Use of proline-rich protein 33 in preparation of a drug for treating cardiomyopathy related to cardiac hypertrophy

Abstract

The application relates to the field of biological medicine, and discloses application of proline-rich protein 33 in preparation of a drug for cardiomyopathy related to cardiac hypertrophy. The application identifies a proline-rich protein 33 highly enriched in myocardial cells. Prr33 Gene deletion can aggravate myocardial cell hypertrophy and ventricular dysfunction, and its overexpression can inhibit cardiac hypertrophy. PRR33 protects the myocardium from pathological hypertrophy by stabilizing the LDB3-MYOZ2-calcineurin complex and inhibiting NFAT activation. These findings reveal a new regulatory level in the sarcomere signaling and highlight that PRR33 is expected to become a therapeutic target for preventing cardiomyopathy related to cardiac hypertrophy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the application of proline-rich protein 33 in the preparation of drugs for cardiomyopathy related to cardiac hypertrophy. Background Technology

[0002] Cardiac hypertrophy is a core pathological feature of many cardiovascular diseases and a major determinant of the progression to heart failure (HF). Initially, cardiac hypertrophy is a compensatory response, but if it persists or becomes dysregulated, it can impair cardiac contractile function, alter myocardial structure, and ultimately lead to malignant ventricular remodeling. Current treatment strategies primarily target the symptoms of hypertrophy or slow disease progression; for pathological myocardial remodeling that has already occurred, there is currently no effective method to reverse the structural and functional abnormalities of cardiomyocytes.

[0003] The cytoskeleton, especially the Z-disc, is a key hub coordinating the structural integrity of cardiomyocytes, sarcomere stability, and hypertrophy signal transduction. Z-disc proteins are not only responsible for anchoring myofilaments and maintaining their contractile alignment, but also participate in the assembly of multi-protein complexes, thereby regulating multiple key pathways, including calcium processing (such as calcineurin-NFAT activation) and MAPK signal transduction. These pathways are crucial for myocardial hypertrophy. Perturbations to these structure-signal associations, such as mutations in Z-disc components (e.g., LDB3, CSRP3, or MYOZ2), are closely associated with the development of dilated and hypertrophic cardiomyopathy. These findings indicate that sarcomere-associated proteins are not only components for maintaining cell structure but also regulators that actively modulate the hypertrophic response.

[0004] Despite some progress in existing research, many sarcomere-related regulatory factors remain uncharacterized, especially small or unannotated proteins that may fine-tune cytoskeleton structure or signal transduction. Therefore, further identification of more sarcomere-related regulatory factors is of positive significance for drug development and disease treatment of hypertrophic cardiomyopathy. Summary of the Invention

[0005] This invention provides the application of proline-rich protein 33 in the preparation of drugs for cardiomyopathy related to cardiac hypertrophy. This invention identifies a novel proline-rich protein 33 that is highly enriched in cardiomyocytes. This invention discovers... Prr33 Gene deletion can exacerbate cardiomyocyte hypertrophy and ventricular dysfunction, while its overexpression can inhibit cardiac hypertrophy and improve cardiac function. Therefore, PRR33 is expected to become a therapeutic target for the prevention of cardiomyopathy related to cardiac hypertrophy.

[0006] The specific technical solution of this invention is as follows: First, this application provides a proline-rich protein 33 containing an amino acid sequence as shown in SEQ ID NO: 1 (human PRR33) or SEQ ID NO: 2 (mouse PRR33).

[0007] This invention identifies a protein highly enriched in cardiac muscle—proline-rich protein 33 (PRR33). This PRR33 differs from the skeletal muscle transcript (Prr33-sk) in both nucleotide and amino acid sequences, and is specifically highly expressed in cardiac muscle. (Prr33-sk) is a myogenic regulatory factor that regulates the cytoskeleton-mitochondrial organization during muscle differentiation, maintaining muscle function. However, the physiological and pathological functions of PRR33 in the heart, its relationship with the cytoskeleton regulatory network, and its potential role in cardiac hypertrophy remodeling remain completely unknown. Based on the central role of Z-disc signaling in hypertrophy regulation and the localized enrichment of PRR33 in skeletal muscle, we hypothesized in our initial research that the PRR33 enriched in cardiac muscle may interact with key cytoskeleton components, influencing cardiac hypertrophy signaling and cardiac function.

[0008] In subsequent studies, this invention identified a heart-specific transcript of PRR33 and found that it exhibits dynamic expression during postnatal cardiac maturation, with its expression trends consistent with the developmental process of key sarcomere genes. This invention further investigated the role of PRR33 in cardiac physiology and pathology, discovering... Prr33 Gene deletion promotes cardiac hypertrophy, while overexpression via the AAV9 system... Prr33 This demonstrates that PRR33 can alleviate pressure-induced cardiac hypertrophy and improve cardiac function, proving its effectiveness in inhibiting cardiac hypertrophy. Furthermore, this invention also reveals that PRR33 interacts with LDB3, regulating the LDB3-MYOZ2-calcineurin complex. This discovery provides novel insights into the mechanism by which the cytoskeleton regulates cardiac hypertrophy and holds promise as a therapeutic target for preventing cardiomyopathy associated with cardiac hypertrophy.

[0009] Second, this application provides a polynucleotide encoding the above-mentioned proline-rich protein 33, which contains the nucleotide sequence shown in SEQ ID NO: 3 (human PRR33) or SEQ ID NO: 4 (mouse PRR33).

[0010] The nucleotide sequences of SEQ ID NO: 3 (human PRR33) or SEQ ID NO: 4 (mouse PRR33) above can encode the human PRR33 protein as shown in SEQ ID NO: 1 and the mouse PRR33 protein as shown in SEQ ID NO: 2, respectively.

[0011] Third, this application provides an adeno-associated virus that overexpresses proline-rich protein 33, which contains the nucleotide sequence shown in SEQ ID NO: 3 (human PRR33) or SEQ ID NO: 4 (mouse PRR33).

[0012] Preferably, the adeno-associated virus vector is AAV9, and its element sequence is: cTNT-Prr33, containing... Prr33 Gene fragments.

[0013] Fourth, this application provides the use of proline-rich protein 33 or substances overexpressing proline-rich protein 33 in the preparation of drugs for cardiomyopathy related to cardiac hypertrophy, wherein the proline-rich protein 33 contains the amino acid sequence shown in SEQ ID NO: 1 (human PRR33) or SEQ ID NO: 2 (mouse PRR33).

[0014] Furthermore, the hypertrophic cardiomyopathy is either hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0015] Preferably, the substance overexpressing proline-rich protein 33 is an adeno-associated virus overexpressing proline-rich protein 33, containing the nucleotide sequence shown in SEQ ID NO: 3 (human PRR33) or SEQ ID NO: 4 (mouse PRR33).

[0016] Fifth, this application provides a drug for the prevention or treatment of cardiomyopathy associated with cardiac hypertrophy, comprising proline-rich protein 33 or a substance overexpressing proline-rich protein 33.

[0017] Preferably, the drug is an injectable or oral formulation; more preferably, it is an intravenous formulation.

[0018] Preferably, the drug further includes one or more pharmaceutically acceptable excipients, carriers, and excipients.

[0019] Sixth, this application provides a method for inhibiting in vitro cardiomyocyte hypertrophy, comprising: adding ad- to the culture medium of an in vitro induced cardiomyocyte hypertrophy model. Prr33 To achieve PRR33 overexpression, PRR33 overexpression inhibits cardiomyocyte hypertrophy, including inhibiting hypertrophy marker genes. Nppa , Nppb and Myh7 The expression of [a substance] and the phenotype of cardiomyocyte hypertrophy were observed, thereby inhibiting cardiomyocyte hypertrophy in vitro.

[0020] Seventh, this application provides a method for constructing a myocardial hypertrophy model, which includes: Inject knockdown / knockout into mice Prr33 AAV9 virus of the gene inhibits the body in micePrr33 Gene expression, thereby obtaining a mouse model of cardiac hypertrophy; or Inhibit cardiomyocytes in vitro Prr33 Gene expression was used to obtain an in vitro model of hypertrophic cardiomyocytes.

[0021] Based on the new discovery that PRR33 overexpression in cardiomyocytes can inhibit cardiomyocyte hypertrophy, mouse models of cardiac hypertrophy and in vitro models of hypertrophic cardiomyocytes can be constructed, which can then be used to assist in the screening of drugs for the treatment of cardiomyopathy related to cardiac hypertrophy or for scientific research for non-therapeutic purposes.

