Application of TRPML1 or agonist thereof in preparation of medicine for inhibiting myocardial cell hypertrophy and heart failure

Abstract

The invention discloses application of TRPML1 or an agonist thereof in preparation of a medicine for inhibiting myocardial cell hypertrophy and heart failure. According to the application disclosed by the invention, it is found through an in-vitro cell experiment and an in-vivo animal experiment that the TRPML1 (TRPML1) has a remarkable effect of resisting myocardial cell hypertrophy and heart failure. In addition, a mechanism of TRPML1 resisting heart failure is explored by adopting a proteomics technology and a molecular docking means, and the application basic theory is firm. On the basis, the invention provides a new strategy for treating myocardial cell hypertrophy and heart failure based on TRPML1 for the first time, the application of TRPML1 in clinical treatment of heart failure is promoted, the time from drug discovery to clinical transformation is greatly shortened, and the application has important clinical significance in treatment of myocardial cell hypertrophy and heart failure.

Description

Technical Field

[0001] The present invention relates to the use of TRPML1 or its agonist in the preparation of a drug for inhibiting myocardial cell hypertrophy and heart failure. The present invention belongs to the field of biomedicine technology. Background Art

[0002] my country currently has approximately 290 million people suffering from cardiovascular disease, ranking first in the world. Recent epidemiological surveys show that the prevalence among residents aged 35 and older is 1.3%. An estimated 8.9 million people currently suffer from heart failure, an increase of nearly 5 million since 2000. Heart failure is the terminal stage of all cardiovascular diseases, with a 5-year mortality rate as high as 50%. Its extremely high mortality and rehospitalization rates pose a heavy global burden and are known as the "last battleground" in the cardiovascular field. Heart failure is a complex clinical syndrome caused by abnormal changes in cardiac structure and / or function due to various factors, resulting in impaired ventricular systolic and / or diastolic function. The pathogenesis of heart failure is complex, involving diverse pathways and pathological processes. Ventricular remodeling, resulting from hemodynamic changes and abnormal activation of the neuroendocrine system, is the primary pathological basis of heart failure. Ventricular remodeling is a chronic, comprehensive compensatory adaptive response regulated by mechanical, genetic, neurohumoral, inflammatory, and oxidative stress mechanisms. This compensatory mechanism is effective but has certain limits. When the heart load continues to be excessive, the heart function shifts from the compensatory stage to the decompensatory stage, eventually leading to heart failure.

[0003] The 2022 AHA / ACC / HFSA Guidelines for the Management of Heart Failure recommend a "new quadruple" regimen for the treatment of chronic heart failure, including RAAS inhibitors (i.e., ARNI / ACEI / ARB drugs), beta-blockers, aldosterone receptor antagonists, and sodium-glucose co-transporter 2 inhibitors. With the advancement of scientific research, new drugs for the treatment of heart failure are constantly being updated. In recent years, new anti-heart failure drugs developed mainly include the following categories: angiotensin receptor neprilysin inhibitors, guanylate cyclase activators, cardiac myosin agonists, potassium binders, and transthyretin stabilizers and silencers. However, the clinical application of many drugs is severely limited by their adverse reactions and toxic side effects. For example, beta-blockers may cause worsening of cardiac function and induce acute heart failure in the early stages of treatment, and can also cause adverse reactions such as bradycardia and hypotension.

[0004] Therefore, a novel, more effective, and more direct drug target is needed to expand current strategies for treating heart failure.

[0005] Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+ The release of cation channels can mediate lysosomal calcium signaling and homeostasis. In response to the new discovery of TRPML1 in the treatment of heart failure, this paper uses TRPML1 small molecule agonists (ML-SA1) and small molecule inhibitors (ML-SI3) as well as molecular pathways through gene overexpression or silencing of TRPML1 as entry points for research. The purpose is to conduct research on the treatment of heart failure with TRPML1 from two aspects: pharmacological inhibition and genetic intervention, to explore whether TRPML1 can effectively inhibit the occurrence of heart failure and whether it can restore the function of damaged mitochondria in cardiomyocytes after heart failure.

[0006] The present invention discovered that TRPML1 can significantly restore cardiac function and reduce the occurrence of fibrosis in mice after heart failure. It also inhibits cardiomyocyte hypertrophy and mitochondrial fragmentation in vitro and improves mitochondrial energy supply. Proteomic analysis and molecular docking techniques also reveal that TRPML1 may target the VDAC1 protein, blocking its oligomerization under pathological conditions. This inhibits mitochondrial dysfunction caused by calcium overload in mitochondria during heart failure, thereby restoring the relaxation and contraction capacity of cardiomyocytes after heart failure. This provides new insights into the clinical treatment of heart failure. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a new target capable of inhibiting myocardial cell hypertrophy and heart failure.

[0008] The purpose of the present invention is to explore the role of TRPML1 in inhibiting heart failure in vivo and in vitro, and to provide a new technical means for the treatment of myocardial hypertrophy and heart failure.

[0009] To achieve the above-mentioned purpose, the present invention adopts the following technical means:

[0010] The present study involved in vitro culture of primary neonatal rat cardiomyocytes. The cells were treated with 1 μM AngⅡ (Sigma-Aldrich, A9525) for 2 hours, followed by the addition of a TRPML1 agonist (ML-SA1, 20 μM, MCE, HY-108462) or inhibitor (ML-SI3, 20 μM, Selleck, E0026). After 24 hours of treatment, Western blotting and qRT-PCR were used to examine the expression of β-MHC and ANP protein and mRNA, markers of cardiomyocyte hypertrophy. Cardiomyocyte hypertrophy was then assessed using α-actinin staining, Rhod-2 assay for changes in mitochondrial calcium, a reactive oxygen species (ROS) detection kit for intracellular ROS levels, Mitochondrial fragmentation using Mitotracker staining, and Oxygraph-2k assay for mitochondrial respiratory capacity. The results demonstrated that the TRPML1 agonist effectively inhibited cardiomyocyte hypertrophy and mitochondrial dysfunction caused by mitochondrial calcium overload.

[0011] In vivo, TRPML1 overexpression and knockout mice were generated using CRISPR / Cas9 gene editing technology. A heart failure model was established using transaortic constriction (TAC) surgery. Eight weeks later, cardiac function was assessed by measuring left ventricular ejection fraction and fractional shortening using a small animal echocardiogram. Changes in cardiac volume were assessed by HE staining, and collagen deposition was determined by Masson staining. Changes in protein and mRNA expression of heart failure markers β-MHC and ANP were analyzed by Western blotting and qRT-PCR. Mitochondrial morphology was assessed by electron microscopy. These results confirmed that TRPML1 overexpression significantly restored cardiac function in heart failure mice, inhibited collagen deposition and cardiac volume increase, and significantly reduced mitochondrial swelling and vacuolation in cardiomyocytes.

