Application of inhibitor targeting Connexin43 gene in preparation of medicine for preventing or treating myocardial fibrosis

Abstract

The invention belongs to the technical field of gene therapy, and particularly relates to application of an inhibitor of a targeted Connexin43 gene in preparation of a medicine for preventing or treating myocardial fibrosis. In order to define the effect of connexin43 in the cardiac hypertrophy process caused by myocardial cell hypertrophy induced by pathological matrix stiffness and pressure overload, the invention provides application of an inhibitor of a targeted Connexin43 gene in preparation of a medicine for preventing or treating myocardial fibrosis. According to the application disclosed by the invention, the Connexin43 gene is found to be used as a potential target for preventing or treating myocardial fibrosis for the first time, and a myocardial cell hypertrophy process induced by pathological matrix hardness and a myocardial hypertrophy process caused by overload of pressure can be relieved by knocking down the expression of the Connexin43 gene or inhibiting the semi-channel activity of the Connexin43. Therefore, the inhibitor targeting the Connexin43 gene can be used for preparing the medicine for preventing or treating the myocardial fibrosis, and a new way is provided for treating the myocardial fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy technology, specifically relating to the use of an inhibitor targeting the Connexin43 gene in the preparation of drugs for the prevention or treatment of myocardial fibrosis. Background Technology

[0002] Cardiomyocyte hypertrophy, characterized by increased volume and weight, is a key mechanism in pathological myocardial hypertrophy. Myocardial hypertrophy often results from myocardial injury factors such as pressure overload and myocardial infarction, and is a common pathological basis and independent risk factor for cardiovascular diseases such as heart failure. Extensive deposition of extracellular matrix (ECM) is a significant characteristic. Deposited ECM increases the matrix stiffness (or rigidity) of the microenvironment surrounding cardiomyocytes by 1-5 times, leading to decreased myocardial tissue compliance and cardiac dysfunction. However, the mechanism by which abnormally increased matrix stiffness is linked to cardiomyocyte hypertrophy remains unclear. Therefore, exploring the role and mechanism of pathological matrix stiffness in cardiomyocyte hypertrophy is of great significance for developing clinical strategies for the prevention and treatment of pathological myocardial hypertrophy.

[0003] Given that intercellular communication is fundamental to the coordinated contractile function of cardiomyocytes, it is urgent to explore targets that mediate cardiomyocyte hypertrophy through intercellular communication under pathological matrix stiffness. The connexin 43 not only mediates gap junctions between cardiomyocytes, facilitating intercellular communication, but its hemichannel protein also mediates communication between cells and the extracellular matrix. However, changes in connexin 43 expression, hemichannel opening, and its relationship with cardiomyocyte hypertrophy under pathological matrix stiffness have not yet been reported. Summary of the Invention

[0004] The technical problem to be solved by this invention is to clarify the role of the connexin43 in the process of cardiomyocyte hypertrophy induced by pathological matrix stiffness and myocardial hypertrophy caused by pressure overload, and to develop a new drug for the treatment of myocardial fibrosis.

[0005] Therefore, the technical solution of the present invention to solve the above-mentioned technical problems is as follows: providing the use of an inhibitor targeting the Connexin43 gene in the preparation of a drug for the prevention or treatment of myocardial fibrosis; the inhibitor is siRNA containing a sequence selected from SEQ ID NO:2-3; the myocardial fibrosis is cardiomyocyte hypertrophy or myocardial hypertrophy.

[0006] The beneficial effects of this invention are as follows: This invention is the first to discover that connexin43 protein expression is upregulated and its half-channel activity is enhanced in a pathological matrix stiffness-induced cardiomyocyte hypertrophy model. Connexin43 expression is also upregulated in the heart tissue of mice with stress overload-induced myocardial hypertrophy, indicating that connexin43 participates in regulating both the pathological matrix stiffness-induced cardiomyocyte hypertrophy process and the stress overload-induced myocardial hypertrophy progression. Subsequently, this invention administered connexin43 half-channel inhibitors to mice via both slow-release pump and tail vein administration, finding that cardiac function was significantly improved in mice with TAC-induced pathological myocardial hypertrophy; the degree of collagen deposition in myocardial tissue was reduced; the myocardial cross-sectional area was significantly decreased; and the expression of marker genes and proteins associated with cardiomyocyte hypertrophy and myocardial fibrosis decreased. Therefore, this invention provides the use of a connexin43-targeting inhibitor in the preparation of drugs for the prevention or treatment of myocardial fibrosis, offering a new treatment approach for myocardial fibrosis. Attached Figure Description