[0022] Compared with existing technologies, the beneficial effects of this invention are: this invention identifies a proline-rich protein 33 (PRR33) that is highly enriched in cardiomyocytes. This invention discovers... Prr33 Gene deletion exacerbates cardiomyocyte hypertrophy and ventricular dysfunction, while its overexpression inhibits cardiac hypertrophy. PRR33 protects the myocardium from pathological hypertrophy by stabilizing the LDB3-MYOZ2-calcineurin complex and inhibiting NFAT activation. These findings reveal a novel regulatory level in sarcomere signaling and highlight PRR33 as a potential therapeutic target for the prevention of hypertrophic cardiomyopathy. Attached Figure Description

[0023] Figure 1 Identifying cardiac enrichment Prr33 Transcription and establishment Prr33 The association between Prr33 and cardiac hypertrophy. Among them: (A) Schematic diagram of different Prr33 transcripts; (B) qPCR detection of relative expression levels of different Prr33 transcripts in the heart, n=3; (C) qPCR detection of different developmental stages (P0-P30). Prr33-C Changes in expression levels, n=3; (D) developmental stage Prr33-C The expression of genes involved in cardiomyocyte growth ( Tnnt2 , Myl2 and Myh6 Correlation analysis of expression, n=3; (E) developmental stage Prr33-C The expression of these genes is related to the reprogramming of genes in hypertrophic hearts. Myh7 , Tnni1 and Myl7 Correlation analysis of the expressed values, n=3; Figure 2 In vitro knockdown Prr33-C Promotes cardiomyocyte hypertrophy. Among them: (A) neonatal mouse cardiomyocytes undergo... si- Prr33 or si-NC(A) Representative images of TNNT2 immunofluorescence staining after processing; (B) Quantitative analysis of neonatal mouse cardiomyocyte size using ImageJ software (https: / / imagej.nih.gov / ij / ), n=6; (C) si-NC and si-Prr33 In transfected neonatal mouse cardiomyocytes, Prr33 and markers of myocardial hypertrophy Nppa , Nppb , Myh7 The expression level, n=6; (D) in the PE (20μM) treatment model, from Prr33 fl / fl Representative images of neonatal mouse cardiomyocytes isolated from mice and infected with Ad-Gfp or Ad-Cre, respectively, after TNNT2 immunofluorescence staining (n=6); (E) Quantitative analysis of the size of neonatal mouse cardiomyocytes in TNNT2 immunofluorescence staining images using ImageJ software (https: / / imagej.nih.gov / ij / ), n=6; (F) Detection of the size of neonatal mouse cardiomyocytes in PE (20 μM) treatment model by qRT-PCR. Prr33 fl / fl Mouse isolated and infected Ad-Gfp or Ad-Cre in neonatal mouse cardiomyocytes Prr33 , Nppa , Nppb , Myh7 The level of expression, n=6; Figure 3 In vitro overexpression Prr33-C Inhibit PE-induced cardiomyocyte hypertrophy. Specifically: (A) In a PE (20 μM)-induced cardiomyocyte hypertrophy model, neonatal mouse cardiomyocytes isolated from wild-type mice were infected with Ad-... Lacz or Ad- Prr33 (A) Representative images of TNNT2 immunofluorescence staining were obtained; (B) Quantitative analysis of the size of neonatal mouse cardiomyocytes (NMCMs) in the TNNT2 immunofluorescence staining images shown in Figure A was performed using ImageJ software (https: / / imagej.nih.gov / ij / ), n=4; (C) qRT-PCR was used to detect the size of Ad-12 cells isolated from wild-type mice and infected with TNNT2 in the PE (20 μM) model. Lacz or Ad- Prr33 In neonatal mouse cardiomyocytes (NMCMs) Prr33 , Nppa , Nppb , Myh7 The level of expression, n=4; Figure 4 In vivo knockdown / knockout Prr33Promotes cardiac hypertrophy and heart failure. Among them: (A) Two months after injection of AAV9 virus, the control group AAV9- scramble Group and AAV9- shPrr33 (B) Hearts of mice in the control group (AAV9-) 2 months after injection of AAV9 virus. scramble ) and AAV9- shPrr33 Sirius red and Fast Green staining results of heart tissue sections from the control group (AAV9-) 2 months after AAV9 virus injection; scramble ) and AAV9- shPrr33 Representative images of transverse cardiac sections of mice were stained with wheat germ agglutinin (WGA) immunostaining; (D) Two months after injection of AAV9 virus, AAV9- scramble Control group and AAV9- shPrr33 (E) Echocardiographic functional testing of mice; FS: Fractional Shortening. Scramble n=9, ShPrr33 n=5; (F) Echocardiographic-based quantitative analysis of cardiac function. EF: Ejection Fraction. Scramble n=9, ShPrr33 : n=5; (G) Two months after injection of AAV9 virus, AAV9- scramble Control group and AAV9- shPrr33 In the heart of the group of mice Prr33 and markers of myocardial hypertrophy Nppa , Nppb , Myh7 level of expression Scramble n=6, ShPrr33 : n=5; (H) after TAC surgery (12 weeks) and 6 months of tamoxifen injection, Prr33 fl / fl mice and Prr33 Hearts of conditionally knockout (cKO) mice; (I) 6 months after TAC surgery (12 weeks) and tamoxifen injection. Prr33 fl / fl mice and Prr33 Sirius red and Fast Green stained sections of heart tissue from conditionally knockout (cKO) mice; (J) after TAC surgery (12 weeks) and 6 months of tamoxifen injection, Prr33 fl / fl mice and Prr33 Heart weight to body weight ratio in conditional knockout (cKO) mice; (K) after TAC surgery (12 weeks) and tamoxifen injection for 6 months, Prr33 fl / fl mice and Prr33Quantitative analysis of cardiac function in conditional knockout (cKO) mice. EF: Ejection Fraction; (L) cardiac function in the heart of sham-operated mice. Prr33 and markers of myocardial hypertrophy Nppa , Nppb , Myh7 Expression levels; (M) TAC surgery group mice heart Prr33 and markers of myocardial hypertrophy Nppa , Nppb , Myh7 The level of expression; Sham- Prr33 fl / fl n=6, Sham- Prr33 cKO n=8, TAC- Prr33 fl / fl n=5, TAC- Prr33 cKO n=5; Figure 5 Overexpression in vivo Prr33-C Inhibits TAC-induced cardiac hypertrophy and heart failure. Among them: (A) AAV9- Luciferase Control group and AAV9- Prr33 Gross cardiac images of mice in the overexpression group 2 months after AAV9 virus injection and TAC surgery; (B) AAV9- Luciferase Control group and AAV9- Prr33 Sirius red and Fast Green stained sections of heart tissue from mice in the overexpression group 2 months after AAV9 virus injection and TAC surgery; (C) AAV9- Luciferase Control group and AAV9- Prr33 Representative transverse images of the heart of mice in the overexpression group 2 months after AAV9 virus injection and TAC surgery, stained with wheat germ agglutinin (WGA); (D) AAV9- Luciferase Control group and AAV9- Prr33 Quantitative analysis of cardiomyocyte cross-sectional area in mice overexpressing AAV9 two months after AAV9 virus injection and TAC surgery; (E) AAV9- Luciferase Control group and AAV9- Prr33 In mice overexpressing AAV9, the ratio of heart weight to body weight was measured 2 months after AAV9 virus injection and TAC surgery; (F) AAV9- Luciferase Control group and AAV9- Prr33 Quantitative analysis of cardiac function in mice overexpressing AAV9 virus two months after AAV9 virus injection and TAC surgery. EF: Ejection Fraction; (G)AAV9- Luciferase Control group and AAV9- Prr33Quantitative analysis of cardiac function in mice overexpressing AAV9 virus two months after AAV9 virus injection and TAC surgery. FS: Fractional Shortening; (H)AAV9- Luciferase Control group and AAV9- Prr33 In mice overexpressing AAV9, two months after AAV9 virus injection and TAC surgery, the heart... Prr33 and markers of myocardial hypertrophy Nppa , Nppb , Myh7 The expression level of AAV9- Luciferase n=5, AAV9- Prr33 n=6; Figure 6 : Prr33 It regulates genes encoding the cytoskeleton and cardiac hypertrophy factors. Among them: (A) AAV9- Scramble Control group and AAV9- ShPrr33 Hierarchical clustering heatmap analysis of differentially expressed genes among groups. (Screening criteria: log2 fold change > 0.5; P-value < 0.05); (B) AAV9- Scramble Control group and AAV9- ShPrr33 KEGG pathway enrichment analysis of differentially expressed genes between groups. (Screening criteria: log2 fold change > 0.5; P value < 0.05); (C) Gene Ontology-Biological Process (GO-BP) enrichment analysis of downregulated genes; (D) Gene Ontology-Cellular Component (GO-CC) enrichment analysis of downregulated genes; Figure 7The study aimed to screen and validate key proteins interacting with PRR33. Specifically: (A) Turbo-ID-based neighborhood marker and proteomics (GO-BP) enrichment analysis; (B) Scatter plot of PRR33-Turbo-ID labeled proteins (screening criteria: iBAQ biotin signal vs. negative control >600, score >100); (C) Bimolecular complementary fluorescence (BiFC-Venus) images of interactions between PRR33 and MYOZ2, PRR33 and LDB3, and MYOZ2 and LDB3; (D) Introduction of Turbo-PRR33 into neonatal mouse cardiomyocytes (NMCMs), enrichment of biotin-labeled neighboring proteins using streptavidin magnetic beads, and Western blotting analysis using antibodies; (E) Co-immunoprecipitation (Co-IP) experiment validating the interaction between LDB3 and PRR33. IB: FLAG-tagged antibody detection of PRR33 expression in both groups; IP: LDB3 antibody dropdown shows the interaction between IgG control and PRR33 overexpression conditions; (F) Representative immunofluorescence staining images of adult mouse cardiomyocytes. α-ACTININ (green), PRR33 (red), DAPI (blue); Figure 8 PRR33 improves cardiac hypertrophy by regulating the NFAT signaling pathway through the modulation of cardiomyocyte cytoskeleton remodeling (MYOZ2-LDB3-CALCINEURIN). Specifically: (A) Prr33 (a) Bimolecular fluorescence complementary (BiFC-Venus) images of the interactions between MYOZ2 and calcineurin, and between MYOZ2 and LDB3 in knockdown or overexpression models; (b) Schematic diagram of the role of PRR33 in regulating the LDB3-MYOZ2-calcineurin complex; (c) Verification in Prr33 Immunoprecipitation (Co-IP) experiment demonstrating the interaction between MYOZ2 and calcineurin in knockdown or overexpression models; (D) Validation in Prr33 Immunoprecipitation (Co-IP) assays of MYOZ2-LDB3 interaction in knockdown or overexpression models; (E) in Prr33 Knock down (Lenti- shPrr33 vs. Lenti- scramble (F) In the calcineurin-NFAT signaling pathway model, the NFAT luciferase reporter gene was detected to verify its activity; (F) In the PE-induced hypertrophy model, the NFAT luciferase reporter gene was detected to verify its activity. Prr33 Overexpression (Ad-) Lacz vs. Ad- Prr33 ) Detection of NFAT luciferase reporter genes for calcineurin-NFAT signaling pathway activity, n=6; Figure 9 Mouse AAV9- shPrr33 plasmid information (knockdown) Prr33 ); Figure 10 Mouse AAV9- Prr33 Plasmid information (overexpression) Prr33 ). Detailed Implementation

