Use of drugs targeting gamma-aminobutyric acid transmitter system in treatment of cardiac diseases

Abstract

The invention provides an application of a drug targeting a gamma-aminobutyric acid transmitter system in heart disease treatment. Specifically, the invention provides an application of a drug targeting a gamma-aminobutyric acid transmitter system (GABA-TS) in preparation of a drug for treating or preventing heart diseases. Specifically, a new intervention strategy is provided for arrhythmia by identifying an endogenous gamma-aminobutyric acid transmitter system and a regulation function contained in the atrioventricular knot pace-making cell.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the use of a drug targeting the γ-aminobutyric acid neurotransmitter system in the treatment of heart disease. Background Technology

[0002] Under physiological conditions, electrical stimulation begins in the sinoatrial node, the source of electrical activity. The electrical excitation first reaches the atria, then travels through the atrioventricular node (AVN) and the His-Purkinje fiber network to the ventricles, ultimately triggering ventricular contraction. The conduction velocity of electrical excitation is slowest in the AVN, a characteristic that ensures sequential contraction of the atria and ventricles and efficient cardiac pumping. Clinically, AVN conduction defects are one of the most common causes of arrhythmias, leading to varying degrees of atrioventricular block (AVB) or even more serious cardiac arrest and sudden cardiac death. Severe AVB is a major clinical indication for pacemaker implantation. Unfortunately, little is known about the mechanisms underlying the slow conduction of electrical excitation within the AVN. Therefore, effective treatments are lacking for cardiac diseases such as slow conduction of electrical excitation within the AVN or arrhythmias related to AVN conduction defects.

[0003] In summary, there is an urgent need in this field to develop a new method for treating or preventing arrhythmias related to AVN conduction defects. Summary of the Invention

[0004] The purpose of this invention is to provide a new treatment or prevention method for cardiac diseases such as arrhythmias related to AVN conduction defects.

[0005] In a first aspect of the invention, there is provided the use of a medicament targeting the γ-aminobutyric acid neurotransmitter system (GABA-TS) in the preparation of a medicament for treating or preventing heart disease.

[0006] In another preferred embodiment, the γ-aminobutyric acid (GABA) neurotransmitter system is the GABA neurotransmitter system in atrioventricular nodal pacemaker cells (AVNPCs).

[0007] In another preferred embodiment, the target γ-aminobutyric acid neurotransmitter system refers to targeting one or more targets within the γ-aminobutyric acid neurotransmitter system.

[0008] In another preferred embodiment, the γ-aminobutyric acid neurotransmitter system includes one or more of the following targets: GABA metabolic enzymes, GABA receptors, and GABA transporters.

[0009] In another preferred embodiment, the γ-aminobutyric acid (GABA) neurotransmitter system includes one or more of the following targets: GAD2, GABRA3, GABRB2, GABRG2, vGAT, GAT-1, GABA-T, and SSADH.

[0010] In another preferred embodiment, the GABA metabolic enzymes include: GABA synthase (GAD2), GABA transferase (GABA-T), and GABA degrading enzyme (SSADH).

[0011] In another preferred embodiment, the GABA receptor is GABA. A GABA receptor A R).

[0012] In another preferred embodiment, the GABA A R subtypes, including GABA receptor GABA A R subunit α3 (GABRA3), GABA receptor GABA A R subunit β2 (GABRB2) and GABA receptor GABA A R subunit γ2 (GABRG2).

[0013] In another preferred embodiment, the GABA receptor comprises: GABA receptor GABA A R subunit α3 (GABRA3), GABA receptor GABA A R subunit β2 (GABRB2) and GABA receptor GABA A R subunit γ2 (GABRG2).

[0014] In another preferred embodiment, the GABA transporter includes: vesicle GABA transporter (vGAT) and GABA transporter 1 (GAT-1).

[0015] In another preferred embodiment, the γ-aminobutyric acid (GABA) neurotransmitter system includes one or more of the following targets: GABA synthase GAD2, GABA receptor GABA A R subunit α3 (GABRA3), GABA receptor GABA A R subunit β2 (GABRB2), GABA receptor GABA A R subunit γ2 (GABRG2), vesicle GABA transporter (vGAT), GABA transporter 1 (GAT-1), GABA transferase (GABA-T), and GABA degrading enzyme (SSADH).

[0016] In another preferred embodiment, the targeted γ-aminobutyric acid neurotransmitter system refers to targeting one or more targets selected from the group consisting of: GABA synthase GAD2, GABA receptor GABA. A R subunit α3 (GABRA3), GABA receptor GABA A R subunit β2 (GABRB2), GABA receptor GABA AR subunit γ2 (GABRG2), vesicle GABA transporter (vGAT), GABA transporter 1 (GAT-1), GABA transferase (GABA-T), and GABA degrading enzyme (SSADH).