[0012] The mechanistic implications of this study were explored through proteomic analysis of primary neonatal rat cardiomyocytes cultured for 48 hours and molecular docking validation. This study demonstrated that VDAC1 is a target of TRPML1. Treatment of cells with the EGS crosslinker revealed that TRPML1 significantly inhibited VDAC1 oligomerization in hypertrophic cardiomyocytes, thereby reducing mitochondrial calcium disturbances in these cells. Using a VDAC1 oligomerization inhibitor, the researchers demonstrated that inhibiting its oligomerization significantly reduced mitochondrial reactive oxygen species (ROS) production and abnormal mitochondrial fragmentation, increased the expression of mitochondrial fusion proteins, and conversely inhibited the expression of mitochondrial fission proteins. Furthermore, the VDAC1 oligomerization inhibitor significantly reversed the decreased cardiac function and cardiomyocyte hypertrophy caused by TRPML1 inhibition, effectively reversing the progression of heart failure. These findings establish TRPML1 as a therapeutic target for heart failure.

[0013] Based on this, the present invention proposes the use of TRPML1 or an agonist thereof in the preparation of a drug for inhibiting myocardial cell hypertrophy.

[0014] Furthermore, the present invention also proposes the use of TRPML1 or an agonist thereof in the preparation of a drug for inhibiting heart failure.

[0015] Among them, preferably, the agonist is the TRPML1 agonist ML-SA1.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention proposes for the first time the use of TRPML1 or its agonist for the treatment of cardiomyocyte hypertrophy and heart failure. Through in vitro cell experiments and in vivo animal experiments, the present invention found that TRPML1 or its agonist has significant anti-cardiomyocyte hypertrophy and heart failure effects. Furthermore, the present invention uses proteomics technology and molecular docking to explore its anti-cardiomyocyte hypertrophy and heart failure mechanism, with a solid application foundation. The present invention proposes for the first time a new strategy based on TRPML1 for the treatment of cardiomyocyte hypertrophy and heart failure. This will promote the application of TRPML1 in the clinical treatment of heart failure, greatly shorten the time from drug discovery to clinical translation, and has important clinical significance for the treatment of cardiomyocyte hypertrophy and heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 In vitro activation of TRPML1 can inhibit the expression of β-MHC and ANP proteins and mRNA, as well as cardiomyocyte hypertrophy markers;

[0019] Among them, (A) Representative graph of β-MHC protein expression, a marker of cardiomyocyte hypertrophy, detected by Western blotting; (B) Statistical graph of β-MHC protein expression, a marker of cardiomyocyte hypertrophy, detected by Western blotting; (C) Representative graph of ANP protein expression, a marker of cardiomyocyte hypertrophy, detected by Western blotting; (D) Statistical graph of ANP protein expression, a marker of cardiomyocyte hypertrophy, detected by Western blotting; (E) Statistical graph of β-MHC mRNA expression, a marker of cardiomyocyte hypertrophy, detected by qRT-PCR; (F) Statistical graph of ANP mRNA expression, a marker of cardiomyocyte hypertrophy, detected by qRT-PCR; (G) Representative graph of α-actinin detection of cardiomyocyte hypertrophy; (H) Statistical graph of α-actinin detection of cardiomyocyte hypertrophy;

[0020] Figure 2 In vitro inhibition of TRPML1 can promote the expression of β-MHC and ANP proteins and mRNA, as well as cardiomyocyte hypertrophy markers;

[0021] Among them, (A) Representative graph of β-MHC protein expression, a marker of cardiomyocyte hypertrophy, detected by Western blotting; (B) Statistical graph of β-MHC protein expression, a marker of cardiomyocyte hypertrophy, detected by Western blotting; (C) Representative graph of ANP protein expression, a marker of cardiomyocyte hypertrophy, detected by Western blotting; (D) Statistical graph of ANP protein expression, a marker of cardiomyocyte hypertrophy, detected by Western blotting; (E) Statistical graph of β-MHC mRNA expression, a marker of cardiomyocyte hypertrophy, detected by qRT-PCR; (F) Statistical graph of ANP mRNA expression, a marker of cardiomyocyte hypertrophy, detected by qRT-PCR; (G) Representative graph of α-actinin detection of cardiomyocyte hypertrophy; (H) Statistical graph of α-actinin detection of cardiomyocyte hypertrophy;

[0022] Figure 3 The effect of TRPML1 on mitochondrial function in cardiomyocytes;

[0023] Among them, (A) Representative graph of total reactive oxygen species detection; (B) Statistical graph of total reactive oxygen species detection; (C) Representative graph of mitochondrial reactive oxygen species detection; (D) Statistical graph of mitochondrial reactive oxygen species detection; (E) Representative graph of Mitotracker staining; (F) Statistical graph of Mitotracker staining; (G) Statistical graph of the ability of Oxygraph-2k to detect mitochondrial respiration;

[0024] Figure 4A-D as well as Figure 4E-H Overexpression of TRPML1 inhibits the occurrence of heart failure in mice;

[0025] Among them, (A) Representative image of mouse cardiac function detected by echocardiography; (B) Statistical graph of mouse cardiac ejection fraction (EF%); (C) Statistical graph of mouse cardiac fractional shortening (FS%); (D) Representative image of mouse heart volume observed by HE staining; (E) Representative image of cardiomyocyte cross-sectional area detected by WGA staining; (F) Statistical graph of cardiomyocyte cross-sectional area detected by WGA staining; (G) Representative image of cardiac tissue collagen deposition detected by Masson staining; (H) Statistical graph of cardiac tissue collagen deposition detected by Masson staining;

[0026] Figure 5A-C as well as Figure 5D-G TRPML1 binds to VDAC1 and inhibits its oligomerization;

[0027] Among them, (A) Proteomic screening of TRPML1 downstream target proteins; (B) Bioinformatics analysis confirmed that VDAC1 is a downstream target protein of TRPML1; (C) Molecular docking confirmed that TRPML1 and VDAC1 bind to each other; (D) Representative image of CO-IP detection of TRPML1 and VDAC1 binding in HEK293T tool cells; (E) Representative image of CO-IP detection of TRPML1 and VDAC1 binding in cardiac tissue; (F) Representative image of TRPML1 agonist ML-SA1 inhibiting VDAC1 oligomerization; (G) Statistical graph of TRPML1 agonist ML-SA1 inhibiting VDAC1 oligomerization;

[0028] Figure 6A-E as well as Figure 6F-I Inhibition of VDAC1 oligomerization reversed the heart failure induced by TRPML1 knockout.

[0029] Among them, (A) NSC15364 administration mode diagram; (B) representative diagram of echocardiography detection; (C) statistical diagram of mouse cardiac ejection fraction (EF%); (D) statistical diagram of mouse cardiac fraction shortening (FS%); (E) representative diagram of mouse cardiac volume detected by HE staining; (F) representative diagram of mouse cardiomyocyte cross-sectional area detected by WGA staining; (G) statistical diagram of mouse cardiomyocyte cross-sectional area detected by WGA staining; (H) representative diagram of mouse cardiac collagen deposition detected by Masson staining; (I) representative diagram of mouse cardiac collagen deposition detected by Masson staining. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0032] Example 1: TRPML1 agonist ML-SA1 can inhibit the protein expression of myocardial hypertrophy markers β-MHC and ANP, thereby inhibiting the occurrence of myocardial hypertrophy

[0033] 1 Experimental methods

[0034] 1.1 Experimental group design:

[0035] Primary neonatal rat cardiomyocytes were cultured in vitro and divided into DMSO, ML-SA1, AngⅡ, and AngⅡ+ML-SA1 groups. The ML-SA1 group was treated with a TRPML1 agonist (ML-SA1, 20 μM, MCE, HY-108462), the AngⅡ group was treated with 1 μM AngⅡ (Sigma-Aldrich, A9525) for 24 hours, the AngⅡ+ML-SA1 group was treated with 1 μM AngⅡ for 2 hours, and then treated with a TRPML1 agonist (ML-SA1, 20 μM, MCE, HY-108462) for 24 hours, and the DMSO group was treated with DMSO for 24 hours.