[0024] First, a proline-rich protein 33 containing an amino acid sequence as shown in SEQ ID NO: 1 (human PRR33) or SEQ ID NO: 2 (mouse PRR33).

[0025] Second, a polynucleotide encoding the above-mentioned proline-rich protein 33, which contains the nucleotide sequence shown in SEQ ID NO: 3 (human PRR33) or SEQ ID NO: 4 (mouse PRR33).

[0026] Third, an adeno-associated virus that overexpresses proline-rich protein 33, containing nucleotide sequences as shown in SEQ ID NO: 3 (human PRR33) or SEQ ID NO: 4 (mouse PRR33).

[0027] In some embodiments, the adeno-associated virus vector is AAV9, whose element sequence is: cTNT-PRR33, containing Prr33 The CDS sequence fragment (SEQ ID NO: 4).

[0028] Fourth, the use of proline-rich protein 33 or substances overexpressing proline-rich protein 33 in the preparation of drugs for cardiomyopathy related to cardiac hypertrophy, wherein the proline-rich protein 33 contains the amino acid sequence shown in SEQ ID NO: 1 (human PRR33) or SEQ ID NO: 2 (mouse PRR33).

[0029] Furthermore, the hypertrophic cardiomyopathy is either hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0030] In some embodiments, the substance overexpressing proline-rich protein 33 is an adeno-associated virus overexpressing proline-rich protein 33, containing the nucleotide sequence shown in SEQ ID NO: 3 (human PRR33) or SEQ ID NO: 4 (mouse PRR33).

[0031] Fifth, a drug for the prevention or treatment of cardiomyopathy associated with cardiac hypertrophy, comprising proline-rich protein 33 or a substance overexpressing proline-rich protein 33.

[0032] In some embodiments, the drug is an injectable or oral formulation; more preferably, it is an intravenous formulation. In some embodiments, the drug further includes one or more pharmaceutically acceptable excipients, carriers, and excipients.

[0033] Sixth, a method for inhibiting in vitro cardiomyocyte hypertrophy, comprising: adding ad- to the culture medium of an in vitro induced cardiomyocyte hypertrophy model. Prr33 To achieve PRR33 overexpression, PRR33 overexpression further suppresses hypertrophy marker genes. Nppa , Nppb and Myh7 The expression of [something] can be used to inhibit cardiomyocyte hypertrophy in vitro.

[0034] Seventh, a method for constructing a myocardial hypertrophy model, comprising: Inject knockdown / knockout into mice Prr33 AAV9 virus of the gene inhibits the body in mice Prr33 Gene expression, thereby obtaining a mouse model of cardiac hypertrophy; or Inhibit cardiomyocytes in vitro Prr33 Gene expression was used to obtain an in vitro model of hypertrophic cardiomyocytes.

[0035] Example 1: Identification of cardiac enrichment Prr33 Transcription and establishment Prr33 The link between it and cardiac hypertrophy (1) Rapid amplification of cDNA ends (RACE) technology to identify cDNA in the heart Prr33 Real transcript (SEQ ID NO: 4): RACE, or rapid amplification of cDNA ends, is a technique based on reverse transcription PCR to rapidly amplify the 5′ and 3′ ends of cDNA from a sample. The experimental sample for RACE was total RNA from heart tissue. First, reverse transcription primers were designed based on the naturally occurring Poly(A) tail at the 3′ end of mRNA to obtain the first cDNA strand through reverse transcription. Based on known... Prr33 The second cDNA strand was synthesized using gene-specific primers (GSPs) designed from the cDNA sequence. Subsequently, [the following was used]... Prr33 A gene-specific primer (GSP) and a 3′ end primer on the positive strand are used as a primer pair to amplify the obtained cDNA strand by PCR, thereby obtaining the 3′ end sequence of the cDNA (gene-specific primer → 3′ end). Based on known... Prr33Gene-specific primers (GSPs) were designed based on the cDNA sequence. The first cDNA strand was obtained through reverse transcription, and a poly(C) tail was added to the 3′ end of the cDNA using terminal deoxynucleotidyl transferase (TdT). A second cDNA strand was synthesized using primers designed based on the poly(C) tail. Subsequently, double-stranded cDNA was synthesized using gene-specific primers, with the second cDNA strand as a template. Finally, PCR amplification was performed using a pair of primers—the gene-specific primer (GSP) and the antisense 3′ end primer—to obtain the 5′ end sequence of the cDNA (gene-specific primer → 5′ end). Nested PCR for 5′ and 3′ RACE was performed using TAKARA's GXL high-fidelity enzyme (GXL Taq® polymerase). The obtained PCR products were recovered using a gel extraction kit, cloned into the Pwd18-T vector, and sequenced.

[0036] (2) qPCR detection of cardiac ions Prr33-C Transcripts and other known Prr33 Transcript expression levels differ: different designs were created based on nucleotide sequences. Prr33 Transcripts ( Prr33-201 , Prr33-202 , Prr33-203 , Prr33-sk , Prr33- Cardiac / C ) Specific qPCR primers were used to detect the expression levels of each transcript in cardiac tissue homogenate samples.

[0037] (3) Collection of heart samples at multiple time points: Wild-type mice were selected on the day of birth (P0), 1 day after birth (P1), 4 days after birth (P4), 7 days after birth (P7), 14 days after birth (P14), 21 days after birth (P21), and 30 days after birth (P30). The mice's hearts were collected at each time point, and the apical part of the heart tissue was cut off with a scalpel for RNA extraction.

