overexpressing lentivirus (OE STC2) and negative control lentivirus (OE NC) for 48 hours, and then replaced with serum containing 1% exosome-free serum (ThermoFisher Scientific, Gibco). TMCells were cultured in a high-glucose medium (A2720803) for 48 hours, and the supernatant was collected. The supernatant was centrifuged at 10000g for 10 minutes to remove dead cells and cell debris. The supernatant was transferred to a 10kb ultrafiltration tube (Merck, Millipore, UFC9010) and centrifuged at 3000g for 30 minutes to obtain a concentrated sample. The concentrated sample was then centrifuged at 100000g for 70 minutes, and the supernatant was discarded. The precipitate was resuspended in 500μl of ultrapure water containing 5% trehalose and 1% mannitol, centrifuged at 10000g for 10 minutes, and the supernatant was further lyophilized in a FreeZone 12L freeze-drying system (Labconco, USA). Before use, the lyophilized sEVs were reconstituted in 100μl of ultrapure water containing 6% hydroxyethyl starch (Vehicle) to restore their original concentration. The obtained human small extracellular vesicles (sEVs) overexpressing STC2 were... OE STC2 Human small extracellular vesicles (sEVs) and their negative control OE NC (This information is intended for use in subsequent research.)

[0032] (3) Western blot detection of sEV OE STC2 STC2 overexpression status: sEV OE STC2 and sEV OE NC Add Pierce TM The protease and phosphatase inhibitors (ThermoFisher Scientific, A32961) were lysed using RIPA lysis buffer (Beyotime, P0013B). After protein extraction and quantification, the protein was separated by 12% SDS-PAGE gel electrophoresis, transferred to a 0.2µm microporous polyvinylidene fluoride (PVDF) membrane (Merck, Millipore, ISEQ00010), blocked at room temperature for 2 hours with Tris-buffered saline-Tween (TBST) containing 5% skim milk, and then coated with STC2 primary antibody (ThermoFisher Scientific, PA5-106379). o Incubate overnight at C. After washing twice with TBST, the membrane is incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour. Protein bands are then visualized using enhanced chemiluminescence and analyzed using Image Lab. TM Quantitative analysis using software version 6.0.

[0033] (4) Myh6-MerCreMer; R26R-Confetti genetically traced mice: such as Figure 2As shown in Figure A, to trace changes in cardiomyocyte proliferation after myocardial infarction, pedigree tracing analysis was performed using Myh6-MerCreMer;R26R-Confetti genetically traced mice. This system contains nuclear green fluorescent protein (nGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), and monomeric cyan fluorescent protein (mCFP) with loxP sites flanking each other. Following a single intraperitoneal injection of tamoxifen (6.7 mg / kg, Merck, Sigma-Aldrich, T5648) into 4-week-old mice, Myh6 expression triggered Cre-loxP recombination, thereby randomly labeling cardiomyocytes and their progeny with a single fluorescent color.

[0034] (5) Preparation of acute myocardial infarction model in mice: Wild-type mice and Myh6-MerCreMer and R26R-Confetti genetically traced mice (all 8 weeks old) injected with tamoxifen for 4 weeks were anesthetized with sodium pentobarbital (60 mg / kg), endotracheally intubated and connected to a small animal ventilator, the chest was opened, and the left anterior descending coronary artery was ligated below the left atrial appendage with 7-0 silk suture. The establishment of the myocardial infarction model was determined by the change in local tissue color. Subsequently, 10 μg (20 μl total volume) of sEV was injected at four points around the infarct. OESTC2 sEV OENC A control of equal volume of solvent (Vehicle) was used, and the chest was closed.

[0035] (6) Assessment of cardiac function in mice: Wild-type mice were placed on an ultrasound imaging platform, and bubble-free ultrasound coupling agent was applied to the shaved area on the chest. The long axis and short axis views of the heart were obtained using a Vevo 3100 ultrasound system (Fujifilm Visual Sonics, Canada). The left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS) were recorded and analyzed under the long axis view in M-mode ultrasound.

[0036] (7) Wild-type mice were euthanized 3 and 28 days after myocardial infarction. Myh6-MerCreMer; R26R-Confetti genetically traced mice were euthanized 28 days after myocardial infarction under sodium pentobarbital anesthesia. Heart samples were collected, embedded, and frozen sectioned.