[0036] 1.2 Western blotting was used to detect the effect of ML-SA1 on the protein expression of β-MHC and ANP.

[0037] The total protein in the cardiomyocytes of neonatal rats in the DMSO group, ML-SA1 group, AngⅡ group and AngⅡ+ML-SA1 group was extracted and subjected to Western blotting experiments.

[0038] 1.3 Data Processing

[0039] The results of the present invention are expressed as mean ± standard deviation. The grayscale values ​​of the protein bands were calculated using Image Studio software and graphed using Graphpadprism 8.0.

[0040] 2 Observation results

[0041] 2.1TRPML1 activation can significantly inhibit the protein expression of myocardial hypertrophy markers β-MHC and ANP

[0042] Western blotting results are as follows Figure 1 As shown in AD, compared with the DMSO group, AngⅡ treatment significantly increased the expression of β-MHC and ANP proteins, while treatment with TRPML1 agonist ML-SA1 for 24 h significantly inhibited the expression of β-MHC and ANP proteins.

[0043] These results indicate that increasing TRPML1 activity can inhibit the occurrence of myocardial hypertrophy by inhibiting the expression of β-MHC and ANP proteins.

[0044] Example 2: TRPML1 agonist ML-SA1 can inhibit the mRNA expression of myocardial hypertrophy markers β-MHC and ANP, thereby inhibiting the occurrence of myocardial hypertrophy

[0045] 1 Experimental methods

[0046] 1.1 Experimental group design:

[0047] Same as Example 1.

[0048] 1.2 qRT-PCR detection of β-MHC and ANP mRNA expression

[0049] Total RNA was extracted from the cardiomyocytes of neonatal rats in the DMSO group, ML-SA1 group, AngⅡ group, and AngⅡ+ML-SA1 group and reverse transcribed into cDNA. qRT-PCR amplification experiments were then performed to detect the mRNA expressions of β-MHC and ANP.

[0050] 1.3 Data Processing

[0051] The results of the present invention are expressed using mean ± standard deviation. -ΔΔCT Methods: The mRNA expression was calculated and plotted using Graphpad Prism 8.0. The correlations between the groups were compared between pairs using one-way analysis of variance.

[0052] 2 Observation results

[0053] 2.1ML-SA1 can inhibit the mRNA expression of β-MHC and ANP

[0054] The results of qRT-PCR experiments are as follows Figure 1 As shown in EF, compared with the DMSO group, AngⅡ treatment significantly increased the expression of β-MHC and ANP mRNA, while treatment with TRPML1 agonist ML-SA1 for 24 h significantly inhibited the expression of β-MHC and ANP mRNA.

[0055] These results indicate that increasing TRPML1 activity can inhibit the occurrence of myocardial hypertrophy by inhibiting the expression of β-MHC and ANP mRNA.

[0056] Example 3: TRPML1 agonist ML-SA1 can inhibit cardiomyocyte hypertrophy

[0057] 1 Experimental methods

[0058] 1.1 Experimental group design:

[0059] Same as Example 1.

[0060] 1.2 α-actinin staining assay to detect cardiomyocyte hypertrophy

[0061] The cardiomyocytes of neonatal rats in the DMSO group, ML-SA1 group, AngⅡ group and AngⅡ+ML-SA1 group were stained for α-actinin, and the cross-sectional area of ​​the cardiomyocytes was observed under a confocal microscope. The specific operations were as follows: first, the culture medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% formaldehyde at 37°C for 15 min, and then the membrane was permeabilized with Triton-X100 for 30 min. The cells were blocked with goat serum for 1 h at room temperature and then incubated with α-actinin primary antibody. After treatment at 4°C overnight, 488 fluorescent secondary antibody was incubated and the cells were stained with DIPA for 15 min. Then, the cross-sectional area of ​​the cells was observed and photographed under a confocal microscope.

[0062] 1.3 Data Processing

[0063] The results of the present invention are expressed as mean ± standard deviation. Graphpadprism 8.0 was used for plotting, and the correlations between the groups were compared between two groups using one-way analysis of variance.

[0064] 2 Observation results

[0065] 2.1 TRPML1 agonist ML-SA1 can inhibit cardiomyocyte hypertrophy

[0066] The results of α-actinin staining experiments are as follows Figure 1 As shown in FG, compared with the DMSO group, the cross-sectional area of ​​cardiomyocytes was significantly increased in the AngⅡ-treated group, while the cross-sectional area of ​​cardiomyocytes was significantly decreased after treatment with the TRPML1 agonist ML-SA1 for 24 h.

[0067] The above results indicate that the TRPML1 agonist ML-SA1 can inhibit cardiomyocyte hypertrophy.

[0068] Example 4: TRPML1 inhibitor ML-SI3 can promote the protein expression of myocardial hypertrophy markers β-MHC and ANP, thereby promoting the occurrence of myocardial hypertrophy

[0069] 1 Experimental methods

[0070] 1.1 Experimental group design:

[0071] Primary neonatal rat cardiomyocytes were cultured in vitro and divided into DMSO, ML-SI3, AngⅡ, and AngⅡ+ML-SI3 groups. The ML-SI3 group was treated with a TRPML1 inhibitor (ML-SI3, 20 μM, Selleck, E0026) for 24 hours, the AngⅡ group was treated with 1 μM AngⅡ (Sigma-Aldrich, A9525) for 24 hours, the AngⅡ+ML-SI3 group was treated with 1 μM AngⅡ for 2 hours and then with a TRPML1 inhibitor (ML-SI3, 20 μM, Selleck, E0026) for 24 hours, and the DMSO group was treated with DMSO for 24 hours.

[0072] 1.2 Western blotting was used to detect the effect of ML-SI3 on ​​the protein expression of β-MHC and ANP.

[0073] The total protein from the neonatal rat cardiomyocytes of the DMSO group, ML-SI3 group, AngⅡ group and AngⅡ+ML-SI3 group was extracted and subjected to Western blotting experiments.

[0074] 1.3 Data Processing

[0075] The results of the present invention are expressed as mean ± standard deviation. The grayscale values ​​of the protein bands were calculated using Image Studio software and graphed using Graphpadprism 8.0.

[0076] 2 Observation results

[0077] 2.1 TRPML1 activity inhibition can significantly promote the protein expression of myocardial hypertrophy markers β-MHC and ANP

[0078] Western blotting results are as follows Figure 2 As shown in AD, compared with the DMSO group, AngⅡ treatment significantly increased the expression of β-MHC and ANP proteins, and treatment with TRPML1 inhibitor ML-SI3 for 24 h further increased the expression of β-MHC and ANP proteins.

[0079] These results indicate that inhibiting TRPML1 activity can promote the occurrence of myocardial hypertrophy by increasing the expression of β-MHC and ANP proteins.