[0038] (4) such as Figure 1 As shown, the heart is enriched with Prr33 Transcripts ( Prr33-C The sequence (SEQ ID NO: 4) is different from other transcripts. Figure 1 A), and the expression level is significantly higher than that of A). Prr33 Other transcripts ( Prr33-201 , Prr33-202 , Prr33-203 , Prr33-sk) ( Figure 1 B). This invention detected Prr33-C The expression level of mRNA was found to gradually increase during heart development. Figure 1 C), and Prr33-C The expression of genes involved in cardiomyocyte growth ( Tnnt2 , Myl2and Myh6 ) shows a coordinated upward adjustment ( Figure 1 D), and genes that are reprogrammed in hypertrophic hearts ( Myh7 , Tnni1 and Myl7 This indicates a coordinated downward adjustment. Figure 1 E).

[0039] Example 2: In vitro knockdown Prr33-C Promotes cardiomyocyte hypertrophy (1) Isolation of cardiomyocytes from P0 day milk mice The Miltenyi Neonatal Heart Dissociation Kit (MILTI) manufactured in Germany was used to isolate neonatal mouse cardiomyocytes. Specifically, the process included: 1) thoroughly mixing premixed enzyme solution 1 (enzyme P 62.5 μL and Buffer X 2300 μL) and premixed enzyme solution 2 (enzyme A 12.5 μL, enzyme D 100 μL and Buffer Y 25 μL).

[0040] 2) Mouse treatment: Immerse P0 day-old mice in 75% alcohol for 10 seconds (to prevent cell contamination). Then, make a small incision in the left sternum of the mouse using surgical scissors. Gently squeeze around the wound with your fingers to expel the heart. Next, place the heart in room temperature PBS buffer in a biosafety cabinet and allow the heart to pump out excess blood through spontaneous beating. At this time, remove blood clots and other tissues around the heart with forceps. Simultaneously, preheat the thoroughly mixed premixed enzyme solution 1 in a 37°C incubator for 5 minutes.

[0041] 3) After 5 minutes, mix the preheated premixed enzyme solution 1 and premixed enzyme solution 2. At the same time, put the suckling mouse heart (with excess blood removed) into the mixture and place it in a 37°C constant temperature incubator for 15 minutes to digest. Remove the mixture and slowly blow and aspirate the heart 20 times with a sterile 5mL dropper.

[0042] 4) Place the mixture back into the 37°C incubator and continue digestion for 15 minutes. Remove the mixture and slowly aspirate the heart 20 times using a sterile 3mL dropper.

[0043] 5) After digesting for another 15 minutes, remove the mixture and slowly aspirate and blow the heart 20 times with a 1 mL pipette. The heart tissue will then become a turbid liquid.

[0044] 6) Neutralize the enzyme solution with DMEM high-glucose medium containing 10% Gibco FBS. Filter the neutralized medium through a 70 μm cell sieve to remove undigested heart tissue. Centrifuge at 1000g for 5 minutes and discard the supernatant. At this point, the precipitate contains all the cells of the mouse heart (cardiomyocytes, cardiac fibroblasts, endothelial cells, etc.).

[0045] 7) To obtain relatively pure cardiomyocytes, the cells were resuspended in fresh DMEM high-glucose medium containing 10% Gibco FBS. The resuspended medium was placed in a 10 cm cell culture dish and incubated at 37°C in a CO2 incubator for differential sedimentation for 20 minutes. At this point, since the cardiomyocytes could not adhere quickly, most of the cardiomyocytes remained in the medium, while fibroblasts and other cells adhered to the bottom of the culture dish. The medium was collected to obtain neonatal mouse cardiomyocytes.

[0046] (2) Establishment of PE-induced mouse cardiomyocyte hypertrophy model First, prepare the stock solution by dissolving 20.3 g of phenylephrine in 1 mL of DMEM high-glucose medium and filtering it through a 0.22 μm filter for sterilization. The stock solution concentration is 100 mM. Prepare the stock solution fresh each time you use it. Dilute the stock solution 5000 times to obtain a 20 μM working solution. After starving the cardiomyocytes for 24 hours, add culture medium containing 20 μM working solution and simultaneously treat the cells with a virus for subsequent experiments.

[0047] (3) Immunofluorescence staining: After fixing with 4% PFA for 15 minutes, wash 3 times with 1x PBS buffer for 3 minutes each time; treat with 0.1% Triton X-100 for 15 minutes, wash 3 times with 1x PBS buffer for 5 minutes each time; block with sheep serum blocking solution for 60 minutes; add primary antibody and incubate overnight at 4°C; after incubating with primary antibody for 16 hours, wash 3 times with 1x PBS buffer for 5 minutes each time; add secondary antibody and incubate at room temperature for 1 hour, wash 3 times with 1x PBS buffer for 5 minutes each time; mount with mounting medium, dry in the dark and observe under a fluorescence microscope; cell well plates can be directly observed in PBS buffer and photographed for recording.

[0048] (4) Knockdown Prr33-C Gene expression: 1) Add to isolated wild-type neonatal mouse cardiomyocytes Prr33 Specific siRNA ( si-Prr33 ), control group added si-NC In order to achieve Prr33 The knockdown.

[0049] 2) In the separation Prr33 fl / fl Adding ad-Cre to the cardiomyocytes of lactating mice to achieve Prr33 The knockdown was performed, and ad-GFP was added to the control group.

[0050] (5) such as Figure 2 As shown, compared with the control group, Prr33 Knockdown leads to increased cardiomyocyte size. Figure 2 (A&B). In addition... Prr33Knockdown promotes hypertrophy marker genes Nppa , Nppb and Myh7 The expression ( Figure 2 C), and Prr33 The absence further enhanced the PE-induced hypertrophic response. Figure 2 DF).

[0051] Example 3: In vitro overexpression Prr33-C Inhibition of PE-induced cardiomyocyte hypertrophy (1) Isolation of cardiomyocytes from P0 day milk mice Miltenyi Neonatal Heart Dissociation Kit (manufactured in Germany) was used to isolate neonatal mouse cardiomyocytes, specifically including: 1) Mix premixed enzyme solution 1 (62.5 μL of enzyme P and 2300 μL of buffer X) and premixed enzyme solution 2 (12.5 μL of enzyme A, 100 μL of enzyme D and 25 μL of buffer Y) thoroughly.

[0052] 2) Mouse treatment: Immerse P0 day-old mice in 75% alcohol for 10 seconds (to prevent cell contamination). Then, make a small incision in the left sternum of the mouse using surgical scissors. Gently squeeze around the incision with your fingers to expel the heart. Next, place the heart in room temperature PBS buffer in a biosafety cabinet and allow the heart to pump out excess blood through spontaneous beating. At this time, remove blood clots and other tissues around the heart with forceps. Simultaneously, preheat the thoroughly mixed premixed enzyme solution 1 in a 37°C incubator for 5 minutes.

[0053] 3) After 5 minutes, mix the preheated premixed enzyme solution 1 and premixed enzyme solution 2. At the same time, put the suckling mouse heart (with excess blood removed) into the mixture and place it in a 37°C constant temperature incubator for 15 minutes to digest. Remove the mixture and slowly blow and aspirate the heart 20 times with a sterile 5mL dropper.

[0054] 4) Place the mixture back into the 37°C incubator and continue digestion for 15 minutes. Remove the mixture and slowly aspirate the heart 20 times using a sterile 3mL dropper.

[0055] 5) After digesting for another 15 minutes, remove the mixture and slowly aspirate and blow the heart 20 times with a 1 mL pipette. The heart tissue will then become a turbid liquid.

[0056] 6) Neutralize the enzyme solution with DMEM high-glucose medium containing 10% Gibco FBS. Filter the neutralized medium through a 70 μm cell sieve to remove undigested heart tissue. Centrifuge at 1000g for 5 minutes and discard the supernatant. At this point, the precipitate contains all the cells of the mouse heart (cardiomyocytes, cardiac fibroblasts, endothelial cells, etc.).

[0057] 7) To obtain relatively pure cardiomyocytes, the cells were resuspended in fresh DMEM high-glucose medium containing 10% Gibco FBS. The resuspended medium was placed in a 10 cm cell culture dish and incubated at 37°C in a CO2 incubator for differential sedimentation for 20 minutes. At this point, since the cardiomyocytes could not adhere quickly, most of the cardiomyocytes remained in the medium, while fibroblasts and other cells adhered to the bottom of the culture dish. The medium was collected to obtain neonatal mouse cardiomyocytes.