[0037] (8) ① Assessment of cardiomyocyte apoptosis: For frozen sections of wild-type mouse hearts 3 days after myocardial infarction, the in situ cell death assay kit (Merck, Roche, 12156792910) and troponin antibody (Abcam, ab47003) were used to evaluate cardiomyocyte apoptosis in the peri-infarct area; ② Assessment of cardiomyocyte proliferation: For frozen sections of wild-type mouse hearts 28 days after myocardial infarction, Ki67 (Abcam, ab15580), pH3 (Merck, Sigma-Aldrich, 06-570) and α-actinin (Merck, Sigma-Aldrich, A7811) were used to evaluate cardiomyocyte proliferation in the peri-infarct area; For frozen sections of Myh6-MerCreMer;R26R-Confetti genetically traced mice 28 days after myocardial infarction, Wheat Germ Agglutinin (WGA, ThermoFisherScientific, Invitrogen™ (W32466) staining was used to outline individual cardiomyocytes to assess the number of RFP-positive and nGFP-positive cardiomyocytes, and the proportion of adjacent RFP-positive and nGFP-positive cardiomyocytes to the total number of RFP-positive and nGFP-positive cells was counted.

[0038] (9) such as Figure 1 As shown in A, with sEV OE NC In comparison, sEV OE STC2 STC2 expression was significantly increased. For example... Figure 1 As shown in B, compared with the solvent control, injected sEV OE NC The LVEF and LVFS of mice were significantly increased, and the injection of sEVs was also observed. OE STC2 Mice injected with sEV OE NC The LVEF and LVFS of mice were further significantly increased, indicating that intramyocardial injection of sEVs OE STC2 It significantly improved cardiac function in mice after acute myocardial infarction. Figure 1 As shown in C, 3 days after the myocardial infarction, sEV was injected. OE NC The proportion of Tunel-positive cardiomyocytes was reduced in mice compared to the solvent control group, and the mice injected with sEVs showed a decrease. OE STC2 The proportion of Tunel-positive cardiomyocytes in mice was further significantly reduced, indicating that intramyocardial injection of sEVs... OE STC2 It significantly increased myocardial survival after myocardial infarction in mice. Figure 1 As shown in D, 28 days after the myocardial infarction, sEV was injected. OE NC Compared with the solvent control group, the proportion of Ki67 / α-actinin double-positive and pH3 / α-actinin double-positive cardiomyocytes in mice was significantly increased, and the injection of sEVs was also observed. OE STC2The proportion of Ki67 / α-actinin double-positive and pH3 / α-actinin double-positive cardiomyocytes in mice was further significantly increased. Figure 2 As shown in B, in Myh6-MerCreMer;R26R-Confetti genetically traced mice, sEV was injected... OE NC Compared with the solvent control group, mice showed an increased proportion of RFP-positive and nGFP-positive cardiomyocytes (two or more adjacent cells) among all RFP-positive and nGFP-positive cells, and were injected with sEVs. OE STC2 In mice, the proportion of adjacent RFP-positive and nGFP-positive cardiomyocytes increased significantly, indicating that intramyocardial injection of sEVs after acute myocardial infarction is beneficial. OE STC2 It significantly promotes the proliferation of cardiomyocytes.

[0039] Example 2: Intramyocardial injection of human small extracellular vesicles with STC2 knockout reduced the effect on improving cardiac function and promoting cardiomyocyte survival and proliferation after acute myocardial infarction in mice. (1) Preparation of human STC2 small interfering RNA: The STC2 small interfering RNA used in this example (Sence: 5'-GGACUUGCUGCUGCACGAA-3', antisense: 5'-UUCGUGCAGCAGCAAGUCC-3') was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0040] (2) Preparation of human small extracellular vesicles with STC2 knockdown: Mesenchymal stem cells derived from human embryonic stem cells were cultured adherently for 24 hours, and then transfected with STC2 small interfering RNA (siSTC2) and negative control (siNC) for 48 hours, respectively. Then, the cells were replaced with serum containing 1% exosome-free serum (ThermoFisher Scientific, Gibco). TM Cells were cultured in a high-glucose medium (A2720803) for 48 hours, and the supernatant was collected. The supernatant was centrifuged at 10000g for 10 minutes to remove dead cells and cell debris. The supernatant was transferred to a 10kb ultrafiltration tube (Merck, Millipore, UFC9010) and centrifuged at 3000g for 30 minutes to obtain a concentrated sample. The concentrated sample was then centrifuged at 100000g for 70 minutes, and the supernatant was discarded. The precipitate was resuspended in 500μL of ultrapure water containing 5% trehalose and 1% mannitol, centrifuged at 10000g for 10 minutes, and the supernatant was further lyophilized in a FreeZone 12L freeze-drying system (Labconco, USA). Before use, the lyophilized sEVs were reconstituted in 100μL of ultrapure water containing 6% hydroxyethyl starch (Vehicle) to restore their original concentration. The obtained STC2-knockdown human small extracellular vesicles (sEVs) were... siSTC2Human small extracellular vesicles (sEVs) and their negative control siNC (This information is intended for use in subsequent research.)