[0007] Figure 1 The following figures show the upregulation of connexin43 (Gja1) expression in a myocardial hypertrophy model induced by pathological stroma stiffness: (a) RNA-seq results of NRCMs in both soft and hard stroma groups, showing the expression sequence of all genes in the connexin family (Gja1, Gjc1, Gja3, Gja5, Gja4, Gjb3, Gjc2, Gjd3, Gjd2, Gjb2, Gjb4, Gjb1, Gjb6, Gjd4, Gja8, Gja10, Gjc3, Gje1, Gjb5); (b) a heatmap of connexin family expression; (c) sequencing results showing the expression pattern of Gja1 in both soft and hard stroma groups; (d) (e) and (f) show the changes in Gja1 levels in cardiomyocytes detected by RT-qPCR; (e) and (f) show the changes in protein levels of connexin43, a marker of cardiomyocyte hypertrophy, detected by Western blot; * indicates statistically significant differences between groups, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, n = 3-6.

[0008] Figure 2 The figure shows the enhanced half-channel activity of connexin43 in a myocardial hypertrophy model induced by pathological matrix stiffness; (a) and (b) represent the changes in half-channel activity of connexin43 in two groups of NRCMs in soft and hard matrix as measured by ethidium bromide (Etbr) uptake, respectively; the scale bar is 50 μm. *** represents p < 0.001, n = 3.

[0009] Figure 3The figures show the upregulation of connexin43 (Gja1) expression in the heart tissue of TAC mice; (a) RNA-seq results of TAC myocardial tissues (Sham, TAC_2w, and TAC_4w) at different time gradients, showing the gene expression sequence of the connexin family (Gja1, Gjc1, Gja4, Gja3, Gjc2, Gjc3, Gjd3, Gjb4, Gjb3, Gjb5, Gjd4, Gja10); (b) a heatmap of connexin family expression; (c) RT-qPCR detection of Gja1 levels in TAC mouse myocardial tissues compared to Sham mouse myocardial tissues (n=7), scale bar 50 μm; (d) immunohistochemical detection of connexin43 expression changes in TAC mouse myocardial tissues compared to Sham mouse myocardial tissues. * indicates statistically significant difference between groups, ***, p < 0.001, n = 5-7. Scale bar 50 μm.

[0010] Figure 4 The results show that specific knockdown of connexin43 expression can alleviate cardiomyocyte hypertrophy induced by pathological matrix stiffness; (a) and (b) show the changes in mRNA levels of cardiomyocyte hypertrophy markers Nppa and Nppb in cardiomyocytes as shown by RT-qPCR, n=5; (c) and (d) show the changes in cardiomyocyte area detected by α-actinin immunofluorescence staining after 24 h of si-connexin43 treatment, n=30-33; (e) show the changes in red fluorescence intensity of connexin43 in cardiomyocytes detected by si-connexin43 immunofluorescence staining after 24 h of si-connexin43 treatment, n=3. DAPI (blue), α-actinin (green), connexin43 (red). Scale bar 20 μm. * indicates statistically significant difference between groups, * represents p<0.05, ** represents p<0.01, *** represents p<0.001.