[0058] (2) Establishment of PE-induced mouse cardiomyocyte hypertrophy model: First, prepare the stock solution by dissolving 20.3 g of phenylephrine in 1 mL of DMEM high-glucose medium and filtering it through a 0.22 μm filter membrane for sterilization. The stock solution concentration was 100 mM at this time, and it was prepared fresh for each use. The stock solution was diluted 5000 times to obtain a working solution with a concentration of 20 μM. After starving the cardiomyocytes for 24 hours, culture medium containing 20 μM working solution was added, and the cells were simultaneously treated with virus for subsequent experiments.

[0059] (3) Immunofluorescence staining: After fixing with 4% PFA for 15 minutes, wash 3 times with 1x PBS buffer for 3 minutes each time; treat with 0.1% Triton X-100 for 15 minutes, wash 3 times with 1x PBS buffer for 5 minutes each time; block with sheep serum (or 5% BSA) blocking solution for 60 minutes; add primary antibody and incubate overnight at 4°C; after incubating with primary antibody for 16 hours, wash 3 times with 1x PBS buffer for 5 minutes each time; add secondary antibody and incubate at room temperature for 1 hour, wash 3 times with 1x PBS buffer for 5 minutes each time; mount with mounting medium, dry in the dark and observe under a fluorescence microscope; cell well plates can be directly observed in PBS buffer and photographed for recording.

[0060] (4) In vitro overexpression Prr33-C Adding ad- to wild-type neonatal mouse cardiomyocytes Prr33 To achieve Prr33 Overexpression, control group added ad- Lacz .

[0061] (5) such as Figure 3 As shown, Prr33 Overexpression significantly reduced PE-induced cardiomyocyte size ( Figure 3 (A&B). In addition... Prr33 Overexpression of hypertrophy marker genes Nppa , Nppb and Myh7 The expression ( Figure 3 C).

[0062] Example 4: In vivo knockdown / knockout Prr33Promotes cardiac hypertrophy and heart failure (1) Preparation of AAV-shRNA: First, design Prr33-C shRNA, using molecular cloning technology to sh-Prr33 and scramble The sequence was introduced into an AAV9 plasmid containing the U6 promoter to obtain AAV9- ShPrr33 (Plasmid information such as) Figure 9 (as shown) and AAV9- scramble The main plasmid of AAV9 and the adeno-associated virus packaging helper plasmids AAV9:Rep-Cap and pHelper (pAd deltaF6, Penn Vector Core) were simultaneously transfected into 293T cells. After 60 hours, the cells were collected and lysed to release viral particles, and the adeno-associated virus was concentrated by gradient centrifugation.

[0063] (2) Subcutaneous injection of AAV9 virus into newborn mice: Newborn wild-type C57 / BL6J mice at day P0 were selected. Hands were first disinfected with alcohol and the odor was masked. AAV9- was then injected subcutaneously into the back of the mouse using an insulin needle. shPrr33 / AAV9- scramble For virus injection, the needle should be inserted subcutaneously on the back with the needle bent, and the injection should be left in place for an additional 5 seconds after injection to prevent leakage. Finally, the suckling mice should be returned to their parents' cages, and the original bedding should be gently covered to remove any residual scent from the injector and prevent the parents from eating the suckling mice. Neonatal mice (day 1 after birth) were injected subcutaneously with 1x10 AAV9-shRNA. 11 Processing of pellets / pups.

[0064] (3) Mouse cardiac ultrasound: Real-time, dynamic, and non-invasive observation of cardiac function in mice after myocardial infarction surgery was performed using a small animal cardiac ultrasound system. A Visual Sonics Vevo2100 imaging system (Visual Sonics, Toronto, Canada) was used for echocardiography, employing an 18-38 MHz probe (model MS-400). Mice were first fixed in a supine position on a 37°C constant-temperature plate using tape. B-mode and M-mode echocardiograms were acquired in the transverse section at the midpoint of the papillary muscles. M-mode echocardiography was used to measure the left ventricular anterior wall, left ventricular diameter, and left ventricular posterior wall thickness at end-systole or end-diastole of the mouse heart for at least five consecutive cardiac cycles. The system's built-in software calculated and statistically analyzed cardiac function indicators in mice, including left ventricular ejection fraction (EF), left ventricular fractional shortening (LVFS), left ventricular internal dimension in end-diastole (LVIDd), and left ventricular internal dimension in end-systole (LVIDs).

[0065] (4) Histomorphological analysis: Mice were injected intraperitoneally with 4% chloral hydrate according to their body weight. After complete anesthesia, the mice were sacrificed. The heart was removed by opening the chest and allowed to beat naturally in PBS buffer at room temperature to pump out excess blood. The heart was then gently dried with absorbent paper and weighed using a 0.01% balance. The heart tissue was fixed directly in 10% neutral formaldehyde solution to prepare for subsequent paraffin sectioning. Sirus red / Fast green staining was performed. The paraffin sections were first dewaxed and rehydrated in a gradient manner, then washed with PBS buffer for 3 minutes, repeated 3 times. Sirus red staining solution prepared with 0.5% picric acid was then added to the sample tissue and stained at room temperature for 2 hours. The tissue was then carefully and slowly rinsed with running water for 3 minutes to remove excess staining. 0.1% Fast green staining solution was added and stained for 15 minutes. The tissue was then slowly rinsed with running water for 5 minutes to remove excess staining. Finally, after staining, the slides were mounted with a neutral resin containing xylene and air-dried before being photographed and observed under a microscope. Normal myocardial tissue appeared green, while fibrotic areas appeared red. Stereoscopic microscopy was used for recording.

[0066] (5) Cardiac cell-specific knockout Prr33 Mouse construction: for further validationPrr33 Regarding its role in cardiac hypertrophy, this invention constructed a heart-specific Prr33 knockout mouse. This invention will... Prr33 fl / fl Mice were crossed with mice that could be induced to express heart-specific Cre recombinase (aMHC-MerCreMer). Cardiomyocyte-specific knockout was achieved by administering five tamoxifen injections to 8-week-old mice to induce Cre recombinase expression. Prr33 mice ( Prr33 cKO ).

[0067] (6) Establishment of a mouse model of aortic coarctation: 4-week-old mice were selected. Prr33 fl / fl aMHC-MerCreMer mice were induced to express Cre recombinase through intraperitoneal injection of tamoxifen (45 mg / kg / day). Four weeks after tamoxifen injection, aortic coarctation (TAC) surgery was performed. Mice were first weighed and anesthetized via intraperitoneal injection of 100 μL of 4% chloral hydrate solution per 10 g of body weight. The mice were disinfected and prepared. The skin was cut along the trachea, and the muscles on both sides of the trachea were bluntly dissected and drawn apart with sutures. A small section of the sternum was carefully cut to expose the field of vision. Glands and adipose tissue were carefully separated to locate the aortic arch. A 27G blunt needle was placed across the aorta and tied with two knots using 6-0 suture. The blunt 27G needle was then removed, narrowing the aortic arch to the thickness of the 27G needle. The muscles and skin were then carefully sutured, and the mice were observed postoperatively. In the same batch of experiments, mice that underwent only open-chest surgery but not ligation were designated as the sham group.

[0068] (7) For example Figure 4 As shown, this invention utilizes AAV9- ShPrr33 In vivo exploration of carriers Prr33 The effect. Subcutaneous injection of 1x10⁻⁶ in newborn mice (P1) 11 AAV9- of each viral particle ShPrr33 Vector, to specifically knock down cardiomyocytes Prr33 The control group was injected with an equal dose of the control vector AAV9- Scramble Two months later, the present invention collected samples and found that knockdown... Prr33 This leads to a significant increase in heart size. Figure 4 A). Histological staining of the heart cross-section showed a significantly increased myocardial cross-sectional area, morphologically confirming the presence of myocardial cells. Prr33 Knockdown induced cardiac hypertrophy ( Figure 4 B). To more directly observe cardiomyocyte hypertrophy, this invention performed wheat germ agglutinin (WGA) staining on cardiac tissue sections ( Figure 4 C). The results showed that, Prr33 Knockdown increased the cross-sectional area of ​​cardiomyocytes and promoted hypertrophic growth of cardiomyocytes. Cardiac function assessment of adult mice (2 months old) using echocardiography revealed significantly impaired cardiac function in the knockdown group. Figure 4 D). Left ventricular fractional shortening (FS) ( Figure 4 E) and ejection fraction (EF) Figure 4 F) were all significantly reduced. Similarly, this invention collected mouse RNA for qPCR gene analysis, and the results showed that the knockdown group included Nppa , Nppb and Myh7 Genes included were significantly upregulated ( Figure 4 G), confirming the occurrence of cardiac hypertrophy at the gene level. This invention detected... Prr33 cKO The mouse heart was significantly enlarged ( Figure 4 H), histological staining further confirmed significant cardiac hypertrophy ( Figure 4 I). Meanwhile, the present invention observed... Prr33 cKO The heart-to-body weight ratio of mice was significantly increased. Figure 4 J), significant decline in cardiac function ( Figure 4 K), and upregulation of hypertrophy marker gene expression (K ...) Figure 4 L).