[0041] (3) Western blot detection of sEV siSTC2 STC2 knockdown details: EV siSTC2 and sEV siNC Add Pierce TM The protease and phosphatase inhibitors (ThermoFisher Scientific, A32961) were lysed using RIPA lysis buffer (Beyotime, P0013B). After protein extraction and quantification, the protein was separated by 12% SDS-PAGE gel electrophoresis and transferred to a 0.2µm microporous polyvinylidene fluoride (PVDF) membrane (Merck, Millipore, ISEQ00010). The membrane was blocked at room temperature for 2 hours with Tris-buffered saline-Tween (TBST) containing 5% skim milk, and then coated with STC2 primary antibody (ThermoFisher Scientific, PA5-106379). o Incubate overnight at C. After washing twice with TBST, the membrane is incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour. Protein bands are then visualized using enhanced chemiluminescence and analyzed using Image Lab. TM Quantitative analysis using software version 6.0.

[0042] (4) Myh6-MerCreMer; R26R-Confetti genetically traced mice: To trace changes in cardiomyocyte proliferation after myocardial infarction, pedigree analysis was performed using Myh6-MerCreMer; R26R-Confetti genetically traced mice. This system contains nuclear green fluorescent protein (nGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), and monomeric cyan fluorescent protein (mCFP) with loxP sites on both sides. After a single intraperitoneal injection of tamoxifen (6.7 mg / kg, Merck, Sigma-Aldrich, T5648) into 4-week-old mice, Myh6 expression triggered Cre-loxP recombination, thereby randomly labeling cardiomyocytes and their progeny with a single fluorescent color.

[0043] (5) Preparation of acute myocardial infarction model in mice: Wild-type mice and Myh6-MerCreMer;R26R-Confetti genetically traced mice (both 8 weeks old) injected with tamoxifen for 4 weeks were anesthetized with sodium pentobarbital (60 mg / kg), endotracheally intubated and connected to a small animal ventilator, the chest was opened, and the left anterior descending coronary artery was ligated below the left atrial appendage with 7-0 silk suture. The establishment of the myocardial infarction model was determined by the change in local tissue color. Subsequently, 10 μg (20 μL total volume) of sEV was injected at four points around the infarct. siSTC2 sEV siNC A control of equal volume of solvent (Vehicle) was used, and the chest was closed.

[0044] (6) Assessment of cardiac function in mice: Wild-type mice were placed on an ultrasound imaging platform, and bubble-free ultrasound coupling agent was applied to the shaved area on the chest. The long axis and short axis views of the heart were obtained using a Vevo 3100 ultrasound system (Fujifilm Visual Sonics, Canada). The left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS) were recorded and analyzed under the long axis view in M-mode ultrasound.

[0045] (7) Wild-type mice were euthanized 3 and 28 days after myocardial infarction. Myh6-MerCreMer; R26R-Confetti genetically traced mice were euthanized 28 days after myocardial infarction under sodium pentobarbital anesthesia. Heart samples were collected, embedded, and frozen sectioned.

[0046] (8) ① Assessment of cardiomyocyte apoptosis: For frozen sections of wild-type mouse hearts 3 days after myocardial infarction, the apoptosis of cardiomyocytes in the peri-infarct area was evaluated using an in situ cell death assay kit (Roche, 12156792910) and troponin antibody (Abcam, ab47003); ② Assessment of cardiomyocyte proliferation: For frozen sections of wild-type mouse hearts 28 days after myocardial infarction, the proliferation of cardiomyocytes in the peri-infarct area was evaluated using Ki67 (Abcam, ab15580), pH3 (Merck, Sigma-Aldrich, 06-570), and α-actinin (Merck, Sigma-Aldrich, A7811), respectively; For frozen sections of Myh6-MerCreMer;R26R-Confetti genetically traced mice 28 days after myocardial infarction, the proliferation of cardiomyocytes in the peri-infarct area was evaluated using WGA (ThermoFisher Scientific, Invitrogen™). W32466 staining was used to outline individual cardiomyocytes to assess the number of RFP-positive and nGFP-positive cardiomyocytes, and the proportion of cardiomyocytes with two or more adjacent RFP-positive or nGFP-positive cells to the total number of RFP-positive and nGFP-positive cells was counted.