[0017] In another preferred embodiment, the targeted γ-aminobutyric acid neurotransmitter system refers to a system targeting one or more targets selected from the group consisting of GABA. A R, vGAT, GABA-T, GAT-1, SSADH.

[0018] In another preferred embodiment, the drug targeting GABA-TS includes one or more of small molecule compounds, antibodies, and shRNA; preferably, it is a small molecule compound.

[0019] In another preferred embodiment, the drug targeting the γ-aminobutyric acid neurotransmitter system includes: GABA. A One or more of the following: R regulator, vGAT regulator, GABA reuptake regulator, GAT-1 regulator, GABA transferase regulator, and SSADH regulator.

[0020] In another preferred embodiment, the drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS inhibitor.

[0021] In another preferred embodiment, the drug targeting the γ-aminobutyric acid neurotransmitter system or the GABA-TS inhibitor is a drug having one or more of the following effects:

[0022] Inhibit GABA A R;

[0023] Suppress vGAT;

[0024] Promotes GABA reuptake;

[0025] Activate GAT-1;

[0026] Activate GABA transferase.

[0027] In another preferred embodiment, the drug targeting the γ-aminobutyric acid (GABA) neurotransmitter system is selected from the group consisting of GABA. A R antagonists, vGAT inhibitors, GABA reuptake promoters, GAT-1 activators, GABA transferase activators, or combinations thereof.

[0028] In another preferred example, GABA AR-antagonists include: Gabazine, Picrotoxinin, Bicuculline, Etbicyphat, Oroxylin A, Songorine, Thiocochicoside, (-)-Securinine, 6,2'-Dihydroxyflavone, etc.

[0029] In another preferred embodiment, the drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS agonist.

[0030] In another preferred embodiment, the drug targeting the γ-aminobutyric acid neurotransmitter system or the GABA-TS agonist is a drug having one or more of the following effects:

[0031] Activate GABA A R;

[0032] Activate vGAT;

[0033] Inhibit GABA reuptake;

[0034] Inhibit GAT-1;

[0035] Inhibit GABA transferase.

[0036] In another preferred embodiment, the drug or GABA-TS agonist targeting the γ-aminobutyric acid neurotransmitter system is selected from the group consisting of GABA. A R agonists, vGAT activators, GABA reuptake inhibitors, GAT-1 inhibitors, GABA transferase inhibitors, or combinations thereof.

[0037] In another preferred embodiment, GABA reuptake inhibitors include tiagabine or its salts.

[0038] In another preferred embodiment, GAT-1 inhibitors include: SKF89976A or a salt thereof, LU-32-176B,

[0039] Guvacine or its salts, CI 966 or its salts, NO-711 or its salts, etc.

[0040] In another preferred embodiment, the heart disease is characterized by heart disease including PR interval abnormalities or heart disease caused by PR interval abnormalities.

[0041] In another preferred embodiment, the PR interval abnormality is PR interval prolongation.

[0042] In another preferred embodiment, the heart disease is characterized by a prolonged PR interval or a heart disease caused by a prolonged PR interval.

[0043] In another preferred embodiment, the PR interval abnormality is a shortened PR interval.

[0044] In another preferred embodiment, the heart disease is characterized by a shortened PR interval or a heart disease caused by a shortened PR interval.

[0045] In another preferred embodiment, the heart disease is a heart disease related to atrioventricular node (AVN) conduction defects.

[0046] In another preferred embodiment, the heart disease is a heart disease associated with slow conduction of the atrioventricular node (AVN).

[0047] In another preferred embodiment, the cardiac disease is selected from the group consisting of: arrhythmias (including tachyarrhythmias and bradycardia), atrioventricular block (AVB), cardiac arrest, sudden cardiac death, atrial fibrillation, atrial flutter, premature atrial contractions, Wolff-Parkinson-White syndrome, short PR syndrome, or combinations thereof.

[0048] In another preferred embodiment, the characterization includes cardiac diseases with prolonged PR interval or cardiac diseases caused by prolonged PR interval, including atrioventricular block (AVB) and bradycardia.

[0049] In another preferred embodiment, the characterization includes cardiac diseases with shortened PR intervals or cardiac diseases caused by shortened PR intervals, including: Wolff-Parkinson-White syndrome, short PR syndrome.

[0050] In another preferred embodiment, the heart disease is atrioventricular block.