[0069] To investigate Prr33 The effect of this invention on cardiac hypertrophy under pathological conditions, compared with a control group ( Prr33 fl / fl )and Prr33 Myocardial-specific knockout mice ( Prr33 cKO Aortic arch coarctation (TAC) was performed to simulate cardiac hypertrophy caused by human pressure overload. At 12 weeks post-procedure, compared to the control group, Prr33 cKO The mouse heart showed significant enlargement ( Figure 4 H). Histological staining of the heart cross-section visually revealed its hypertrophic phenotype ( Figure 4 I), and Prr33 cKO The heart weight to body weight ratio (HW / BW) of mice was significantly increased. Figure 4 J). Echocardiographic analysis showed that after TAC, Prr33 The cardiac function of knockout mice continued to decline. Figure 4 K), at the same time Nppa , Nppb and Myh7 The expression of was significantly upregulated ( Figure 4 M).

[0070] Example 5: In vivo overexpression Prr33-C Inhibiting TAC-induced cardiac hypertrophy and heart failure (1) Overexpression Prr33 Preparation of adeno-associated virus: First, specific PCR primers were used to clone... Prr33-C The CDS sequence was obtained, and molecular cloning technology was used to obtain the CDS sequence and the control sequence ( luciferase The AAV9 plasmid containing the myocardial-specific promoter cTNT was introduced to obtain AAV9- Prr33 (Plasmid information such as) Figure 10 (as shown) and AAV9- luciferase The main plasmid of AAV9 and the adeno-associated virus packaging helper plasmids AAV9:Rep-Cap and pHelper (pAd deltaF6, Penn Vector Core) were simultaneously transfected into 293T cells. After 60 hours, the cells were collected and lysed to release viral particles, and the adeno-associated virus (vector AAV9, whose original sequence is cTNT-PRR33) was concentrated by gradient centrifugation.

[0071] (2) Establishment of a mouse model of aortic coarctation: Four-week-old wild-type mice were selected for aortic coarctation (TAC) surgery. The mice were weighed and anesthetized by intraperitoneal injection of 100 μL of 4% chloral hydrate solution per 10g of body weight. The mice were disinfected and prepared. The skin was cut along the trachea, and the muscles on both sides of the trachea were bluntly dissected and pulled apart with sutures. A small section of the sternum was carefully cut to expose the field of vision. The glands and adipose tissue were carefully separated, and the aortic arch was located. A 27G blunt needle was placed across the aorta and tied with two knots using 6-0 suture. The blunt 27G needle was then removed, at which point the aortic arch was narrowed to the thickness of the 27G needle. The muscles and skin were then carefully sutured, and the mice were observed postoperatively. In the same batch of experiments, mice that underwent only open-chest surgery but not ligation were designated as the sham group.

[0072] (3) Ocular injection of AAV9 virus in adult mice: Adult mice that received AAV9 treatment were injected intraocularly during TAC surgery. luciferase / Prr33 1x10 per mouse 12 Particle. Deeply anesthetize the animal, gently open the eyelids, and under a surgical microscope or high-powered magnifying glass, gently grasp the target area with micro-forceps. Insert the tip of a 30G insulin needle into the subconjunctival space at a shallow angle of less than 15 degrees, slowly inject the medication, and slowly withdraw the needle, applying slight pressure for a moment to prevent backflow. Routinely observe the animal after recovery.

[0073] (3) Mouse cardiac ultrasound: Real-time, dynamic, and non-invasive observation of cardiac function in mice after myocardial infarction surgery was performed using a small animal cardiac ultrasound system. Echocardiography was conducted using a Visual Sonics Vevo 2100 imaging system (Visual Sonics, Toronto, Canada) with an 18-38 MHz probe (modelMS-400). Mice were first fixed in a supine position on a 37°C constant-temperature plate using tape. B-mode and M-mode echocardiograms were acquired in the transverse section at the midpoint of the papillary muscles. M-mode echocardiography was used to measure the left ventricular anterior wall, left ventricular diameter, and left ventricular posterior wall thickness at end-systole or end-diastole of the mouse heart for at least five consecutive cardiac cycles. The system's built-in software calculated and statistically analyzed cardiac function indicators in mice, including left ventricular ejection fraction (EF), left ventricular fractional shortening (LVFS), left ventricular internal dimension in end-diastole (LVIDd), and left ventricular internal dimension in end-systole (LVIDs).

[0074] (4) Histomorphological analysis: Mice were injected intraperitoneally with 4% chloral hydrate according to their body weight. After complete anesthesia, the mice were sacrificed. The heart was removed by opening the chest and allowed to beat naturally in PBS buffer at room temperature to pump out excess blood. The heart was then gently dried with absorbent paper and weighed using a 0.01% balance. The heart tissue was fixed directly in 10% neutral formaldehyde solution to prepare for subsequent paraffin sectioning. Sirus red / Fast green staining: First, the paraffin sections were dewaxed and rehydrated in a gradient manner. Then, they were washed with PBS buffer for 3 minutes, 3 times. Sirus red staining solution prepared with 0.5% picric acid was then added to the sample tissue and stained at room temperature for 2 hours. Then, the tissue was carefully and slowly rinsed with running water for 3 minutes to remove excess staining. After staining with 0.1% Fast green staining solution for 15 minutes, the tissue was slowly rinsed with running water for 5 minutes to remove excess staining. Finally, after staining, the slides were mounted with a neutral resin containing xylene and air-dried before being photographed and observed under a microscope. Normal myocardial tissue appeared green, while fibrotic areas appeared red. Stereoscopic microscopy was used for recording.

[0075] (5) such as Figure 5 As shown, the present invention was tested in vivo. Prr33Overexpression protects against TAC-induced hypertrophy. Injection of AAV9- Prr33 The mouse heart size was significantly reduced ( Figure 5 A), histological staining confirmed that it significantly inhibited cardiac hypertrophy ( Figure 5 B). Wheat germ agglutinin (WGA) staining showed that after TAC surgery, injection of AAV9- Prr33 The cross-sectional area of ​​the myocardial cells in the mice was smaller than that in the control group ( Figure 5 C&D). Compared with control virus (AAV9- Luciferase Compared to the injection group, AAV9- Prr33 The heart-to-body weight ratio decreased in the injection group mice after TAC surgery. Figure 5 E). Furthermore... Prr33 Overexpression significantly improved cardiac function in mice. Figure 5 F&G), while the expression of hypertrophic marker genes in response to hypertrophic stimulation is reduced ( Figure 5 H).

[0076] Example 6: Prr33 Regulates genes encoding cytoskeleton and cardiac hypertrophy factor. (1) Transcriptome Sequencing: The RNA samples used for sequencing were obtained from heart tissues of adeno-associated virus knockdown PRR33 and control groups. RNA transcriptome sequencing was performed by Novogene's Illumina platform. The main process is as follows: First, all RNA was extracted from the heart tissues using Trizol solution. Based on the structural feature that all mRNAs have polyA tails, mRNAs with polyA tails were enriched using Oligo(dT) magnetic beads. After quantification, 1 μg of RNA was used as total RNA for sequencing. The library was constructed using NEB's Ultra™ RNA Library Prep Kit and then sequenced using Illumina. Fluorescently labeled dNTPs, DNA polymerase, and adapter primers were added to the sequencing flow cell, and then amplification was performed. After the sequencer captured the fluorescence signal, it was converted into a quantifiable sequencing peak, and the sequence information of the fragment to be tested was obtained. Bioinformatics Analysis Process: The core of RNA-seq data analysis is to perform bioinformatics analysis on genes with significant expression differences. By comparing the differences in gene expression, the biological functions of these differentially expressed genes can be further analyzed. The analysis process includes quality control, quantification, comparison, differential significance analysis, functional enrichment, variant sites, alternative splicing, and fusion genes. After obtaining RNA-seq data, these differentially expressed genes are enriched using methods such as GO or KEGG to uncover potential mechanisms and screen for therapeutic targets.