[0047] (9) such as Figure 3 As shown in A, with sEV siNC In comparison, sEV siSTC2 STC2 expression was significantly reduced. For example... Figure 3 As shown in B, compared with injected sEV siNC Compared to mice, intramyocardial injection of sEVs siSTC2 The mice showed a significant decrease in LVEF and LVFS, suggesting that intramyocardial injection of sEVs may have contributed to the decline. siSTC2 The ability to improve cardiac function after acute myocardial infarction in mice was subsequently weakened. For example... Figure 3 As shown in C, the patient had a myocardial infarction 3 days prior, and was receiving an injection of sEV. siNC Compared to mice, injection of sEV siSTC2 The significantly increased proportion of Tunel-positive cardiomyocytes in mice indicates that intramyocardial injection of sEVs after acute myocardial infarction... siSTC2 Its ability to promote cardiomyocyte survival decreases afterward. For example... Figure 3 As shown in D, 28 days after the myocardial infarction, following the injection of sEV... siNC Compared to mice, injection of sEV siSTC2 The proportion of Ki67 / α-actinin double-positive and pH3 / α-actinin double-positive cardiomyocytes in mice was significantly reduced; for example Figure 4 As shown, in Myh6-MerCreMer; R26R-Confetti genetically traced mice, compared with those injected with sEVs... siNC Compared to mice, injection of sEVsiSTC2 The proportion of adjacent RFP-positive and nGFP-positive cardiomyocytes in mice was also significantly reduced compared to the total RFP-positive and nGFP-positive cardiomyocytes, indicating that intramyocardial injection of sEVs after acute myocardial infarction... siSTC2 Its ability to promote cardiomyocyte proliferation decreases afterward.

[0048] Example 3: Recombinant human STC2 protein improves the survival and proliferation of ventricular myocytes derived from human induced pluripotent stem cells under in vitro ischemic conditions. (1) Preparation of recombinant human STC2 protein and ventricular myocytes derived from human induced pluripotent stem cells: The recombinant human STC2 protein (hrSTC2, HY-P76096) used in this embodiment was purchased from MedChemExpress, and its amino acid sequence is shown in SEQ ID NO.2. The ventricular myocytes (hiPSC-vCM, HELP4111) derived from human induced pluripotent stem cells used in this embodiment were purchased from Nanjing Airpu Regenerative Medicine Technology Co., Ltd.

[0049] (2) HiPSC-vCM culture: hiPSC-vCM stored in liquid nitrogen was cultured at 37°C. o Thaw rapidly in a C-water bath, then transfer to cardiomyocyte resuscitation solution, centrifuge at 300g for 5 minutes, resuspend the cell pellet in cardiomyocyte resuscitation solution, and centrifuge at 1×10⁻⁶. 5 cells / cm 2 The cells were seeded at a density of 100% in 24-well plates coated with cardiomyocyte plating medium and cultured for 24 hours before being replaced with cardiomyocyte maintenance medium.

[0050] (3) In vitro ischemic stress treatment and hrSTC2 intervention: After the hiPSC-vCM stabilized, the cell culture medium was replaced with cardiomyocyte maintenance culture medium supplemented with hrSTC2 (100 ng / ml) or an equal volume of solvent control (PBS) and cultured in a hypoxic incubator with 1% oxygen concentration for 24 hours. hiPSC-vCM cultured under normoxic conditions for 24 hours served as a control.

[0051] (4) ① Apoptosis assessment of hiPSC-vCM: Cardiac cell apoptosis was evaluated by co-staining with an in situ cell death detection kit (Roche, 12156792910) and α-actinin antibody (Merck, Sigma-Aldrich, A7811); ② Proliferation assessment of hiPSC-vCM: Cardiac cell proliferation was evaluated by co-staining with Ki67 (Abcam, ab15580) and pH3 (Merck, Sigma-Aldrich, 06-570) and α-actinin.

[0052] (5) such as Figure 5As shown, compared with the normoxic group, the proportion of Tunel-positive hiPSC-vCM was significantly increased and the proportion of Ki67 / α-actinin double-positive and pH3 / α-actinin double-positive cardiomyocytes was significantly decreased under in vitro ischemic conditions. However, after hrSTC2 intervention, the proportion of Tunel-positive hiPSC-vCM was significantly reduced and the proportion of Ki67 / α-actinin double-positive and pH3 / α-actinin double-positive cardiomyocytes was significantly increased, indicating that hrSTC2 can significantly improve the survival and proliferation of hiPSC-vCM under in vitro ischemic conditions.