[0011] Figure 5The results show that specific inhibition of connexin43 hemichannel function can alleviate cardiomyocyte hypertrophy induced by pathological matrix stiffness; (a), (b), and (c) show the changes in mRNA levels of cardiomyocyte hypertrophy markers Nppa, Nppb, and My7 in cardiomyocytes as shown by RT-qPCR, n=4; (d) and (e) show the changes in cardiomyocyte area detected by immunofluorescence staining against α-actinin after inhibiting the hemichannel function of connexin43 with the specific inhibitor Xentry-gap19 for 24 hours, with cell count n greater than 50, scale bar 20 μm; (f) shows the changes in green fluorescence intensity of connexin43 in cardiomyocytes detected by immunofluorescence staining against connexin43 after inhibiting the hemichannel function of connexin43 with the specific inhibitor Xentry-gap19 for 24 hours, n=3. DAPI (blue), α-actinin (red), connexin43 (green). Scale bar 10 μm. * indicates a statistically significant difference between groups, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0012] Figure 6 The diagram shows that the sustained-release pump-induced TAT-gap19 (a connexin43 hemichannel inhibitor) significantly improved cardiac function impairment and myocardial fibrosis in mice induced by pressure overload. (a) is a time flow chart of the animal experimental protocol; (b) is a representative image of echocardiographic detection of cardiac function parameters in mice, showing changes in ejection fraction (EF), left ventricular fractional shortening (FS), interventricular septal thickness (IVSd), left ventricular mass (LV Mass), diastolic left ventricular diameter (LVIDd), and left ventricular posterior wall thickness (LVPWd); (c) is a representative image of cardiac anatomy in each group of mice, showing changes in the heart weight / body weight ratio (HW / BW, mg / g) and heart weight / tibia length ratio (HW / TL, mg / mm) among the three groups. Ns indicates no statistically significant difference between groups, p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, n = 8-10. Detailed Implementation

[0013] This invention provides the use of an inhibitor targeting the connexin43 gene in the preparation of drugs for the prevention or treatment of myocardial fibrosis. Specifically, this invention is the first to discover that the connexin43 gene can serve as a potential target for the prevention or treatment of myocardial fibrosis. By knocking down the expression of the connexin43 gene or inhibiting its half-channel activity, the process of myocardial hypertrophy induced by pathological matrix stiffness and the progression of myocardial hypertrophy caused by pressure overload can be alleviated. Therefore, an inhibitor targeting the connexin43 gene can be used to prepare drugs for the prevention or treatment of myocardial fibrosis, providing a new approach to the treatment of myocardial fibrosis.

[0014] This invention first prepared a matrix of pathological stiffness in vitro and observed the hypertrophic phenotype of neonatal rat cardiomyocytes (NRCMs) to construct a cardiomyocyte hypertrophy model induced by pathological matrix stiffness. In this model, this invention detected changes in the expression and half-channel activity of connexin43, finding that connexin43 protein expression was upregulated and its half-channel activity was enhanced in the cardiomyocyte hypertrophy model induced by pathological matrix stiffness. Simultaneously, it was found that connexin43 expression was upregulated in the heart tissue of mice with myocardial hypertrophy caused by stress overload.

[0015] Furthermore, this invention also constructed a pathological myocardial hypertrophy model induced by stress overload in mice. The model was constructed using transverse aortic constriction (TAC). Twenty-eight days post-surgery, tissue samples were collected to measure the heart-to-body weight ratio and the left ventricular mass-to-tibia length ratio to assess gross cardiac changes. Masson's staining, Sirius red, hematoxylin-eosin (HE), and wheat germ agglutinin (WGA) staining were used to observe collagen deposition and cardiomyocyte hypertrophy. Changes in the expression of markers for cardiomyocyte hypertrophy and myocardial fibrosis were also detected to comprehensively evaluate the success of the myocardial hypertrophy model. After successful model construction, RT-qPCR and immunohistochemistry were used to verify changes in connexin43 (Gja1) expression. Simultaneously, combining previous data from the research group's whole transcriptome sequencing of mice after TAC surgery and single-cell data of cardiomyocytes from Gene Expression Omnilus (GEO) (GSE95143), the expression sequence of the connexin family and changes in connexin43 (Gja1) expression were further clarified. The results showed that connexin43 (Gja1) expression was upregulated in the stress overload-induced pathological myocardial hypertrophy model.