[0051] In another preferred embodiment, the heart disease is atrioventricular block caused by AVN conduction defects.

[0052] In another preferred embodiment, the drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS inhibitor, and the heart disease is characterized by heart disease including PR interval prolongation or heart disease caused by PR interval prolongation.

[0053] In another preferred embodiment, the drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS inhibitor, and the heart disease is atrioventricular block or bradycardia.

[0054] In another preferred embodiment, the drug targeting the γ-aminobutyric acid neurotransmitter system is GABA. A R antagonists and / or vGAT inhibitors, and the cardiac disease is atrioventricular block or bradycardia.

[0055] In another preferred embodiment, the drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS agonist, and the heart disease is characterized by a shortened PR interval or a heart disease caused by a shortened PR interval.

[0056] In a second aspect of the invention, a pharmaceutical combination is provided comprising: (1) a drug targeting the γ-aminobutyric acid neurotransmitter system (GABA-TS), and (2) an additional drug.

[0057] In another preferred embodiment, the additional drug is selected from the group consisting of: drugs for treating or preventing heart disease, drugs for treating or preventing diseases caused by heart disease, and drugs for relieving symptoms caused by heart disease.

[0058] In another preferred embodiment, the additional drug is not a drug that targets the γ-aminobutyric acid neurotransmitter system (GABA-TS).

[0059] In a third aspect of the invention, a kit is provided comprising: (1) a drug targeting the γ-aminobutyric acid neurotransmitter system (GABA-TS), and (2) additional drugs.

[0060] In another preferred embodiment, the additional drug is as defined above.

[0061] In a fourth aspect of the invention, a pharmaceutical composition is provided comprising: (1) a first active substance, the first active substance being a drug targeting the γ-aminobutyric acid neurotransmitter system (GABA-TS), and (2) a pharmaceutically acceptable carrier or excipient.

[0062] In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for treating or preventing heart disease.

[0063] In another preferred embodiment, the pharmaceutical composition further includes a second active substance, which is another drug.

[0064] In another preferred embodiment, the additional drug is as defined above.

[0065] In a fifth aspect of the invention, a method for regulating the PR interval of a subject is provided, comprising the step of contacting the subject with a drug targeting GABA-TS, thereby regulating the PR interval.

[0066] In another preferred embodiment, the regulation of PR period refers to shortening the PR period, and the drug targeting GABA-TS refers to a GABA-TS inhibitor (as previously defined).

[0067] In another preferred embodiment, the regulation of PR period refers to prolonging the PR period, and the drug targeting GABA-TS refers to a GABA-TS agonist (as previously defined).

[0068] In another preferred embodiment, the object is a cell (such as an atrioventricular node pacemaker cell (AVNPC)) or tissue derived from the atrioventricular node (AVN).

[0069] In another preferred embodiment, the method is non-therapeutic in vitro.

[0070] In a sixth aspect of the invention, a method for modulating the electrical excitability of atrioventricular nodal pacemaker cells (AVNPCs) is provided, comprising the step of contacting the AVNPCs with a drug targeting GABA-TS, thereby modulating the electrical excitability of the AVNPCs.

[0071] In another preferred embodiment, the regulation of the electrical excitability of the atrioventricular nodal pacemaker cells is to increase the regulation of the electrical excitability of the atrioventricular nodal pacemaker cells, and the drug targeting GABA-TS refers to a GABA-TS inhibitor (as previously defined).

[0072] In another preferred embodiment, the regulation of the electrical excitability of the atrioventricular nodal pacemaker cells is to reduce the regulation of the electrical excitability of the atrioventricular nodal pacemaker cells, and the drug targeting GABA-TS refers to a GABA-TS agonist (as previously defined).

[0073] In another preferred embodiment, the method is non-therapeutic in vitro.

[0074] In a seventh aspect of the invention, a method for modulating AVN electrical signal conduction is provided, comprising the step of contacting an atrioventricular nodal pacing object with a drug targeting GABA-TS, thereby modulating AVN electrical signal conduction.

[0075] In another preferred embodiment, the regulation of AVN electrical signal transduction is to accelerate AVN electrical signal transduction, and the drug targeting GABA-TS refers to a GABA-TS inhibitor (as previously defined).

[0076] In another preferred embodiment, the regulation of AVN electrical signal transduction is to slow down AVN electrical signal transduction, and the drug targeting GABA-TS refers to a GABA-TS agonist (as previously defined).

[0077] In another preferred embodiment, the object is a cell (such as an atrioventricular node pacemaker cell (AVNPC)) or tissue derived from the atrioventricular node (AVN).