[0053] Example 4: STC2 in human small extracellular vesicles binds to PURA in cardiomyocytes. (1) Preparation of human STC2 overexpression lentivirus: The STC2 overexpression lentivirus used in this embodiment (vector name GV492; element sequence Ubc-MCS-3FLAG-CBh-gcGFP-IRES-puromycin; constructed by Shanghai Jikai Gene Technology Co., Ltd.) is shown in SEQ ID NO.1. The sequence of calcitonin 2 is the CDS sequence of the NM_003714.3 gene.

[0054] (2) Preparation of human small extracellular vesicles overexpressing STC2: Mesenchymal stem cells derived from human embryonic stem cells were cultured adherently for 24 hours, transfected with STC2 overexpressing lentivirus (OE STC2) and negative control lentivirus (OE NC) and cultured for 48 hours, and then replaced with serum containing 1% exosome-free serum (ThermoFisher Scientific, Gibco). TM Cells were cultured in a high-glucose medium (A2720803) for 48 hours, and the supernatant was collected. The supernatant was centrifuged at 10000g for 10 minutes to remove dead cells and cell debris. The supernatant was transferred to a 10kb ultrafiltration tube (Merck, Millipore, UFC9010) and centrifuged at 3000g for 30 minutes to obtain a concentrated sample. The concentrated sample was then centrifuged at 100000g for 70 minutes, and the supernatant was discarded. The precipitate was resuspended in 500μL of ultrapure water containing 5% trehalose and 1% mannitol, centrifuged at 10000g for 10 minutes, and the supernatant was further lyophilized in a FreeZone 12L freeze-drying system (Labconco, USA). Before use, the lyophilized sEVs were reconstituted in 100μL of ultrapure water containing 6% hydroxyethyl starch (Vehicle) to restore their original concentration. The obtained human small extracellular vesicles (sEVs) overexpressing STC2 were... OE STC2 Human small extracellular vesicles (sEVs) and their negative control OE NC (This information is intended for use in subsequent research.)

[0055] (3) Isolation and culture of neonatal rat cardiomyocytes: Hearts of neonatal rats 24 hours after birth were rinsed with PBS and digested using a neonatal rat cardiomyocyte isolation kit (Miltenyi Biotec, 130-098-373). Digestion was performed for 15 minutes each time, three times in total. Digestion was then terminated by adding high-glucose DMEM (complete culture medium) containing 10% fetal bovine serum. The cells were filtered through a 70 μm filter, and the filtrate was centrifuged at 1000 rpm for 5 minutes. After removing the supernatant, the cell pellet was resuspended in erythrocyte lysis buffer. Erythrocytes were lysed on ice for 2 minutes, and the cell suspension was centrifuged again at 1000 rpm for 5 minutes. The cell pellet was then resuspended in complete culture medium. The cell suspension was subjected to differential adhesion to remove as many adherent cardiac fibroblasts as possible to obtain suspended cardiomyocytes. The supernatant was collected, and the cardiomyocytes were then cultured at 2 × 10⁻⁶. 5 / cm 2 Inoculate into well plates and culture using complete culture medium in a routine manner.

[0056] (4) Extracorporeal ischemia treatment and sEV OE STC2 Intervention: Replace the culture medium for neonatal rat cardiomyocytes with one containing sEVs. OE STC2 (5μg / 2×10) 5 Cells), sEV OE NC (5μg / 2×10) 5 Cells or an equal volume of vehicle serum-free, low-glucose DMEM were cultured in a hypoxic incubator containing 1% oxygen for 24 hours. Neonatal rat cardiomyocytes cultured under normoxic conditions for 24 hours served as a control (Normoxia).

[0057] (5) Immunoprecipitation sample preparation: The sample was subjected to in vitro ischemia and then sEV was added. OE STC2 After intervention, neonatal rat cardiomyocytes were washed with PBS, then lysed thoroughly with a lysis buffer containing protease and phosphatase inhibitors, and incubated at 4°C. o After centrifugation at 12000g for 10 minutes at C, collect the cell lysis supernatant. Use an immunoprecipitation kit (Beyotime, P2179S) to extract STC2-binding proteins. Add STC2 antibody (Abcam, ab255610) or normal IgG to Protein A+G beads and incubate at room temperature for 1 hour. Add Protein A+G beads bound to STC2 antibody or Protein A+G beads bound to IgG to the cell lysis supernatant, and incubate for 4 hours. o Incubate overnight at C20°C. After incubation, remove the supernatant, wash three times with lysis buffer containing protease and phosphatase inhibitors, then add Acid Elution Buffer, incubate at room temperature for 5 minutes, separate on a magnetic rack for 10 seconds to remove the magnetic beads, collect the supernatant, add Neutralization Buffer to neutralize and mix, and obtain the protein sample.