[0016] Subsequently, we administered the connexin43 hemichannel inhibitor TAT-gap19 to mice via both slow-release pump and tail vein administration. In both cases, we found that cardiac function was significantly improved in mice with TAC-induced pathological myocardial hypertrophy; the degree of collagen deposition in myocardial tissue was reduced; the cross-sectional area of ​​myocardium was significantly reduced; and the expression of marker genes and proteins associated with cardiomyocyte hypertrophy and myocardial fibrosis decreased.

[0017] The specific implementation of the present invention will be further explained and described below through examples, but this does not mean that the scope of protection of the present invention is limited to the scope described in the examples.

[0018] Unless otherwise specified, the instruments and reagents used in the embodiments of this invention are all commercially available products.

[0019] Example 1: Pathological matrix stiffness induces hypertrophic phenotype in NRCMs (1) To construct pathological matrix hardness and observe the occurrence of hypertrophic phenotypes in NRCMs, we first prepared PDMS matrices with gradient hardness (curing agent to gel ratios of 1:5, 1:15, 1:30, 1:45, and 1:60) to explore the matrix hardness suitable for subsequent experiments. Dow Corning 184 PDMS curing agent was mixed with the gel at different ratios (1:5, 1:15, 1:30, 1:45, and 1:60), and the mixture was thoroughly stirred and agitated with a glass rod to remove air bubbles before being laid on the plates. The well plates were thoroughly shaken to ensure a smooth surface, and the plates were left to stand overnight at room temperature. The next day, they were placed in an oven for curing (60 ℃, 10 h / day, one week). After curing, the plates were stored at room temperature in a clean storage cabinet for later use.

[0020] NRCMs from newborn rats were isolated using primary cell isolation methods. The isolated and cultured NRCMs were seeded into PDMS substrates of varying hardness and cultured for 48 h for further screening. Total RNA was extracted from mouse left ventricular tissue or NRCMs using Trizol. 1 μg of total RNA was used to prepare a 20 μL qPCR RT kit (Rever Tra Ace qPCR RT Kit). The RNA was reverse transcribed into cDNA according to the experimental protocol of 37℃ for 15 min, 98℃ for 5 min. The 20 μL qPCR RT kit consisted of: 4 μL 5×RT buffer, 1.0 μL primer mixture, 1.0 μL Rever Tra Ace, 1 μg RNA, and 14 μL of RNase-free ddH2O.

[0021] Real-time fluorescent RT-qPCR was performed using a 20 μL reaction system prepared with SYBR Green to detect the relative expression levels of genes (using GAPDH as an internal reference gene). The reaction system consisted of: 2 μL cDNA, 6 μL ddH2O, 10 μL SYBR Green Real-time PCR Master Mix (2×), 1 μL Primer 1 (10 µM), and 1 μL Primer 2 (10 µM). The reaction conditions were: 95℃ for 2 min; 95℃ for 10 s; 59℃ for 10 s; and 72℃ for 15 s (40 cycles).

[0022] PCR results showed that on gradient PDMS substrates (1:5, 1:15, 1:30, 1:45, 1:60), the gene expression levels of cardiomyocyte hypertrophy markers Nppa, Nppb, and My7 gradually increased with increasing curing agent / gel ratio and substrate stiffness. Therefore, substrates with curing agent / gel ratios of 1:60 (soft substrate, soft group) and 1:5 (hard substrate, stiff group) could be selected as the control group and the pathological substrate stiffness group, respectively. Considering the insufficient curing and deformation characteristics of the 1:60 PDMS substrate, we selected a 1:50 PDMS substrate to simulate the in vitro culture environment of healthy myocardium and a 1:5 PDMS substrate to simulate the in vitro culture environment of pathological myocardium.

[0023] (2) NRCMs from both soft and stiff matrix groups were collected to investigate the occurrence of cardiomyocyte hypertrophy phenotype. RT-qPCR results showed that, compared with the soft group, the levels of hypertrophy markers Nppa, Nppb, and Myh7 in the stiff group increased by 2.1, 2.0, and 1.7 times, respectively. Western blot results showed that the protein levels of Nppb and Myh7 in the stiff group were approximately 1.7 and 2.4 times higher than those in the soft group. These results suggest that pathological matrix stiffness can induce hypertrophy phenotype in NRCMs.