[0078] In another preferred embodiment, the method is non-therapeutic in vitro.

[0079] In an eighth aspect of the invention, a method for treating or preventing heart disease is provided, comprising: administering a safe and effective amount of a drug targeting the gamma-aminobutyric acid neurotransmitter system (GABA-TS) to a subject in need.

[0080] In another preferred embodiment, the method further includes administering a safe and effective amount of another drug.

[0081] In another preferred embodiment, the additional drug is as defined above.

[0082] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0083] Figure 1 The presence of GABA vesicles and GABA-induced ligand-gated currents are shown in rat atrioventricular node pacemakers. a: Transmission electron microscopy (TEM) images show numerous vesicles subcellularly in rat atrioventricular node pacemakers (AVNPCs). The right image is a magnified version of the white dashed box in the left image. White arrows indicate vesicles. Scale bar, 500 nm. b: Immunofluorescence images show the colocalization of GABA and the vesicle marker CAST in a single rat AVNPC. Scale bar, 10 μm. c: Patch-clamp recordings of currents induced by different concentrations of GABA (0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) in rat AVNPCs. Holding potential set to -60 mV. Horizontal bar, 500 ms; vertical bar, 10 pA.

[0084] Figure 2 To demonstrate the mouse (Hcn4) CreERT2(+) Rosa26 TomRed+ A heatmap of GABA neurotransmitter system gene expression in a single atrioventricular node pacemaker cell (AVNPC) in a mouse. Rows represent samples of AVNPC cells, and columns represent Ct values ​​of GABA neurotransmitter system genes. The color scale shows the relative expression of the genes (measured by Ct values). A score of -1 (red) indicates high expression levels, and a score of 1 (blue) indicates low expression levels.

[0085] Figure 3The images show the expression of key components of the GABA neurotransmitter system in rat atrioventricular node (AVN) tissue. a: Immunofluorescence staining of GABA metabolic enzymes (GAD2, GABA-T, and SSADH) in rat AVN tissue. Scale bar, 25 μm. b: Immunofluorescence staining of GABAA receptors (GABRA3, GABRB2, and GABRG2) in rat AVN tissue. Scale bar, 25 μm. c: Immunofluorescence staining of GABA transport proteins (vGAT and GAT-1) in rat AVN tissue. Scale bar, 25 μm.

[0086] Figure 4 The images show the expression of key components of the GABA neurotransmitter system in rat atrioventricular node pacemakers. a: Immunofluorescence staining shows the expression and localization of GABA-metabolizing enzymes (GAD2, GABA-T, and SSADH) in rat atrioventricular node pacemakers (AVNPCs). Scale bar, 10 μm. b: Immunofluorescence staining shows the expression and localization of GABAA receptors (GABRA3, GABRB2, and GABRG2) in rat AVNPCs. Scale bar, 10 μm. c: Immunofluorescence staining shows the expression and localization of GABA transporters (vGAT and GAT-1) in rat AVNPCs. GABA A R, GABA A R receptor. Scale bar, 10 μm.

[0087] Figure 5 It shows GABA and GABA A Receptor activation reduces the excitability of atrioventricular nodal pacemaker cells. Pooled data of maximum diastolic potentials (MDPs) recorded using current patch-clamp techniques in adult rat atrioventricular nodal pacemaker cells (AVNPCs). n = 7. Data are presented as mean ± sd. P-values ​​were calculated using one-way ANOVA and Dunnett's multiple comparison test.