[0080] Example 5: TRPML1 inhibitor ML-SI3 can increase the mRNA expression of myocardial hypertrophy markers β-MHC and ANP, thereby promoting the occurrence of myocardial hypertrophy

[0081] 1 Experimental methods

[0082] 1.1 Experimental group design:

[0083] Same as Example 4.

[0084] 1.2 qRT-PCR detection of β-MHC and ANP mRNA expression

[0085] Total RNA was extracted from the cardiomyocytes of neonatal rats in the DMSO group, ML-SI3 group, AngⅡ group, and AngⅡ+ML-SI3 group and reverse transcribed into cDNA. qRT-PCR amplification experiments were then performed to detect the mRNA expressions of β-MHC and ANP.

[0086] 1.3 Data Processing

[0087] The results of the present invention are expressed using mean ± standard deviation. -ΔΔCT Methods: The mRNA expression was calculated and plotted using Graphpad Prism 8.0. The correlations between the groups were compared between pairs using one-way analysis of variance.

[0088] 2 Observation results

[0089] 2.1ML-SI3 can increase the mRNA expression of β-MHC and ANP

[0090] The results of qRT-PCR experiments are as follows Figure 2 As shown in EF, compared with the DMSO group, AngⅡ treatment significantly increased the expression of β-MHC and ANP mRNA, and after 24h treatment with TRPML1 inhibitor ML-SI3, the expression of β-MHC and ANP mRNA was further increased.

[0091] These results indicate that inhibiting TRPML1 activity can promote the occurrence of myocardial hypertrophy by increasing the expression of β-MHC and ANP mRNA.

[0092] Example 6: TRPML1 inhibitor ML-SI3 can promote cardiomyocyte hypertrophy

[0093] 1 Experimental methods

[0094] 1.1 Experimental group design:

[0095] Same as Example 4.

[0096] 1.2 α-actinin staining assay to detect cardiomyocyte hypertrophy

[0097] The cardiomyocytes of neonatal rats in the DMSO group, ML-SI3 group, AngⅡ group and AngⅡ+ML-SI3 group were stained for α-actinin, and the cross-sectional area of ​​the cardiomyocytes was observed under a confocal microscope. The specific operations were as follows: first, the culture medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% formaldehyde at 37°C for 15 min, and then the membrane was permeabilized with Triton-X100 for 30 min. The cells were blocked with goat serum for 1 h at room temperature and then incubated with α-actinin primary antibody. After treatment at 4°C overnight, 488 fluorescent secondary antibody was incubated and the cells were stained with DIPA for 15 min. The cross-sectional area of ​​the cells was observed and photographed under a confocal microscope.

[0098] 1.3 Data Processing

[0099] The results of the present invention are expressed as mean ± standard deviation. Graphpadprism 8.0 was used for plotting, and the correlations between the groups were compared between two groups using one-way analysis of variance.

[0100] 2 Observation results

[0101] 2.1 TRPML1 inhibitor ML-SI3 can inhibit cardiomyocyte hypertrophy

[0102] The results of α-actinin staining experiments are as follows Figure 2 As shown in FG, compared with the DMSO group, AngⅡ treatment significantly increased the cross-sectional area of ​​cardiomyocytes, and treatment with the TRPML1 inhibitor ML-SI3 for 24 h further increased the cross-sectional area of ​​cardiomyocytes.

[0103] The above results indicate that TRPML1 inhibitor ML-SI3 can promote cardiomyocyte hypertrophy.

[0104] Example 7: TRPML1 agonist ML-SA1 can inhibit the production of reactive oxygen species in myocardial cells

[0105] 1 Experimental methods

[0106] 1.1 Experimental group design:

[0107] Same as Example 1.

[0108] Effects of ML-SA1 on the production of reactive oxygen species in cardiomyocytes

[0109] After primary neonatal rat cardiomyocytes were treated with drugs for 24 hours, reactive oxygen species (ROS) staining was performed using a reactive oxygen species detection kit according to the reagent manufacturer's instructions. The production of reactive oxygen species in the cardiomyocytes was observed under a confocal microscope. The specific operation was as follows: first, the culture medium was discarded, and the cells were washed three times with DMEM medium. Then, the cells were incubated at 37°C for 30 minutes using culture medium containing DCHF-DA probe. After incubation, the cells were washed three times, and the excitation wavelength of 488 nm was selected under a confocal microscope to obtain the reactive oxygen species detection signal.

[0110] 1.3 Data Processing

[0111] The results of the present invention are expressed as mean ± standard deviation. Fluorescence intensity analysis was performed using Image J, graphing was performed using Graphpadprism 8.0, and correlations between groups were compared between pairs using one-way analysis of variance.

[0112] 2 Observation results

[0113] 2.1TRPML1 agonist ML-SA1 can inhibit the production of reactive oxygen species in hypertrophic cardiomyocytes.

[0114] The results of the active oxygen staining experiment are as follows Figure 3 As shown in AB, compared with the DMSO group, AngⅡ treatment significantly increased the production of reactive oxygen species in cardiomyocytes. At the same time, after 24 hours of treatment with TRPML1 agonist ML-SA1, the production of reactive oxygen species in cardiomyocytes was significantly reduced.

[0115] The above results indicate that the TRPML1 agonist ML-SA1 can reduce oxidative stress in hypertrophic cardiomyocytes by inhibiting the production of intracellular reactive oxygen species, thereby restoring the normal physiological function of hypertrophic cardiomyocytes.

[0116] Example 8: TRPML1 agonist ML-SA1 can inhibit the production of reactive oxygen species in myocardial mitochondria

[0117] 1 Experimental methods

[0118] 1.1 Experimental group design:

[0119] Same as Example 1.

[0120] Effects of ML-SA1 on mitochondrial reactive oxygen species production in cardiomyocytes

[0121] After primary neonatal rat cardiomyocytes were treated with drugs for 24 hours, mitochondrial reactive oxygen species (ROS) staining was performed using a mitochondrial reactive oxygen species (ROS) detection kit according to the manufacturer's instructions. The production of reactive oxygen species in cardiomyocytes was observed under a confocal microscope. The specific operation was as follows: first, the culture medium was discarded, and the cells were washed three times with DMEM medium. Then, the cells were incubated at 37°C for 30 minutes using culture medium containing Mitosox probe. After incubation, the cells were washed three times, and the excitation wavelength of 594 nm was selected under a confocal microscope to obtain the reactive oxygen species detection signal.

[0122] 1.3 Data Processing

[0123] The results of the present invention are expressed as mean ± standard deviation. Fluorescence intensity analysis was performed using Image J, graphing was performed using Graphpadprism 8.0, and correlations between groups were compared between pairs using one-way analysis of variance.

[0124] 2 Observation results

[0125] 2.1TRPML1 agonist ML-SA1 can inhibit the production of mitochondrial reactive oxygen species in hypertrophic cardiomyocytes.

[0126] The results of mitochondrial reactive oxygen species staining experiments are as follows Figure 3 As shown in CD, compared with the DMSO group, AngⅡ treatment significantly increased the production of mitochondrial reactive oxygen species in cardiomyocytes. At the same time, after 24 hours of treatment with TRPML1 agonist ML-SA1, the production of mitochondrial reactive oxygen species in cardiomyocytes was significantly reduced.