[0024] Example 2: Exploring the expression of Connexin43 in cardiomyocyte hypertrophy / myocardial hypertrophy To explore the expression changes of connexin43 in myocardial hypertrophy at both in vivo and in vitro levels, we conducted subsequent experiments in a pathological matrix stiffness-induced myocardial hypertrophy model and a pressure overload-induced myocardial hypertrophy mouse model constructed by TAC surgery.

[0025] (1) In the pathological matrix stiffness-induced cardiomyocyte hypertrophy model, the expression of connexin43 was upregulated and the half-channel activity was enhanced.

[0026] After successfully establishing the NRCMs hypertrophy model caused by pathological matrix stiffness, RNA-seq was performed to analyze gene expression changes in the connexin family, and core molecules were screened based on expression levels. Two μg of total RNA was extracted from each sample for the construction of a whole transcriptome library. The ribosomal RNA removal kit (Ribo-Zero™ Gold Kits) was used to remove rRNA from the samples, and the remaining RNA was recovered and used to construct chain-specific libraries using the NEBNext® Chain-Specific Library Construction Kit (NEB). The libraries were detected and quantified using an Agilent 2100 bioanalyzer and RT-qPCR. After passing quality control, Illumina sequencing was performed.

[0027] Gene expression levels of the connexin family (Gja1, Gjc1, Gja3, Gja5, Gja4, Gjb3, Gjc2, Gjd3, Gjd2, Gjb2, Gjb4, Gjb1, Gjb6, Gjd4, Gja8, Gja10, Gjc3, Gje1, Gjb5) were extracted and sorted according to expression levels. It was found that connexin43 (Gja1) had the highest expression abundance in cardiomyocytes. Figure 1 ab); and compared to the soft group, Gja1 expression showed an upregulation trend in the stiff group ( Figure 1 c). Subsequently, the expression of connexin43 was validated in a cardiomyocyte hypertrophy model induced by pathological matrix stiffness. RT-qPCR results showed that, compared with the soft group, the expression level of connexin43 in cardiomyocytes of the stiff group increased by approximately 1.4 times ( Figure 1 d). Western blot results showed that the protein level of connexin43 in the stiff group was approximately 1.3 times higher than that in the soft group ( Figure 1 e, f).

[0028] Given that connexin43, located on the cell membrane, not only mediates gap junctions and facilitates intercellular communication between cardiomyocytes, but its hemichannel proteins also mediate communication between cells and the extracellular matrix, we determined the changes in connexin43 hemichannel activity under different matrix stiffnesses using ethidium bromide (Etbr) staining. Laser confocal microscopy results showed that, compared to the soft group, the red fluorescence intensity of ethidium bromide in cardiomyocytes in the stiff group increased by approximately 2.3 times (…). Figure 2 a, b).

[0029] This indicates that in the pathological matrix stiffness-induced myocardial hypertrophy model, the expression of connexin43 is upregulated and its half-channel activity is enhanced.

[0030] (2) Upregulation of connexin43 expression in hypertrophic heart tissue of mice induced by stress overload A mouse model of myocardial hypertrophy / fibrosis induced by stress overload was constructed. Specifically, C57BL / 6 mice underwent transcatheter arteriosclerosis (TAC) surgery. Sampling, pathological examination, and subsequent experiments were performed on day 28 post-surgery. Sham mice served as controls. Ultrasound results showed that the hearts of mice in the TAC_4w group were significantly larger than those in the Sham group. RT-qPCR was used to detect the mRNA expression levels of myocardial hypertrophy markers Nppa and Nppb, and myocardial fibrosis markers Connective tissue growth factor (CTGF), Periostin (or POSTN), alpha smooth muscle actin (a-SMA), and type I collagen (COL-1). The results showed increased levels of Nppa and Nppb, and CTGF and COL-1, indicating that the mouse model of myocardial hypertrophy / fibrosis induced by TAC surgery was successfully established.