[0088] Figure 6 This shows the electrical signal conduction in the atrioventricular junction controlled by the GABA neurotransmitter system. a: Representative activation plots are shown using solvent controls DMSO and GABA. AElectrical activation and conduction in isolated rat atrioventricular node (AVN) tissue perfused with the R-specific agonist Afloqualone (320 μM), the GABA reuptake inhibitor Tiagabine (64 μM), or the GAT-1 inhibitor SKF89976A (128 μM). Magnified activation maps show electrical activation and conduction within the central region of the AVN. Electrical activity in rat AVN tissue was recorded by optical mapping using the fluorescent dye Di-4-ANBDQBS. Activation time, conduction velocity, and vector maps were obtained during continuous pacing with a stimulating electrode located at the crista terminalis (5 Hz, 2 V). b: Statistical analysis of conduction time within the AVN in different treatment groups (n = 6 samples for the control group, n = 7 samples for the Afloqualone treatment group, n = 9 samples for the Tiagabine treatment group, and n = 8 samples for the SKF89976A treatment group). Data are presented as mean ± sd. P-values ​​were calculated using one-way ANOVA and Dunnett's multiple comparison test. c, f, and i: Representative electrocardiogram (ECG) records of perfused rat hearts treated with different concentrations of Afloqualone (0–640 μM) (c), Tiagabine (0–64 μM) (f), or SKF89976A (0–128 μM). Arrows point to typical ECGs of second-degree type I atrioventricular block (AVB) (c, f) and second-degree AVB (2:1) (i). Scale bar is 100 ms horizontally and 2 mV vertically. d, g, and j: Dose-response of Afloqualone (d), Tiagabine (g), and SKF89976A (j) to the PR interval in perfused rat hearts. n = 5 for the Afloqualone and SKF89976A treatment groups, and n = 6 for the Tiagabine treatment group. Data are presented as mean ± sd. P-values ​​were calculated using one-way ANOVA and Dunnett's multiple comparison test. e, h, and k: Concentration-response curves of PR interval alterations induced by Afloqualone (EC50, 165.70 μM) (e), Tiagabine (IC50, 20.22 μM) (h), and SKF89976A (IC50, 37.57 μM) (k). Concentration-response curves were fitted to the Hill equation using nonlinear regression and normalized to the PR interval with the greatest variation. Data are presented as mean ± SD. n = 5 for the Afloqualone and SKF89976A treatment groups, and n = 6 for the Tiagabine treatment group. l: Representative ECGs of conscious rats were recorded via telemetry after injection of AAV2 / 9-Control, AAV2 / 9-Gabrb2, AAV2 / 9-Slc32a1, or AAV2 / 9-Abat viruses. Scale bars were set horizontally at 100 ms and vertically at 0.5 mV.m and n are statistical plots of the mean PR interval (m) and P wave duration (n) in rats within the specified groups. For the AAV2 / 9-Control group, n = 7; for the AAV2 / 9-Gabrb2 group, n = 6; for the AAV2 / 9-Slc32a1 group, n = 6; and for the AAV2 / 9-Abat group, n = 6. Data are shown as mean ± SD. P-values ​​were calculated using one-way ANOVA and Dunnett's multiple comparison test.

[0089] Figure 7 This study demonstrates that intervention targeting the GABA neurotransmitter system induced severe atrioventricular block in isolated rat hearts. a: Representative electrocardiogram (ECG) recordings show that when the heart was perfused with 640 μM GABA... A a: Cardiac arrest occurred with the receptor agonist Afloqualone (top image), and normal atrioventricular conduction was restored after the cessation of Afloqualone perfusion (bottom image). Scale bar: horizontal 100ms, vertical 2mV. b: Representative ECG recordings show that high concentrations of the GABA reuptake inhibitor Tiagabine (64μM) can cause third-degree atrioventricular block (AVB) in the perfused heart (top image), and normal PR interval cessation is restored after the cessation of Tiagabine perfusion (bottom image). Scale bar: horizontal 100ms, vertical 2mV. c: Representative ECG recordings show that high concentrations of the GABA transporter-1 inhibitor SKF89976A (128μM) can cause third-degree atrioventricular block in the perfused heart (top image), and normal PR interval is restored after the cessation of SKF89976A perfusion (bottom image). Scale bar: horizontal 100ms, vertical 2mV.

[0090] Figure 8 This study demonstrates that GABA agonists or inhibitors do not alter the QRS and QT intervals in isolated rat hearts. (The text also mentions different concentrations of GABA, but the connection to the preceding sentence is unclear.) A Statistical plots of QRS and QT intervals in perfused rat hearts treated with receptor agonist (Afloqualone) (a), GABA reuptake inhibitor (Tiagabine) (b), and GABA transporter inhibitor (SKF89976A) (c). n=5 for the Afloqualone and SKF89976A treatment groups, and n=6 for the Tiagabine treatment group. Data are expressed as mean ± sd. P-values ​​were calculated using one-way ANOVA and Dunnett's multiple comparison test.

[0091] Figure 9The intervention of the GABA neurotransmitter system showed that it did not affect the QRS interval, QT interval, and heart rate in vivo. The bar charts (a), (b), and (c) plots show the statistical distribution of mean QRS interval (a), QT interval (b), and heart rate (beats per minute, BPM) in the hearts of rats infected with AAV2 / 9-Control, AAV2 / 9-Gabrb2, AAV2 / 9-Slc32a1, and AAV2 / 9-Abat viruses, respectively. For the AAV2 / 9-Control group, n = 7; for the AAV2 / 9-Gabrb2 group, n = 6; for the AAV2 / 9-Slc32a1 group, n = 6; and for the AAV2 / 9-Abat group, n = 6. Data are shown as mean ± SD. P-values ​​were calculated using one-way ANOVA and Dunnett's multiple comparison test.