[0127] The above results indicate that the TRPML1 agonist ML-SA1 can reduce oxidative stress in hypertrophic cardiomyocytes by inhibiting the production of intracellular mitochondrial reactive oxygen species, thereby restoring the normal physiological function of hypertrophic cardiomyocytes.

[0128] Example 9: TRPML1 agonist ML-SA1 can inhibit abnormal mitochondrial fragmentation in cardiomyocytes

[0129] 1 Experimental methods

[0130] 1.1 Experimental group design:

[0131] Same as Example 1.

[0132] Effects of ML-SA1 on mitochondrial fragmentation in cardiomyocytes

[0133] After primary neonatal rat cardiomyocytes were treated with drugs for 24 hours, Mitotracker staining was used to detect mitochondrial morphological changes, and mitochondrial fragmentation in the cardiomyocytes was observed under a confocal microscope. The specific operation was as follows: after primary neonatal rat cardiomyocytes were treated with drugs for 24 hours, the culture medium was first discarded, and the cells were washed three times with DMEM culture medium. Then, the cells were incubated at 37°C for 30 minutes using culture medium containing Mitotracker probes. After incubation, the cells were washed three times, and the excitation wavelength of 594 nm was selected under a confocal microscope to obtain the Mitotracker detection signal.

[0134] 1.3 Data Processing

[0135] The results of the present invention are expressed as mean ± standard deviation. Image J was used to calculate the length of mitochondria, and Graphpadprism 8.0 was used for plotting. The correlation between the groups was compared between two groups using one-way analysis of variance.

[0136] 2 Observation results

[0137] 2.1TRPML1 agonist ML-SA1 can inhibit mitochondrial fragmentation in hypertrophic cardiomyocytes.

[0138] Mitotracker staining results are shown in Figure 3 As shown in EF, compared with the DMSO group, AngⅡ treatment significantly increased the number of abnormally broken mitochondria in cardiomyocytes. At the same time, after 24h treatment with TRPML1 agonist ML-SA1, the number of abnormally broken mitochondria in cardiomyocytes was significantly reduced.

[0139] The above results indicate that the TRPML1 agonist ML-SA1 can maintain normal mitochondrial function by inhibiting the abnormal fragmentation of mitochondria in cells, thereby restoring the energy supply of hypertrophic cardiomyocytes.

[0140] Example 10: TRPML1 agonist ML-SA1 can promote mitochondrial respiration in hypertrophic cardiomyocytes

[0141] 1 Experimental methods

[0142] 1.1 Experimental group design:

[0143] Same as Example 1.

[0144] Effects of ML-SA1 on mitochondrial respiration in cardiomyocytes

[0145] After 24 hours of drug treatment, primary neonatal rat cardiomyocytes were tested for mitochondrial respiration using an Oxygraph-2k mitochondrial respiration monitor. The following steps were performed: First, cells were counted using a cell counter to control the number of cells in each group. Then, when basal respiration was achieved, oligomycin was added to measure mitochondrial ATP synthesis. After 10 minutes of oligomycin treatment, the mitochondrial uncoupling agent FCCP was immediately added to determine the maximal respiratory capacity of the cells. Upon reaching maximal respiration, rotenone (Rot) and Ama (Ama) were immediately added to restore cellular respiration to normal levels.

[0146] 1.3 Data Processing

[0147] The results of the present invention are expressed as mean ± standard deviation. Statistics were performed using DatLab, graphs were drawn using Graphpadprism 8.0, and correlations between groups were compared between two groups using one-way analysis of variance.

[0148] 2 Observation results

[0149] 2.1TRPML1 agonist ML-SA1 can promote mitochondrial respiration in hypertrophic cardiomyocytes.

[0150] Oxygraph-2k mitochondrial respiration assay results Figure 3 As shown in G, compared with the DMSO group, AngⅡ treatment significantly inhibited the basal respiration, ATP production and maximal respiration of mitochondria in cardiomyocytes. At the same time, after treatment with the TRPML1 agonist ML-SA1 for 24 hours, the basal respiration, ATP production and maximal respiration of mitochondria in cardiomyocytes were significantly restored.

[0151] The above results indicate that the TRPML1 agonist ML-SA1 can restore the energy supply of hypertrophic cardiomyocytes by stimulating mitochondrial respiration in hypertrophic cardiomyocytes.

[0152] Example 11: Cardiomyocyte-specific overexpression of TRPML1 can restore cardiac function in TAC mice

[0153] 1 Experimental methods

[0154] 1.1 Construction of TRPML1 cardiomyocyte-specific overexpression transgenic mice and TAC mice

[0155] First, the CRISPR design tool CRISPOR was used to design a guide RNA targeting the mouse TRPML1 gene (gRNA, Forward: 5'-TTCCTACAGCTCCTGGGCAACG-3'; Reverse: 5'-ATGCTCCAGACTGCCTTGGG-3'), followed by linearization of the SV40pA vector with a restriction endonuclease (BsmBI). The annealed gRNA was ligated into the vector using T4 DNA ligase, and the DH5α ligation product was transformed into Escherichia coli after insertion into the vector. Positive clones were then screened with antibiotics (ampicillin). Single clones were picked and the plasmids were extracted and sent for sequencing to check whether the gRNA sequence was correct. After verification, the constructed vector and Cas9 mRNA were co-injected into mouse fertilized eggs to produce targeted conditional knock-in offspring. The F0 generation animals were identified by polymerase chain reaction and sequence analysis, and then hybridized with wild-type mice to confirm germline transmission and generate conditional TRPML1 knock-in mice (TRPML1 fl / fl KI:Forward:

[0156] 5'-AGTCATAGCTGTCCTCTTCTCT-3'; Reverse:

[0157] 5'-CCATTTAAGCCATGGGAAGTTAGT-3'. TRPML1 WT:Forward:

[0158] 5'-GTCTGCAGCCATTACTAAACATGAA-3'; Reverse: 5'-AAAGGTAGACGGATTTAGCCACA-3') F1 offspring. Then TRPML1 fl / flKI mice were crossed with α-MHC-cre (Forward: 5'-ATGACAGACAGATCCCTCCTATCTCC-3'; Reverse: 5'-CTCATCACTCGTTGCATCATCGAC-3', reference: Agah, R., et al. (1997). "Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo." The Journal of Clinical Investigation, 100(1), 169-179.) mice. After multiple generations of breeding, myocardial-specific overexpression of TRPML1 transgenic (TRPML1) was successfully established. cKI ) mice.

[0159] After the mice reached adulthood, a TAC-induced heart failure mouse model was established. The mice were anesthetized using tribromoethanol avertin (2%, 0.01 ml / g). After the mouse's blink reflex disappeared, they were fixed on the operating table in a supine position for chest skin preparation. A small animal ventilator was then inserted into the mouse's trachea to ventilate it (at a rate of 100 times / minute, a tidal volume of 0.3 ml). After the mouse's breathing stabilized, the prepared skin was disinfected using iodine tincture. Sterile surgical scissors were used to make an oblique incision in the mouse's neck skin, and then the mouse's sternum was cut open with sterile surgical scissors. After exposing the thymus, a chest expander was used to separate the thymus to expose the aorta. A 27G needle was then used as a guide to tie a knot around the aortic arch with 6-0 polypropylene suture. After the knot was tied, the 27G needle was carefully withdrawn, and the muscle and skin were closed with 6-0 sutures after observing the vigorous pulsation of the mouse's left common carotid artery. Mice in the sham operation group underwent the same surgical procedure, but without aortic constriction.