[0031] RNA-seq was performed on TAC myocardial tissues constructed at different time gradients, and KEGG analysis was performed on differentially expressed genes. It was found that the differentially expressed genes were mainly enriched in pathways such as ECM-receptor interaction, indicating that changes in matrix stiffness caused by extracellular matrix deposition are related to myocardial hypertrophy / fibrosis, which is accompanied by increased expression of connexin43 (Gja1).

[0032] Further analysis of gene expression in the connexin family (Gja1, Gjc1, Gja4, Gja3, Gjc2, Gjc3, Gjd3, Gjb4, Gjb3, Gjb5, Gjd4, Gja10) revealed that connexin43 (Gja1) was most abundant in mouse myocardial tissue. Furthermore, compared to the Sham group, Gja1 expression showed a gradient increase in the TAC_2w and TAC_4w groups. Figure 3 (a, b) are consistent with the in vitro experimental data.

[0033] RT-qPCR was used to verify the reliability of the sequencing results, and it was found that the expression level of connexin43 in TAC_4w was approximately 1.7 times higher than that in the Sham group. Figure 3 c). Subsequent immunohistochemical experiments also showed that the expression level of connexin43 increased by approximately 1.3 times. Figure 3d). In summary, the upregulation of connexin43 expression in hypertrophic mouse heart tissue induced by stress overload suggests that it may be involved in the process of myocardial hypertrophy in mice.

[0034] Example 3: Specific knockdown of connexin43 expression can alleviate cardiomyocyte hypertrophy induced by pathological matrix stiffness. Connexin43 expression was specifically inhibited using siRNAs. Three siRNAs were named si-connexin43-001, si-connexin43-002, and si-connexin43-003, with nucleotide sequences shown in SEQ ID NO:1-3 (SEQ ID NO:1's nucleotide sequence is CCGCAATTACAACAAGCAA; SEQ ID NO:2's is CCTGATGACCTGGAGATTT; SEQ ID NO:3's is CTGAGAACCTACATCATCA). The siRNAs for four other genes were named si-itgav-001 (GGAGACTTCCAGACTACAA), si-itgav-002 (GCTGAGCTCATCGTTTCTA), si-itga5-001 (CGGCACAGCCATGGAAAAA), and si-itga5-002 (CTCGGCTTCTTCAAACGCT), with nucleotide sequences shown in SEQ ID NO:4-7. Meanwhile, disordered siRNA was set up as a negative control group si-nc.

[0035] RT-qPCR results showed that all three si-connexin43 sequences could efficiently inhibit connexin43 expression, with knockdown efficiencies of 91%, 95%, and 92%, respectively. Sequence 2 showed the highest relative knockdown efficiency and was used in all subsequent experiments.

[0036] After clarifying the knockdown efficiency of si-connexin43 (si-cx43), we examined the changes in myocardial hypertrophy markers with and without knockdown of connexin43 under pathological matrix stiffness. Compared with the stiff+si-nc group, the stiff+si-cx43 group showed a decrease of approximately 43% and 65% in the cardiomyocyte hypertrophy markers Nppa and Nppb, respectively. Figure 4 a, 4b). Furthermore, immunofluorescence assays showed that, compared to the stiff+si-nc group, the area of ​​NRCMs in the stiff+si-cx43 group was reduced by approximately 11% (a, 4b). Figure 4c, 4d). After the addition of si-connexin43, compared with the stiff+si-nc group, the expression of connexin43 in NRCMs of the stiff+si-cx43 group decreased by approximately 23% ( Figure 4 e).

[0037] The results showed that specific knockdown of connexin43 expression could alleviate cardiomyocyte hypertrophy induced by pathological matrix stiffness.

[0038] Example 4: Specific inhibition of the hemichannel function of connexin43 can alleviate cardiomyocyte hypertrophy caused by pathological matrix stiffness. The Xentry-gap19 (XG19) polypeptide, based on the sequence provided in the reference: lclrpvGGKQIEIKKFK (SEQ ID NO: 8, lowercase letters indicate that it is a D amino acid), was synthesized by Shanghai Chutai Biotechnology Co., Ltd., with a molecular weight of 1957.42 and a purity greater than 95%.