[0092] Figure 10 This shows that intervention targeting the GABA neurotransmitter system can prevent the occurrence and development of atrioventricular block. On the left, representative electrocardiogram (ECG) recordings show successful induction of severe atrioventricular block (AVB) by perfusion of verapamil (250 nM) into isolated hearts injected with AAV2 / 9-Control virus. Arrows indicate representative second-degree AVB (2:1). Hearts injected with AAV2 / 9-Gabrb2 and AAV2 / 9-Slc32a1 viruses did not develop second-degree or high-degree AVB. Scale bars are 100 ms horizontally and 2 mV vertically. On the right, the proportion of second-degree or high-degree AVB occurring in hearts injected with AAV2 / 9-Control, AAV2 / 9-Gabrb2, or AAV2 / 9-Slc32a1 viruses. AAV2 / 9-Control: n = 10, AAV2 / 9-Gabrb2: n = 6, AAV2 / 9-Slc32a1: n = 8. P-values ​​were calculated using Fisher's exact test. Detailed Implementation

[0093] Through long-term and in-depth research, the inventors unexpectedly discovered that the main functional cells in the atrioventricular node (AVN), namely the atrioventricular node pacemaker cells (AVNPCs), possess an endogenous γ-aminobutyric acid neurotransmitter system (GABA-TS) and its key components, such as GABA metabolic enzymes and GABA receptors (GAB). A R) and GABA transporters such as vesicular GABA transporters (vGAT) and GABA transporter 1 (GAT-1); furthermore, the present invention also found that intervention on GABA-TS, for example targeting key elements therein, can significantly modulate electrical signal conduction in AVN, thereby preventing the occurrence and development of severe AVB. Based on this, the inventors completed the present invention.

[0094] the term

[0095] Unless otherwise stated, the abbreviations used herein have the meanings well known to those skilled in the art.

[0096] In this article, the term "PR interval" refers to the period from the start of the P wave to the start of the QRS complex in an electrocardiogram.

[0097] Atrioventricular nodal pacemaker cells (AVNPC)

[0098] Atrioventricular node (AVN)

[0099] The atrioventricular node (AV node) is located in the lower part of the atrioventricular septum, on the right side of the atrium, below the endocardium and anterosuperior to the coronary sinus ostium. It is flattened and oval-shaped, smaller than the sinoatrial node, and its anteroinferior end continues as the atrioventricular bundle. Its function is to transmit impulses from the sinoatrial node to the ventricles. The electrical impulse is briefly delayed within the AV node to ensure sequential contraction of the atrial and ventricular myocardium; that is, the atria contract first, followed by the ventricles.

[0100] Pacemaker cell (PC)

[0101] Pacemakers are capable of spontaneously exciting themselves and transmitting the generated current to all or part of the non-automatic myocardial cells or cell groups. They are the smallest constituent units of automatic myocardium and the tissue basis of the pacemaker mechanism. A key characteristic of pacemakers is the slope of their four-phase action potential; they can spontaneously and slowly depolarize during diastole, thus possessing automaticity. Under physiological conditions, pacemakers are mainly distributed in the cardiac conduction system, with a very small number dispersed in ordinary myocardium, especially atrial myocardium.

[0102] γ-Aminobutyric acid (GABA) and the γ-aminobutyric acid neurotransmitter system (GABA-TS)

[0103] Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. The GABA-TS system refers to a series of molecules and cellular structures, including GABA, GABA receptors, and GABA transporters, which work together to maintain homeostasis and information transmission between neurons. Specifically: 1) GABA: GABA is the main inhibitory neurotransmitter, widely distributed in the brain. It promotes inhibitory signal transmission in neurons by binding to GABA receptors; 2) GABA receptors: GABA receptors are the core components for GABA's function. They are divided into three categories: GABA-A receptors, GABA-B receptors, and GABA-C receptors. The first two are the main functional types. GABA-A receptors are ion channel receptors; when GABA binds, they allow Cl ions to pass through, increasing intracellular hyperpolarization and leading to an inhibitory effect in neurons. GABA-B receptors are G protein-coupled receptors; when GABA binds, they activate related signal transduction pathways, thereby producing an inhibitory effect. 3) GABA transporters: GABA transporters are responsible for regulating the concentration of GABA in the synaptic cleft. They can recycle GABA from the synaptic cleft back into the neuron or remove excess GABA from the synaptic cleft to maintain appropriate neurotransmitter levels.