[0160] 1.2 Experimental group design:

[0161] The experiment was divided into 4 groups: Sham+TRPML1 fl / fl group, Sham+TRPML1 cKI group, TAC+TRPML1 fl / fl group and TAC+TRPML1 cKI Group.

[0162] 1.3 Cardiomyocyte-specific overexpression of TRPML1 can increase EF% and FS%, thereby restoring cardiac function in mice.

[0163] TAC+TRPML1 fl / fl group and TAC+TRPML1 cKI Eight weeks after induction, the ejection fraction (EF%) and fractional shortening (FS%) of the mice were measured using a small animal echocardiogram. Mice were anesthetized with avertin and placed supine on an ultrasound table. EF% and FS% were recorded using a 10 Hz ultrasound probe in M-mode to determine changes in cardiac function.

[0164] 1.4 Data Processing

[0165] The results of the present invention are expressed as mean ± standard deviation. Graphpadprism 8.0 was used for plotting, and the correlations between the groups were compared between two groups using one-way analysis of variance.

[0166] 2 Observation results

[0167] 2.1 Cardiomyocyte-specific overexpression of TRPML1 inhibited the progression of heart failure by restoring EF% and FS% in heart failure mice.

[0168] The results of echocardiography are shown in Figure 4A-C. fl / fl Group comparison, TAC+TRPML1 fl / fl The TAC group significantly reduced the EF% and FS% of the mouse heart. However, when TRPML1 was specifically overexpressed, it was able to significantly increase the EF% and FS% of the TAC mice, thereby restoring the heart function of the mice.

[0169] The above results indicate that cardiomyocyte-specific overexpression of TRPML1 can inhibit the decrease of EF% and FS% in the hearts of heart failure mice, thereby restoring the cardiac function of heart failure mice.

[0170] Example 12: Cardiomyocyte-specific overexpression of TRPML1 can inhibit cardiac volume enlargement in TAC mice

[0171] 1 Experimental methods

[0172] 1.1 Experimental group design:

[0173] The experiment was divided into 4 groups: Sham+TRPML1 fl / fl group, Sham+TRPML1 cKI group, TAC+TRPML1 fl / fl group and TAC+TRPML1 cKI Group.

[0174] 1.2 Cardiomyocyte-specific overexpression of TRPML1 can inhibit the increase in cardiac volume in mice with heart failure

[0175] TAC+TRPML1 fl / fl group and TAC+TRPML1 cKI After 8 weeks of induction, the mice were killed under anesthesia, and the hearts were quickly removed and fixed with 4% paraformaldehyde for 48 hours before being embedded in paraffin. The heart tissue was cut into 5 μm slices using a paraffin slicer. After the slices were dewaxed with gradient ethanol, they were stained with hematoxylin for 10 minutes and the staining of the cell nuclei was observed; then, they were stained with eosin for 1-2 minutes, the slices were sealed and photographed.

[0176] 1.3 Data Processing

[0177] The results of the present invention are expressed as mean ± standard deviation. Graphpadprism 8.0 was used for plotting, and the correlations between the groups were compared between two groups using one-way analysis of variance.

[0178] 2 Observation results

[0179] 2.1 Cardiomyocyte-specific overexpression of TRPML1 significantly inhibited the increase in cardiac volume in heart failure mice.

[0180] The results of HE staining experiments are shown in Figure 4D. fl / fl Group comparison, TAC+TRPML1 fl / fl The TAC group significantly increased the heart volume of mice, but when TRPML1 was specifically overexpressed, it was able to significantly inhibit the heart volume of TAC mice and thus inhibit the progression of heart failure.

[0181] The above results indicate that cardiomyocyte-specific overexpression of TRPML1 can inhibit the heart volume of heart failure mice, thereby restoring the heart function of heart failure mice.

[0182] Example 13: Cardiomyocyte-specific overexpression of TRPML1 can inhibit cardiomyocyte hypertrophy in TAC mice

[0183] 1 Experimental methods

[0184] 1.1 Experimental group design:

[0185] The experiment was divided into 4 groups: Sham+TRPML1 fl / fl group, Sham+TRPML1 cKI group, TAC+TRPML1 fl / fl group and TAC+TRPML1 cKI Group.

[0186] 1.2 Cardiomyocyte-specific overexpression of TRPML1 can inhibit cardiomyocyte hypertrophy in mice with heart failure

[0187] TAC+TRPML1 fl / fl group and TAC+TRPML1 cKI After 8 weeks of induction, the mice were killed under anesthesia, and the hearts were quickly removed and fixed with 4% paraformaldehyde for 48 hours before being embedded in paraffin. The heart tissue was cut into 5 μm slices using a paraffin slicer. The slices were dewaxed with gradient ethanol, stained with WGA dye for 1 hour, and then sealed and photographed.

[0188] 1.3 Data Processing

[0189] The results of the present invention are expressed as mean ± standard deviation. Graphpadprism 8.0 was used for plotting, and the correlations between the groups were compared between two groups using one-way analysis of variance.

[0190] 2 Observation results

[0191] 2.1 Cardiomyocyte-specific overexpression of TRPML1 significantly inhibited the increase in cardiac volume in heart failure mice.

[0192] The results of WGA staining experiments are shown in Figure 4E-F. fl / fl Group comparison, TAC+TRPML1 fl / fl The TAC group significantly increased the cross-sectional area of ​​myocardial cells in mice. However, when TRPML1 was specifically overexpressed, it was able to significantly inhibit the cross-sectional area of ​​myocardial cells in TAC mice, thereby inhibiting the progression of heart failure.

[0193] The above results indicate that cardiomyocyte-specific overexpression of TRPML1 can inhibit cardiomyocyte hypertrophy in heart failure mice, thereby restoring cardiac function in heart failure mice.

[0194] Example 14: Cardiomyocyte-specific overexpression of TRPML1 can inhibit cardiac collagen deposition in TAC mice

[0195] 1 Experimental methods

[0196] 1.1 Experimental group design:

[0197] The experiment was divided into 4 groups: Sham+TRPML1 fl / fl group, Sham+TRPML1 cKI group, TAC+TRPML1 fl / fl group and TAC+TRPML1 cKI Group.

[0198] 1.2 Cardiomyocyte-specific overexpression of TRPML1 inhibits cardiac collagen deposition in heart failure mice

[0199] TAC+TRPML1fl / fl group and TAC+TRPML1 cKI After 8 weeks of induction, the mice were killed under anesthesia, and the hearts were quickly removed and fixed with 4% paraformaldehyde for 48 h before being embedded in paraffin. The heart tissue was cut into 5 μm slices using a paraffin slicer. The slices were dewaxed with gradient ethanol, stained with a Masson dye kit, and then sealed and photographed.

[0200] 1.3 Data Processing

[0201] The results of the present invention are expressed as mean ± standard deviation. Graphpadprism 8.0 was used for plotting, and the correlations between the groups were compared between two groups using one-way analysis of variance.