[0058] (6) Mass spectrometry detection: The obtained protein samples were concentrated and denatured using ultrafiltration tubes. Then, the samples were denatured, reduced, alkylated, enzymatically digested, and eluted using a Barocycler high-pressure sample pretreatment system. Subsequently, the samples were acquired using a Vanquish nano-liquid chromatography system and an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific) equipped with a FAIMSPro™ (ThermoFisher Scientific) interface. The mobile phase was an acetonitrile-water-formic acid system, where mobile phase A was a 0.1% formic acid and 2% acetonitrile solution, and phase B was a 0.1% formic acid and 80% acetonitrile solution. During sample acquisition, peptides were injected into the pre-column (3µm, 100Å, 20mm×75µm id) at a flow rate of 6μL / min, followed by the analytical column (1.9µm, 120Å, 150mm×75µm id) at a flow rate of 300nL / min. Analysis was performed using a 60-minute effective liquid chromatography gradient (8% to 35% mobile phase B). The mass spectrometer's primary scan range was 350–1200 m / z. The Swissprot_Mouse database was searched using the MSFragger search engine.

[0059] (7) For example Figure 6 As shown in Figure A, to identify proteins that bind to STC2, non-specific binding proteins (i.e., proteins present in the IgG control group) were first excluded. Subsequently, the set of common proteins identified in all three independent STC2 immunoprecipitations was defined as candidate proteins. Among these, five were transcription-translation related proteins, and PURA was identified as a sEV in ischemic injury. OE STC2 The transcription-translation related protein that interacts most strongly with STC2 in treated neonatal rat cardiomyocytes.

[0060] (8) Western blot verification of the interaction between STC2 and PURA: ischemic in vitro samples were treated and sEVs were added. OE STC2 Using neonatal rat cardiomyocytes after intervention as samples, immunoprecipitation kits were used with STC2 antibody (Abcam, ab255610), PURA antibody (Proteintech, 17733-1-AP) and corresponding IgG antibodies to pull down proteins that bind to STC2 or PURA, respectively. The expression of PURA and STC2 in the pulled-down proteins was verified by Western blot and with STC2 primary antibody (ThermoFisher Scientific, PA5-106379) or PURA primary antibody and corresponding HRP-labeled secondary antibody.

[0061] (9) such as Figure 6 As shown in Figure B, PURA protein signals can be detected in samples that underwent immunoprecipitation using STC2 antibody, and STC2 protein signals can be detected in samples that underwent immunoprecipitation using PURA antibody, suggesting that STC2 in human small cell extracellular vesicles binds to PURA in neonatal rat cardiomyocytes.

[0062] (10) Molecular docking prediction: The amino acid sequences of human STC2 and mouse PURA were obtained from the UniProt database (https: / / www.uniprot.org / ). The core three-dimensional structure of the protein and its molecular docking interaction sites were predicted using AlphaFold 3 (https: / / alphafoldserver.com / ). Subsequent hydrogen bond addition and structural visualization were performed using PyMOL software.

[0063] (11) such as Figure 6 As shown in Figure C, in the interaction system between human STC2 and mouse PURA, multiple key amino acid residues form a stable binding interface through hydrogen bonds, indicating that human STC2 and mouse PURA bind to each other.

[0064] (12) Subcellular localization of STC2 and PURA: The nuclear protein and cytoplasmic protein extraction kit (Beyotime, P0028) was used to analyze the localization of STC2 and PURA under normal oxygen conditions and with the addition of Vehicle and sEVs. OE NC or sEV OE STC2 The cytoplasm and nucleus of neonatal rat cardiomyocytes were separated after in vitro ischemia treatment. The obtained cytoplasmic and nuclear proteins were verified by Western blot to determine the subcellular localization of STC2 and PURA in each group.

[0065] (13) such as Figure 6 As shown in D, with the addition of sEV OE NC Compared to cardiomyocytes, the addition of sEV OE STC2 In postcardiomyocytes, STC2 and PURA translocate extensively from the cytoplasm to the nucleus, indicating that STC2 and PURA interact to promote PURA's entry into the nucleus and exert its function.