[0104] The gamma-aminobutyric acid (GABA) neurotransmitter system is crucial for maintaining brain stability and proper signal transmission between neurons. It participates in multiple physiological and behavioral processes, including mood regulation, motor control, and cognitive function. Abnormal GABA neurotransmitter system function may be associated with various neurological disorders, such as anxiety, depression, and epilepsy. Drugs targeting the GABA neurotransmitter system are widely used clinically to treat various neurological and mental illnesses. These drugs are mainly divided into two categories: those that increase GABA levels and those that modulate GABA receptor function.

[0105] Drugs that increase GABA levels: Benzodiazepines Benzodiazepines, such as lorazepam and alprazolam, produce sedative, anxiolytic, and antidepressant effects by increasing the activity of GABA-A receptors. Baclofen is a GABA-B receptor agonist used to treat muscle spasms and movement disorders.

[0106] Drugs that regulate GABA receptor function: Antiepileptic drugs, such as sodium valproate and gabapentin, inhibit abnormal nerve excitation by regulating GABA receptors or increasing GABA levels, thereby preventing and controlling epileptic seizures. Pregabalin: Similar to gabapentin, it is also used to treat epilepsy and neuropathic pain. Ketoconazole: It is an NMDA receptor antagonist, but it can also affect the GABA neurotransmitter system and is used to treat depression and other mental illnesses.

[0107] The GABA-TS system and its function in atrioventricular nodal pacemaker cells (AVNPCs)

[0108] In this study, single-cell qPCR and immunofluorescence staining were used to identify the core components of GABA-TS in AVNPC, such as GAD2, vGAT, and ionic GABA. A The presence of R and GABA-degrading enzymes (GABA-T and SSADH). Importantly, using TEM and confocal microscopy, the inventors discovered that AVNPCs not only express the molecular elements of GABA-TS, but also possess abundant GABA vesicles beneath the AVNPC cell membrane and in the intercellular spaces. This study is the first to demonstrate that AVNPCs possess intact endogenous GABA-TS.

[0109] The in vitro and in vivo data presented in this paper demonstrate that endogenous GABA-TS in AVNPCs play a crucial role in AVN electrical signal transduction. The slow conduction characteristic of AVNs and the resulting physiological "conduction delay" between the atria and ventricles have remained unsolved problems in the field of cardiac arrhythmia. The presence of unique gap junction proteins in AVNPCs is considered a significant factor contributing to the slow conduction of AVNs. Here, the inventors discovered that intervention targeting multiple molecules of endogenous GABA-TS in AVNPCs can significantly alter the electrical signal transduction velocity of AVNs, implying that GABA-TS is another functional bioelectrical regulatory system controlling AVN electrical signal transduction. Furthermore, the findings of this study also provide a unique mechanism for regulating the sequential contraction of the atria and ventricles.

[0110] AVB can develop into a fatal arrhythmia. Currently, drug treatments for AVB are largely ineffective, and severe AVB is primarily treated with pacemakers. Due to the extremely limited number of AVNPCs and the difficulty in sampling, research on AVNs at the cellular level has been relatively scarce, resulting in a lag in AVB research. Therefore, these factors significantly hinder the progress of clinical prevention and treatment of AVB. This study found that intervention with GABA using a tool-based drug approach... AR or GAT-1 can significantly affect the conduction of electrical excitation within the AVN. Knocking out the GABA-TS encoding genes Gabrb2, Slc32a1, or Abat can significantly alter the PR interphase in rats. Importantly, the inventors have demonstrated that inhibiting GABA... A R or vGAT can prevent the occurrence and development of severe AVB. These data suggest that GABA-TS may be a promising intervention target for AVB.

[0111] In summary, the inventors have discovered that endogenous GABA-TS in AVNPC regulates the excitability and electrical signal transduction of AVNs, which is a crucial basis for electrical conduction between the atria and ventricles. This research reveals a novel electrophysiological mechanism of AVN conduction based on an intrinsic neurotransmitter system, providing a new intervention strategy for cardiac arrhythmias.

[0112] Drugs or compounds targeting the gamma-aminobutyric acid neurotransmitter system (GABA-TS), pharmaceutical compositions containing them, and methods of administration.