[0202] 2 Observation results

[0203] 2.1 Cardiomyocyte-specific overexpression of TRPML1 significantly inhibits cardiac collagen deposition in heart failure mice

[0204] The results of WGA staining experiments are shown in Figure 4G-H. fl / fl Group comparison, TAC+TRPML1 fl / fl The TAC group significantly increased cardiac collagen deposition in mice. However, when TRPML1 was specifically overexpressed, it was able to significantly inhibit cardiac collagen deposition in TAC mice, thereby inhibiting the occurrence of fibrosis after heart failure.

[0205] The above results indicate that cardiomyocyte-specific overexpression of TRPML1 can inhibit cardiac collagen deposition and fibrosis in heart failure mice, thereby restoring cardiac function in heart failure mice.

[0206] Example 15: Proteomic screening of downstream target proteins of TRPML1

[0207] 1 Experimental methods

[0208] 1.1 Experimental group design:

[0209] The experiment was divided into two groups: IgG group and IP-TRPML1 group.

[0210] 1.24D-FastDIA screening of target proteins interacting with TRPML1.

[0211] After 48 hours of culture, primary neonatal rat cardiomyocytes were collected and total cell proteins were extracted for protein immunoprecipitation experiments. Mouse IgG primary antibody was added to the IgG group, and mouse TRPML1 primary antibody was added to the IP group. Then, equal amounts of the extracted total proteins were added to the above-mentioned magnetic beads containing the two antibodies and incubated overnight at 4°C. Finally, the two groups of magnetic beads were placed in dry ice and sent to Hangzhou Jingjie Biotechnology Co., Ltd. for 4D-Fast DIA proteomics analysis. The results of the test were screened for proteins that interacted with TRPML1 through GO analysis and KEGG enrichment analysis. The crystal structures of TRPML1 and VDAC1 were obtained using the UniProt knowledge base database. TRPML1 and VDAC1 were docked together using the AutoDock tool. The official recommendation is to dock at least 50 times, and the number of dockings in our study was set to 100 times. PyMOLWin was then used to visualize the docking results.

[0212] 2 Observation results

[0213] 2.1VDAC1 is a downstream target protein directly acted upon by TRPML1 in cardiomyocytes.

[0214] Mass spectrometry results are as follows Figure 5A-C As shown, a total of 263 proteins interacting with TRPML1 were screened. GO analysis of these 263 proteins revealed that the majority were localized to mitochondria. Functional assays of these proteins identified VDAC1 as a direct target protein of TRPML1, and molecular docking results revealed that TRPML1 and VDAC1 can bind to each other.

[0215] The above results indicate that TRPML1 can directly bind to VDAC1 on mitochondria to regulate mitochondria.

[0216] Example 16: Direct binding of TRPML1 to VDAC1 in vitro

[0217] 1 Experimental methods

[0218] 1.1 Experimental group design:

[0219] The experiment was divided into two groups: HA-TRPML1 group and HA-TRPML1+Flag-VDAC1 group.

[0220] 1.2 Direct binding of TRPML1 to VDAC1 in HEK293T cells

[0221] By adding an HA tag to the C segment of the TRPML1 sequence and a Flag tag to the C segment of VDAC1, the recombinant expression plasmids HA-TRPML1 and Flag-VDAC1 were constructed. The two plasmids were transfected into HEK293T cells via Lipo3000 liposomes and then cultured for 48 hours before immunoprecipitation experiments.

[0222] 2 Observation results

[0223] The results of Western blotting experiments after co-immunoprecipitation are shown in Figure 5D . In HEK293T cells, HA-tagged TRPML1 can directly bind to Flag-tagged VDAC1.

[0224] The above results indicate that TRPML1 can bind to VDAC1 on mitochondria in HE293T cells in vitro.

[0225] Example 17: TRPML1 directly binds to VDAC1 under physiological conditions, and this binding is weakened by TAC surgery

[0226] 1 Experimental methods

[0227] 1.1 Experimental group design:

[0228] The experiment was divided into 4 groups: Sham+IgG group, TAC+IgG group, Sham+IP-TRPML1 group and TAC+IP-TRPML1 group.

[0229] 1.2TRPML1 directly binds to VDAC1.

[0230] Eight weeks after TAC induction, total protein from heart tissue was extracted for immunoprecipitation. Mouse IgG primary antibodies were added to the IgG group, while mouse TRPML1 primary antibodies were added to the IP group. Equal amounts of extracted total protein were then added to magnetic beads containing the two antibodies and incubated overnight at 4°C. The beads were eluted and then subjected to Western blotting.

[0231] 2 Observation results

[0232] 2.1TRPML1 directly binds to VDAC1, and this binding is weakened by TAC.

[0233] The results of co-immunoprecipitation are shown in Figure 5E . In the sham group, TRPML1 strongly bound to VDAC1, but this binding was disrupted after 8 weeks of TAC induction.

[0234] The above results indicate that TRPML1 can directly bind to VDAC1 on mitochondria.

[0235] Example 18: TRPML1 agonist ML-SA1 inhibits VDAC1 oligomerization

[0236] 1 Experimental methods

[0237] 1.1 EGS cross-linking experiment

[0238] After collecting cardiomyocytes treated with AngⅡ and / or ML-SA1 for 24 hours, EGS crosslinker (200uM, MCE, E3257) was added to the EP tube in which the cells were collected and the samples were boiled at 30℃ for 30 minutes before Western blotting experiments.

[0239] 1.2 Immunoblotting analysis

[0240] The treated cells were lysed with RIPA and centrifuged. The supernatant was aspirated and protein concentration was quantified using a BCA protein assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and incubated with primary antibodies against VDAC1 and β-actin overnight at 4°C. After washing three times with 1× PBST, the membrane was incubated with enzyme-linked anti-mouse or anti-rabbit secondary antibodies for 1–2 hours. The blot was scanned using an Odyssey CLx imaging system and quantified using LI-COR Image Studio software.

[0241] 2 Observation results

[0242] 2.1TRPML1 agonist inhibits VDAC1 oligomerization.

[0243] As shown in Figure 5F-G, Western blotting experiments revealed that the expression of VDAC1 dimers increased under AngⅡ treatment, but this increase was inhibited by ML-SA1.

[0244] The above results indicate that the increase in TRPML1 activity can inhibit the occurrence of VDAC1 oligomerization, thereby reducing the disorder of calcium ions in mitochondria and restoring mitochondrial function.

[0245] Example 19: Inhibition of VDAC1 oligomerization can reverse the decreased cardiac function in TRPML1 knockout-induced heart failure mice

[0246] 1 Experimental methods

[0247] 1.1 VDAC1 oligomerization inhibitor dosing regimen and grouping:

[0248] NSC15364 is a direct inhibitor of VDAC1 oligomerization. VDAC1 oligomerization was inhibited by intraperitoneal injection of NSC15364 (2.5 mg / kg, Selleck, S0231) twice a week for 8 consecutive weeks. fl / fl +Vehicle group, TAC+TRPML1 fl / fl +NSC15364 group, TAC+TRPML1 cKO +Vehicle group and TAC+TRPML1 cKO +NSC15364 group. TAC+TRPML1 fl / fl +Vehicle group: TRPML1 fl / fl After TAC surgery, mice were intraperitoneally injected with a vehicle [400 μl DMSO + 1600 μl PEG 300 (MCE, HY-Y0873) + 200 μl Tween 80 (Beyotime, ST2789) + 1800 μl ultrapure water] twice a week for 8 consecutive weeks; TAC + TRPML1 fl / fl +NSC15364 group: TRPML1 fl / fl After TAC surgery, mice were intraperitoneally injected with NSC15364 twice a week for 8 consecutive weeks; TAC+TRPML1 cKO +Vehicle group: TRPML1 cKO After TAC surgery, mice were intraperitoneally injected with Vehicle twice a week for 8 consecutive weeks; TAC+TRPML1 cKO +NSC15364 group: TRPML1 cKO After TAC surgery, mice were intraperitoneally injected with NSC15364 twice a week for 8 consecutive weeks.