[0077] (2) such as Figure 6 As shown, compared to the control heart,Prr33 Two hundred differentially expressed genes were found in the missing heart (log2 fold change > 0.5 and P < 0.05), of which 89 genes were upregulated and 111 genes were downregulated. Figure 6 A). KEGG pathway analysis showed that, Prr33 In the missing heart, genes associated with dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and myocardial contraction were significantly altered, consistent with the hypertrophic phenotype observed in vitro and in vivo in this invention. Figure 6 B). GO enrichment showed that many downregulated genes were associated with cytoskeleton and sarcomere structure (B). Figure 6 C&D).

[0078] Example 7: Screening and Validation of Key Proteins Interacting with PRR33 (1) Novel Proximity Marker Turbo ID: PRR33-Turbo-ID is generated by fusing PRR33 with TurboID sequences. In short, in Ad-Prr33-TurboID Approximately 48 hours after infecting NMCMs, 400 μM biotin was added and the cells were incubated for another 30 minutes. Samples were then collected, and biotinylated proteins were enriched using streptavidin magnetic beads. The captured proteins were subsequently separated by SDS-PAGE. The entire gel channel was excised, and the gel was systematically sliced ​​from top to bottom into small fragments, which were then processed for mass spectrometry analysis.

[0079] (2) Bimolecular fluorescence complementarity, BiFC: The BiFC template plasmid CSII-EF-MCS-IRES2-Venus-PGK1 was purchased from the plasmid resource platform of Suzhou Research Institute (No.: SP-2446). Based on the main chain plasmid, PRR33-VN, MYOZ2-VN, PRR33-VC, LDB3-VC and calcineurin-VC were constructed. About 24 hours after transfecting the BiFC plasmid into 293T cells using Lipofectamine 3000 (Invitrogen), fluorescence signals were examined and imaged.

[0080] (3) Immunoprecipitation, Co-IP: MYOZ2-FLAG, calcineurin-HA, and LDB3-HA plasmids were constructed based on pcDNA3.1. Lentiviral knockdown was then performed. Prr33 (Lenti- shPrr33 Lenti- scramble As a control, adenovirus was used for overexpression in the HL1 cell line. Prr33 (Ad- Prr33 Ad- Lazz(As a control). Two days later, MYOZ2-FLAG and calcineurin-HA plasmids were transfected to test the interaction between MYOZ2 and calcineurin. MYOZ2-FLAG and LDB3-HA plasmids were transfected to test the interaction between MYOZ2 and LDB3. Twenty-four hours later, M2-Flag magnetic beads were used to enrich proteins tagged with HA. A detailed list of antibodies used in this study is shown in Table 1.

[0081] Table 1 (4) Immunofluorescence staining: After fixing with 4% PFA for 15 minutes, wash 3 times with 1x PBS buffer for 3 minutes each time; treat with 0.1% Triton X-100 for 15 minutes, wash 3 times with 1x PBS buffer for 5 minutes each time; block with sheep serum blocking solution for 60 minutes; add primary antibody and incubate overnight at 4°C; after incubating with primary antibody for 16 hours, wash 3 times with 1x PBS buffer for 5 minutes each time; add secondary antibody and incubate at room temperature for 1 hour, wash 3 times with 1x PBS buffer for 5 minutes each time; mount with mounting medium, dry in the dark and observe under a fluorescence microscope; cell well plates can be directly observed in PBS buffer and photographed for recording.

[0082] (5) such as Figure 7 As shown, this invention designed a PRR33 proximity labeling experiment, identifying a total of 110 proteins labeled with PRR33-Turbo-ID. GO enrichment analysis showed that these proteins are mainly associated with the cytoskeleton and cardiac hypertrophy. Figure 7 A). Notably, in this category, two cytoskeletal proteins, MYOZ2 and LDB3, were significantly enriched in the PRR33-TURBO-ID sample. Figure 7 B). Therefore, this invention will focus subsequent research on these two candidate proteins.

[0083] To further investigate the possible interactions between PRR33 and these two candidate proteins, bimolecular fluorescence complementation (BiFC) experiments were performed in live cells. The results showed that LDB3 could form a complex with PRR33, while MYOZ2 and PRR33 only showed weak fluorescence signals. Figure 7 C). To verify these findings, the present invention independently replicated the Turbo-ID experiment and confirmed that LDB3 can be tagged by PRR33-Turbo-ID ( Figure 7 D). Furthermore, this invention also verified the interaction between LDB3 and PRR33 through co-immunoprecipitation (Co-IP) experiments. Figure 7E). These results clearly confirm that PRR33 can form a complex with LDB3. LDB3 is a skeletal protein located in the Z-disc of the sarcomere. LDB3 forms stable complexes with various myosin proteins (including α-actin, CYRP3, and MYOZ) through its PDZ domain, playing a crucial role in maintaining the structural integrity and mechanical stability of cardiac myocyte sarcomeres. Knockout Ldb3 It caused severe congenital cardiomyopathy in mice, which died shortly after birth due to muscle failure during the perinatal period. Ldb3 Myocardial-specific knockout of PRR33 impairs cardiac function and induces dilated cardiomyopathy. This is similar to what was observed in the loss of PRR33 function in cardiomyocytes in this invention. More importantly, this invention uses immunostaining techniques to confirm the localization of PRR33, finding that PRR33 co-localizes with Z-disc-labeled α-actin in cardiomyocytes (…). Figure 7 F).

[0084] Example 8: Prr33 Improving cardiac hypertrophy by influencing the NFAT signaling pathway by regulating cardiomyocyte cytoskeleton remodeling (MYOZ2-LDB3-CALCINEURIN). (1) Bimolecular fluorescence complementarity, BiFC: The BiFC template plasmid CSII-EF-MCS-IRES2-Venus-PGK1 was purchased from the plasmid resource platform of Suzhou Research Institute (No.: SP-2446). Based on the main chain plasmid, PRR33-VN, MYOZ2-VN, PRR33-VC, LDB3-VC and calcineurin-VC were constructed. About 24 hours after transfecting the BiFC plasmid into 293T cells using Lipofectamine 3000 (Invitrogen), fluorescence signals were examined and imaged.

[0085] (2) Co-immunoprecipitation, Co-IP: MYOZ2-FLAG, calcineurin-HA, and LDB3-HA plasmids were constructed based on pcDNA3.1. Lentiviral knockdown was then performed. Prr33 (Lenti- shPrr33 Lenti- scramble As a control, adenovirus was used for overexpression in the HL1 cell line. Prr33 (Ad- Prr33 Ad- Lazz(As a control). Two days later, MYOZ2-FLAG and calcineurin-HA plasmids were transfected to test the interaction between MYOZ2 and calcineurin. MYOZ2-FLAG and LDB3-HA plasmids were transfected to test the interaction between MYOZ2 and LDB3. Twenty-four hours later, M2-Flag magnetic beads were used to enrich proteins tagged with HA. A detailed list of antibodies used in this study is shown in Table 1.

[0086] (3) The lentivirus knockdown plasmid vector was purchased from SBI (System Biosciences). This invention employs a seamless cloning method to design... Prr33 shRNA sequence and control scramble The sequence was cloned into the plasmid. Sequencing was then used to verify the correctness of the cloning. A large number of master and helper plasmids for virus construction were prepared using the MN plasmid large-scale extraction kit.

[0087] 1) Virus packaging: 293T cells were cultured in a cell culture incubator using 15 cm cell culture dishes. The amount of virus used for cell-level experiments was approximately the amount obtained from the purification of virus solution from 10 15 cm cell culture dishes. After the cells reached a density of 70-80%, lentivirus packaging-related plasmids were transfected using PEI transfection reagent.

[0088] 2) Virus Collection and Concentration: 72 hours post-transfection, obvious cytopathic effects and cell rounding are observed. Collect the supernatant and discard the cell pellet. Centrifuge the obtained supernatant at 2000g for 10 min, then filter through a 0.45 μm filter membrane to remove cell debris. Prepare a 10% glucose solution for virus purification. First, add the virus purification solution to the bottom of the centrifuge flask, then slowly add the filtered lentivirus supernatant on top of the virus purification solution. Note that the virus solution and purification solution should not be mixed; the ratio of virus solution to purification solution is 4:1. Centrifuge at 15000g for 4 hours to obtain purified lentivirus. After centrifugation, visible white plates can be seen at the bottom of the test tube; this is the lentivirus particle precipitate. Collect the virus, aliquot, and store at -80℃.

[0089] 3) Virus titer determination: Lentiviral titer was detected using quantitative real-time PCR combined with a standard curve.