[0066] Example 5: PURA knockdown inhibits cardiomyocyte survival and proliferation driven by STC2-overexpressing human small extracellular vesicles. (1) Preparation of mouse PURA small interfering RNA: The mouse PURA small interfering RNA (Sence: 5'-GCGACUUCAUCGAGCACUA-3', antisense: 5'-UAGUGCUCGAUGAAGUCGC-3') used in this example was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0067] (2) Preparation of human small extracellular vesicles overexpressing STC2: After adhering to mesenchymal stem cells derived from human embryonic stem cells for 24 hours, they were transfected with STC2 overexpressing lentivirus (OE STC2) and negative control lentivirus (OE NC) for 48 hours, and then replaced with serum containing 1% exosome-free serum (ThermoFisher Scientific, Gibco). TM Cells were cultured in a high-glucose medium (A2720803) for 48 hours, and the supernatant was collected. The supernatant was centrifuged at 10000g for 10 minutes to remove dead cells and cell debris. The supernatant was transferred to a 10kb ultrafiltration tube (Merck, Millipore, UFC9010) and centrifuged at 3000g for 30 minutes to obtain a concentrated sample. The concentrated sample was then centrifuged at 100000g for 70 minutes, and the supernatant was discarded. The precipitate was resuspended in 500μl of ultrapure water containing 5% trehalose and 1% mannitol, centrifuged at 10000g for 10 minutes, and the supernatant was further lyophilized in a FreeZone 12L freeze-drying system (Labconco, USA). Before use, the lyophilized sEVs were reconstituted in 100μl of ultrapure water containing 6% hydroxyethyl starch (Vehicle) to restore their original concentration. The obtained human small extracellular vesicles (sEVs) overexpressing STC2 were... OE STC2 Human small extracellular vesicles (sEVs) and negative controls OE NC (This information is intended for use in subsequent research.)

[0068] (3) Isolation and culture of neonatal rat cardiomyocytes: Hearts of neonatal rats 24 hours old were rinsed with PBS and digested using a neonatal rat cardiomyocyte isolation kit (Miltenyi Biotec, 130-098-373). Digestion was performed for 15 minutes each time, three times in total. Digestion was then terminated by adding high-glucose DMEM (complete culture medium) containing 10% fetal bovine serum. The cells were filtered through a 70 μm filter, and the filtrate was centrifuged at 1000 rpm for 5 minutes. After removing the supernatant, the cell pellet was resuspended in erythrocyte lysis buffer. Erythrocytes were lysed on ice for 2 minutes, and the cell suspension was centrifuged again at 1000 rpm for 5 minutes. The cell pellet was resuspended in complete culture medium. The cell suspension was subjected to differential adhesion to remove as many adherent cardiac fibroblasts as possible to obtain suspended cardiomyocytes. The supernatant was collected, and the cardiomyocytes were then cultured at 2 × 10⁻⁶ cells / mL. 5 / cm 2 Inoculate into well plates and culture using complete culture medium in a routine manner.

[0069] (4) Knockdown of PURA in neonatal rat cardiomyocytes: Neonatal rat cardiomyocytes were cultured adherently for 24 hours, and then transfected with PURA small interfering RNA (siPURA) and negative control (siNC) respectively and cultured for 48 hours.

[0070] (5) Experimental groups: ① Normoxia group: The culture medium for neonatal rat cardiomyocytes was replaced with high-glucose DMEM containing 10% fetal bovine serum and cultured under normoxic conditions for 24 hours; ② CM siNC +Vehicle: After transfecting neonatal rat cardiomyocytes with siNC for 48 hours, the medium was changed to serum-free, low-glucose DMEM with an equal volume of solvent control, and the cells were incubated in a hypoxic incubator containing 1% oxygen for 24 hours; ③CM siNC +sEV OE NC 48 hours after transfecting neonatal rat cardiomyocytes with siNC, the medium was changed to include sEVs. OE NC (5μg / 2×10) 5 Cells were cultured in serum-free, low-glucose DMEM in a hypoxic incubator containing 1% oxygen for 24 hours; ④CM siNC +sEV OE STC2 48 hours after transfecting neonatal rat cardiomyocytes with siNC, the medium was changed to include sEVs. OE STC2 (5μg / 2×10) 5 Cells were cultured in serum-free, low-glucose DMEM and placed in a hypoxic incubator containing 1% oxygen for 24 hours; ⑤CM siPURA +sEV OE STC2 48 hours after transfecting neonatal rat cardiomyocytes with siPURA, the medium was changed to contain sEVs. OE STC2 (5μg / 2×10) 5 Cells were cultured in serum-free, low-glucose DMEM and placed in a hypoxic incubator containing 1% oxygen for 24 hours.