[0249] 1.2 Echocardiography to detect cardiac function in mice

[0250] After 8 weeks of TAC and NSC15364 treatment, the mice were anesthetized with avertin and placed on an ultrasound table. The ejection fraction (EF%) and fractional shortening (FS%) of the mouse hearts were measured using a 10 Hz ultrasound probe in M ​​mode.

[0251] 1.3 Data Processing

[0252] The results of the present invention are expressed as mean ± standard deviation. Graphpadprism 8.0 was used for plotting, and the correlations between the groups were compared between two groups using one-way analysis of variance.

[0253] 2 Observation results

[0254] 2.1 Inhibiting VDAC1 oligomerization can restore the decreased cardiac function in heart failure mice caused by TRPML1 knockout.

[0255] As shown in Figure 6A-D, TAC+TRPML1 fl / fl Compared with the TAC+TRPML1 group, the inhibition of VDAC1 oligomerization by NSC15364 could restore the decrease of EF% and FS% in mice. cKO Compared with the +Vehicle group, inhibition of VDAC1 oligomerization reversed the decline in cardiac function caused by TRPML1 knockout, as manifested by increased EF% and FS% values.

[0256] The above results indicate that inhibiting VDAC1 oligomerization can restore the decreased cardiac function in heart failure mice caused by TRPML1 knockout, thereby reversing heart failure.

[0257] Example 20: Inhibition of VDAC1 oligomerization can reverse the increase in cardiac volume in TRPML1-knockout heart failure mice

[0258] 1 Experimental methods

[0259] 1.1 Experimental Grouping:

[0260] The experiment was divided into TAC+TRPML1 fl / fl +Vehicle、TAC+TRPML1 fl / fl +NSC15364 group, TAC+TRPML1 cKO +Vehicle group and TAC+TRPML1 cKO +NSC15364 group.

[0261] 1.2 HE staining to detect mouse heart volume

[0262] After the mice were modeled with TAC for 8 weeks, the samples were collected and sectioned in paraffin. The sections were dewaxed with gradient ethanol and stained with hematoxylin for 10 minutes to observe the staining of the cell nuclei. The sections were then stained with eosin for 1-2 minutes and sealed for photographing and recording.

[0263] 2 Observation results

[0264] 2.1 Inhibiting VDAC1 oligomerization can restore the enlarged heart volume in heart failure mice caused by TRPML1 knockout.

[0265] As shown in Figure 6E, TAC+TRPML1 fl / fl Compared with the TAC+TRPML1 group, the inhibition of VDAC1 oligomerization by NSC15364 significantly inhibited the increase in mouse heart volume.cKO Compared with the +Vehicle group, inhibition of VDAC1 oligomerization reversed the increase in heart volume caused by TRPML1 knockout.

[0266] The above results indicate that inhibiting VDAC1 oligomerization can restore cardiac enlargement in heart failure mice caused by TRPML1 knockout, thereby reversing heart failure.

[0267] Example 21: Inhibition of VDAC1 oligomerization can reverse cardiomyocyte hypertrophy in heart failure mice induced by TRPML1 knockout.

[0268] 1 Experimental methods

[0269] 1.1 Experimental Grouping:

[0270] The experiment was divided into TAC+TRPML1 fl / fl +Vehicle、TAC+TRPML1 fl / fl +NSC15364 group, TAC+TRPML1 cKO +Vehicle group and TAC+TRPML1 cKO +NSC15364 group.

[0271] 1.2 WGA staining to detect the cross-sectional area of ​​mouse cardiomyocytes

[0272] After the mice were modeled with TAC for 8 weeks, the samples were collected and sectioned in paraffin. The sections were dewaxed with gradient ethanol, stained with WGA dye for 1 hour, and then mounted for photographing and recording.

[0273] 2 Observation results

[0274] 2.1 Inhibiting VDAC1 oligomerization can restore the enlarged cross-sectional area of ​​cardiomyocytes in heart failure mice caused by TRPML1 knockout.

[0275] As shown in Figure 6F-G, TAC+TRPML1 fl / fl Compared with the TAC+TRPML1 group, the inhibition of VDAC1 oligomerization by NSC15364 significantly inhibited the increase in the cross-sectional area of ​​mouse cardiomyocytes. cKO Compared with the +Vehicle group, inhibition of VDAC1 oligomerization reversed the increase in cardiomyocyte cross-sectional area in mice caused by TRPML1 knockout.

[0276] The above results indicate that inhibiting VDAC1 oligomerization can restore the enlarged cross-sectional area of ​​cardiomyocytes in heart failure mice caused by TRPML1 knockout, thereby reversing heart failure.

[0277] Example 22: Inhibition of VDAC1 oligomerization can reverse the increase in cardiac collagen deposition in heart failure mice induced by TRPML1 knockout

[0278] 1 Experimental methods

[0279] 1.1 Experimental Grouping:

[0280] The experiment was divided into TAC+TRPML1 fl / fl +Vehicle、TAC+TRPML1 fl / fl +NSC15364 group, TAC+TRPML1 cKO +Vehicle group and TAC+TRPML1 cKO +NSC15364 group.

[0281] 1.2 WGA staining to detect the cross-sectional area of ​​mouse cardiomyocytes

[0282] After the mice were modeled with TAC for 8 weeks, the samples were collected and sectioned in paraffin. The sections were dewaxed with gradient ethanol, stained with Masson stain, and then sealed for photographing and recording.

[0283] 2 Observation results

[0284] 2.1 Inhibiting VDAC1 oligomerization can restore the increased collagen deposition in the hearts of mice with heart failure caused by TRPML1 knockout.

[0285] As shown in Figure 6H-I, TAC+TRPML1 fl / fl Compared with the TAC+TRPML1 group, the inhibition of VDAC1 oligomerization by NSC15364 significantly inhibited the collagen deposition in the mouse heart. cKO Compared with the +Vehicle group, inhibition of VDAC1 oligomerization reversed the increase in cardiac collagen deposition in mice induced by TRPML1 knockout.

[0286] The above results indicate that inhibiting VDAC1 oligomerization can restore the occurrence of cardiac fibrosis in heart failure mice caused by TRPML1 knockout, thereby reversing heart failure.

Claims

1. Application of transient receptor potential mucolipin 1 (TRPML1) or its agonist in the preparation of drugs for inhibiting cardiomyocyte hypertrophy.

2. The use according to claim 1, characterized in that The agonist is TRPML1 agonist ML-SA1.

3. Use of transient receptor potential mucolipin 1 (TRPML1) or its agonist in the preparation of drugs for inhibiting heart failure.

4. The use according to claim 3, characterized in that The agonist is TRPML1 agonist ML-SA1.

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