[0090] (4) Immunofluorescence staining: After fixing with 4% PFA for 15 minutes, wash 3 times with 1x PBS buffer for 3 minutes each time; treat with 0.1% Triton X-100 for 15 minutes, wash 3 times with 1x PBS buffer for 5 minutes each time; block with sheep serum blocking solution for 60 minutes; add primary antibody and incubate overnight at 4°C; after incubating with primary antibody for 16 hours, wash 3 times with 1x PBS buffer for 5 minutes each time; add secondary antibody and incubate at room temperature for 1 hour, wash 3 times with 1x PBS buffer for 5 minutes each time; mount with mounting medium, air dry in the dark and observe under a fluorescence microscope; cell well plates can be directly observed in PBS buffer and photographed for recording.

[0091] (5) NFAT luciferase activation experiment: Mouse cardiomyocyte cell line HL-1 cells were seeded in 96-well plates and Lenti- shPrr33 and control group virus Lenti- scramble Twelve hours later, the NFAT reporter plasmid and internal control plasmid were transfected into cells using Lipo3000 liposomes. Twenty-four hours later, the fluorescence values ​​(Firefly Luciferase and Renilla Luciferase) were measured using a luciferase reporter gene system (Promega Dual-Glo). When the intracellular NFAT signaling pathway is activated, the activated NFAT transcription factor binds to the response element of the reporter plasmid, driving the expression of the downstream luciferase gene. By detecting luciferase activity, the activation intensity of the NFAT signaling pathway can be quantitatively reflected.

[0092] (6) For example Figure 8 As shown, BiFC data confirm that MYOZ2 can interact with both calcineurin and LDB3. Figure 8 A). These three proteins form a complex to regulate cardiac hypertrophy. Based on these results, this invention identified PRR33 as a binding protein of LDB3. This invention hypothesizes that PRR33 regulates cardiac hypertrophy by modulating this complex. To verify this hypothesis, this invention knocked down or overexpressed PRR33 in the BiFC system and examined the interactions between MYOZ2 and calcineurin, as well as between MYOZ2 and LDB3. The results showed that knocking down PRR33 inhibited the binding of MYOZ2 to calcineurin while promoting the interaction between MYOZ2 and LDB3, leading to the release of calcineurin and activation of the hypertrophy signaling pathway. Conversely, overexpression of PRR33 enhanced the binding of MYOZ2 to calcineurin while inhibiting the interaction between MYOZ2 and LDB3, indicating that more calcineurin was immobilized to inhibit the hypertrophy signaling pathway. Figure 8 A&B). Furthermore, this invention further validated this conclusion using a mouse cardiomyocyte cell line (HL-1) via co-immunoprecipitation (Co-IP) experiments. Figure 8 C&D). To explore Prr33 To investigate the activation of the calcineurin-NFAT signaling pathway under conditions of deletion or overexpression, this invention employed an NFAT luciferase reporter gene assay. The results showed that... Prr33 Knockdown significantly enhanced NFAT luciferase signaling ( Figure 8 E), while in the PE-induced hypertrophy model, Prr33 Overexpression significantly weakened NFAT luciferase signaling. Figure 8 F). Based on this data, the present invention concludes that: knockdown Prr33 It releases calcineurin, thereby activating the NFAT signaling pathway and promoting cardiac hypertrophy; conversely, overexpression... Prr33 This will fix calcineurin, leading to inhibition of the NFAT signaling pathway and prevention of cardiac hypertrophy.

[0093] Summarize This invention reveals the link between PRR33 and the cytoskeleton signaling network that regulates cardiac hypertrophy remodeling. This invention discovers... Prr33 The deletion of this gene downregulates genes involved in cytoskeleton arrangement, sarcomere structure, and myocardial contraction, indicating that PRR33 plays a crucial role in maintaining the structural integrity and function of cardiomyocytes. This finding aligns with current mainstream view that cytoskeleton dysfunction is a major driver of pathological hypertrophy and cardiomyopathy.

[0094] From a mechanistic perspective, this invention identifies LDB3 as a direct interacting protein of PRR33. LDB3 is a core Z-disc protein maintaining sarcomere stability; its deficiency leads to severe cardiomyopathy and premature death in mice. The phenotypic similarity between the LDB3 and PRR33 loss-of-function models reinforces the view that PRR33 maintains Z-disc homeostasis by binding to LDB3. Furthermore, proteomics screening and BiFC experiments located PRR33 on the LDB3-MYOZ2-calcineurin regulatory axis—a known molecular switch controlling NFAT signaling and hypertrophic growth. MYOZ2 anchors calcineurin to the Z-disc, inhibiting its activation; disruption of this anchoring mechanism enhances calcineurin-NFAT signaling, accelerating the progression of pathological hypertrophy.

[0095] The data from this invention show that PRR33 regulates the balance of this complex by modulating the interaction preference of MYOZ2. PRR33 deficiency weakens the binding of MYOZ2 to calcineurin while enhancing the association between MYOZ2 and LDB3, thereby releasing calcineurin and activating NFAT signaling. Conversely, PRR33 overexpression promotes MYOZ2-calcineurin interaction and reduces MYOZ2-LDB3 binding, thereby inhibiting NFAT signaling. These bidirectional regulatory effects on the calcineurin-NFAT pathway, combined with its alterations in cytoskeleton gene expression, provide a plausible mechanistic explanation for the observed structural disorder and hypertrophic phenotype. Importantly, these findings reveal a previously unknown regulatory level in Z-disk-based signal integration: PRR33 precisely regulates the spatial availability of calcineurin not by altering protein abundance, but by reshaping the dynamics of the protein complex.

[0096] The gain-of-function protective effect of PRR33 observed in the TAC model further supports its potential for clinical translational therapy. In a disease context characterized by increased mechanical load, disrupted sarcomere stability, and aberrant activation of calcineurin signaling, PRR33 overexpression can reduce cardiac enlargement, normalize the expression of hypertrophy-related genes, and maintain ventricular function. These findings suggest that enhancing PRR33 expression or stabilizing its interaction with the LDB3-MYOZ2-calcineurin complex may represent a novel therapeutic approach to alleviating pathological hypertrophy.

[0097] In summary, this invention identifies PRR33 as a key cytoskeleton-related regulator that protects the myocardium from pathological hypertrophic remodeling. PRR33 inhibits NFAT activation and maintains sarcomere homeostasis by regulating the LDB3-MYOZ2-calcineurin complex, integrating structural and biochemical signals at the Z-disc. These findings reveal a novel regulatory mechanism in the cytoskeleton signaling network and highlight PRR33 as a potential therapeutic target for cardiac hypertrophy and heart failure.

Claims

1. A proline-rich protein 33, characterized in that: It contains an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A polynucleotide encoding proline-rich protein 33 as described in claim 1, characterized in that: It contains a nucleotide sequence as shown in SEQ ID NO:3 or SEQ ID NO:

4.

3. An adeno-associated virus overexpressing proline-rich protein 33, characterized in that: It contains nucleotide sequences as shown in SEQ ID NO:3 or SEQ ID NO:

4.

4. The application as described in claim 3, characterized in that: The vector of the adeno-associated virus is AAV9, and its original sequence is cTNT-Prr33.

5. The application of proline-rich protein 33 or substances overexpressing proline-rich protein 33 in the preparation of drugs for cardiomyopathy related to cardiac hypertrophy, characterized in that: The proline-rich protein 33 contains an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

6. The application as described in claim 5, characterized in that: The aforementioned cardiac hypertrophy-related cardiomyopathy refers to hypertrophic cardiomyopathy or dilated cardiomyopathy.

7. The application as described in claim 5, characterized in that: The substance overexpressing proline-rich protein 33 is an adeno-associated virus overexpressing proline-rich protein 33, containing the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

8. A drug for the prevention or treatment of cardiomyopathy associated with cardiac hypertrophy, characterized in that: This includes substances that express or overexpress proline-rich protein 33.

9. A method for inhibiting cardiomyocyte hypertrophy in vitro, characterized in that... include: Add ad- to the culture medium of the in vitro induced cardiomyocyte hypertrophy model Prr33 To achieve PRR33 overexpression, PRR33 overexpression inhibits cardiomyocyte hypertrophy, including inhibiting hypertrophy marker genes. Nppa , Nppb and Myh7 The expression of [a substance] and the phenotype of cardiomyocyte hypertrophy were observed, thereby inhibiting cardiomyocyte hypertrophy in vitro.

10. A method for constructing a myocardial hypertrophy model, characterized in that... include: Inject knockdown / knockout into mice Prr33 AAV9 virus of the gene inhibits the body in mice Prr33 Gene expression was used to obtain a mouse model of cardiac hypertrophy. or Inhibit cardiomyocytes in vitro Prr33 Gene expression was used to obtain an in vitro model of hypertrophic cardiomyocytes.

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