[0071] (6) ① Assessment of cardiomyocyte apoptosis: Cardiomyocyte apoptosis was evaluated by co-staining with an in situ cell death assay kit (Roche, 12156792910) and α-actinin antibody (Merck, Sigma-Aldrich, A7811); ② Assessment of cardiomyocyte proliferation: Cardiomyocyte proliferation was evaluated by co-staining with Ki67 (Abcam, ab15580) and pH3 (Merck, Sigma-Aldrich, 06-570) and α-actinin.

[0072] (7) For example Figure 7 As shown, compared with the normoxic group, the proportion of TUNEL-positive cardiomyocytes was significantly increased under in vitro ischemic conditions, with the addition of sEVs. OE NC The percentage of positive Tunel test results decreased significantly afterward, and sEVs were added. OE STC2 The proportion of Tunel-positive cardiomyocytes further decreased; however, even with the addition of sEVs, the percentage of PURA knockout in cardiomyocytes did not decrease. OE STC2The proportion of Tunel-positive cells was similar to that in the solvent control group, suggesting that STC2 drives cardiomyocyte survival by binding to PURA under in vitro ischemic conditions. Compared with the normoxic group, the proportion of Ki67 / α-actinin double-positive and pH3 / α-actinin double-positive cardiomyocytes was significantly reduced under in vitro ischemic conditions. However, under in vitro ischemic conditions, the addition of sEVs... OE NC Subsequently, the proportion of Ki67 / α-actinin double-positive and pH3 / α-actinin double-positive cardiomyocytes significantly increased, and the addition of sEVs further enhanced the effect. OE STC2 Subsequently, the proportion of Ki67 / α-actinin double-positive and pH3 / α-actinin double-positive cardiomyocytes further increased significantly. However, after knocking down PURA in cardiomyocytes, even with the addition of sEVs... OE STC2 The proportion of Ki67 / α-actinin double positive and pH3 / α-actinin double positive cardiomyocytes decreased significantly, suggesting that STC2 promotes cardiomyocyte proliferation by binding to PURA under in vitro ischemic conditions.

Claims

1. Application of calcitonin 2 or substances that overexpress calcitonin 2 in the preparation of drugs for the treatment of myocardial infarction.

2. The application as described in claim 1, characterized in that: The myocardial infarction mentioned refers to acute myocardial infarction.

3. The application of calcitonin 2 or substances overexpressing calcitonin 2 in the preparation of reagents that promote the survival and proliferation of cardiomyocytes after myocardial ischemia, characterized in that: The reagents are for non-therapeutic purposes.

4. The application as described in claim 1 or 3, characterized in that: The substance overexpressing calcitonin 2 is a virus overexpressing calcitonin 2 or a small extracellular vesicle overexpressing calcitonin 2.

5. The application as described in claim 4, characterized in that: The virus overexpressing calcitonin 2 is an adeno-associated virus, and its vector is GV571; the element sequence is cTNTp-MCS-3Flag-T2A-EGFP. The small extracellular vesicles overexpressing calcitonin 2 were obtained by transfecting stem cells with a lentivirus overexpressing calcitonin 2; the lentiviral vector overexpressing calcitonin 2 was GV492; the element sequence was Ubc-MCS-3FLAG-CBh-gcGFP-IRES-puromycin.

6. The application of recombinant human calcitonin 2 protein in the preparation of drugs for the treatment of myocardial infarction, characterized in that: The amino acid sequence of the recombinant human calcitonin 2 protein is shown in SEQ ID NO.

2.

7. The application of recombinant human calcitonin 2 protein in the preparation of reagents that promote the survival and proliferation of cardiomyocytes after myocardial ischemia, characterized in that: The amino acid sequence of the recombinant human calcitonin 2 protein is shown in SEQ ID NO.2; the reagent is for non-therapeutic use.

8. The application as described in claim 1 or 6, characterized in that: The drug is an injectable preparation.

9. A method for promoting the survival and proliferation of cardiomyocytes in vitro, characterized in that: Under hypoxic and hyposeral conditions, recombinant human calcitonin 2 protein or small extracellular vesicles overexpressing calcitonin 2 were added to the culture medium of in vitro cardiomyocytes to promote the survival and proliferation of in vitro cardiomyocytes; the amino acid sequence of the recombinant human calcitonin 2 protein is shown in SEQ ID NO.

2.

10. The method as described in claim 9, characterized in that: The recombinant human calcitonin 2 protein or small extracellular vesicles overexpressing calcitonin 2 promote the survival and proliferation of in vitro cardiomyocytes by binding to the PURA protein in in vitro cardiomyocytes.

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