[0039] First, we used ethidium bromide staining to verify the inhibitory efficiency of Xentry-gap19 on the half-channel activity of connexin43. The results showed that, compared with the soft-control group, the ethidium bromide uptake rate in the stiff-control group was significantly increased (approximately 56%); and compared with the stiff-control group, the EtBr uptake rate in the stiff-gap19 group was significantly decreased (approximately 76%). These results suggest that the half-channel activity of connexin43 can be significantly inhibited after the addition of Xentry-gap19.

[0040] After confirming the inhibitory effect of Xentry-gap19 on the connexin43 half-channel function, we examined changes in myocardial hypertrophy markers to investigate whether inhibiting the half-channel function of connexin43 could alleviate myocardial hypertrophy caused by pathological stromal stiffness. The results showed that after the addition of Xentry-gap19, the myocardial hypertrophy markers Nppa, Nppb, and Myh7 decreased by approximately 37%, 25%, and 30%, respectively. Figure 5 Furthermore, immunofluorescence results showed that the area of ​​NRCMs decreased by approximately 24% after the addition of Xentry-gap19. Figure 5 d, e). After the addition of Xentry-gap19, the expression of connexin43 in NRCMs was also suppressed, decreasing by approximately 22% ( Figure 5 f).

[0041] The results show that specific inhibition of the half-channel function of connexin43 can alleviate cardiomyocyte hypertrophy induced by pathological matrix stiffness.

[0042] Example 5: A slow-release pump-induced half-channel inhibitor of connexin43 (TAT-gap19) improved the degree of myocardial hypertrophy / fibrosis in mice induced by stress overload. To verify whether specific inhibition of Cx43 hemichannel function can affect pressure overload-induced myocardial hypertrophy and myocardial fibrosis in vivo, we implanted a TAT-gap19 peptide sustained-release pump (4 mg / kg / day) into the peritoneum of mice on day 7 after TAC surgery, and performed echocardiography and sample analysis on day 28 postoperatively. Figure 6 a). The experimental groups were Sham group, TAC+saline group (TAC+con group) and TAC+TAT-gap19 peptide treatment group (TAC+gap19 group), with 8 mice in each group.

[0043] Echocardiography revealed changes in cardiac function in mice. Compared to the Sham group, the TAC group showed a significant decrease in cardiac function, with markedly lower ejection fraction (EF) and left ventricular fractional shortening (FS), while interventricular septal thickness (IVSd) remained relatively unchanged. However, left ventricular mass (LV Mass), diastolic left ventricular diameter (LVIDd), and systolic left ventricular diameter (LVIDd) were significantly increased. After sustained-release TAT-gap19, compared to the TAC+con group, the TAC+gap19 group showed improvement in some parameters such as EF, FS, and LVIDd, while changes in LV Mass and LVIDd were not significant. Figure 6 b).

[0044] Mouse hearts were collected and their morphology compared. Compared to the Sham group, the hearts of mice in the TAC+con group were significantly larger, and decreased in size after sustained release of TAT-gap19. Compared to the Sham group, the HW / BW ratio in the TAC+con group increased 1.7-fold, and the HW / TL ratio increased 2-fold; compared to the TAC+con group, the HW / BW ratio in the TAC+gap19 group decreased by approximately 30%, and the HW / TL ratio decreased by approximately 23%. Figure 6 c).

[0045] In conclusion, TAT-gap19 administration via a sustained-release pump significantly improved cardiac function impairment and myocardial fibrosis in mice induced by stress overload.

Claims

1. Use of an inhibitor targeting the Connexin43 gene in the preparation of a medicament for the prevention or treatment of myocardial fibrosis; wherein the inhibitor is an siRNA comprising a sequence selected from SEQ ID NO:2-3; and wherein the myocardial fibrosis is cardiomyocyte hypertrophy or myocardial hypertrophy.

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