[0113] As used herein, the term "medicine targeting the γ-aminobutyric acid (GABA) neurotransmitter system" refers to substances (such as small molecule compounds, antibodies, etc.) that target the GABA neurotransmitter system and have the function of intervening in or regulating the GABA neurotransmitter system. Preferably, the compound targeting the GABA neurotransmitter system can target one or more key components in the GABA neurotransmitter system (such as GABA). A (R, vGAT, GAT-1, SSADH). In some alternatives, the compound targeting the γ-aminobutyric acid (GABA) neurotransmitter system is specific or non-specific. In some embodiments, the "medicine targeting the GABA neurotransmitter system" is as defined in the first aspect.

[0114] As used herein, the term "GABA-TS inhibitor" refers to a drug or substance that can modulate the γ-aminobutyric acid (GABA) neurotransmitter system by targeting one or more targets within the GABA neurotransmitter system, reducing the inhibitory effect of GABA, thereby promoting / accelerating electrical signal transduction, increasing atrioventricular node excitability, and shortening the PR interval, for example. According to this article, a GABA-TS agonist can be GABA... A GABA antagonists, vGAT inhibitors, GABA reuptake promoters, GAT-1 activators, GABA transferase activators, etc. Similarly, the term "GABA-TS agonist" refers to a drug or substance that can modulate the γ-aminobutyric acid (GABA) neurotransmitter system by targeting one or more sites within the GABA neurotransmitter system, thereby enhancing the inhibitory effect of GABA, and thus, for example, slowing electrical signal transduction, reducing atrioventricular node excitability, and prolonging the PR interval. According to this article, GABA-TS inhibitors can be GABA... AR agonists, vGAT activators, GABA reuptake inhibitors, GAT-1 inhibitors, GABA transferase inhibitors, etc.

[0115] Because AVNPCs contain GABA-TS, and intervention in GABA-TS within AVNPCs has excellent effects on regulating the excitability and electrical signal transduction of AVNs, it can effectively mediate electrical conduction between the atria and ventricles. Therefore, drugs or compounds with the ability to intervene in / regulate GABA-TS or target GABA-TS (especially those with GABA-TS-targeting effects) are of great interest. A Drugs or compounds that regulate GAT, vGAT, GABA reuptake, GAT-1, and GABA transferase can be used to treat or prevent heart diseases. According to existing technology, these heart diseases include, but are not limited to: arrhythmias, atrioventricular block (AVB), cardiac arrest, sudden cardiac death, heart failure, cardiomyopathy, congenital heart disease, myocardial infarction, etc. In particular, drugs or compounds that, for example, inhibit GABA... A Drugs or compounds that inhibit vGAT, promote GABA reuptake, activate GAT-1, or activate GABA transferase are particularly suitable for the treatment or prevention of heart diseases such as bradycardia that are related to atrioventricular node (AVN) dysfunction (e.g., slow AVN conduction).

[0116] In some implementations, drugs or compounds capable of intervening in GABA-TS, or drugs or compounds targeting GABA-TS, can be drugs or compounds capable of intervening in GABA-TS in the cardiovascular, nervous, and / or immune systems, or targeting GABA-TS in the cardiovascular, nervous, and / or immune systems. Therefore, antiepileptic drugs, sedatives, and benzodiazepines acting on GABA-TS are examples. Class II drugs, benzothiadiazepines Drugs such as anti-sleep medications, muscle relaxants, etc., can be used to treat or prevent heart disease.

[0117] In this document, the pharmaceutical compositions of the present invention comprise a drug targeting the γ-aminobutyric acid (GABA) neurotransmitter system within a safe and effective range. "Safe and effective range" refers to an amount of drug sufficient to significantly improve the condition without causing serious side effects.

[0118] The drugs targeting the γ-aminobutyric acid neurotransmitter system in this invention can be administered alone or in combination with other drugs (such as other treatments or preventions of heart disease or diseases or conditions related to or caused by heart disease).

[0119] The main advantages of this invention include:

[0120] The technical solution of this invention provides a new and effective intervention strategy for heart diseases, especially arrhythmias.

[0121] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0122] Experimental methods

[0123] Method 1: AVNPCs separation

[0124] Rats were heparinized (>200 units per animal, ip) and anesthetized (sodium pentobarbital, 30 mg / kg). The heart was then rapidly removed and perfused retrogradely through the aorta with solution A (mM): NaCl 140, KCl 5.4, CaCl2 1.8, KH2PO4 1.2, D-glucose 5.5, Hepes 5, with the pH adjusted to 7.4 using NaOH. The heart was then perfused with solution B, containing (mM): NaCl 140, KCl 5.4, CaCl2 0.2, KH2PO4 1.2, taurine 50, D-glucose 18.5, Hepes 5, with the pH adjusted to 6.9 using KOH. Next, the heart was perfused with solution B containing 1 mg / ml type 2 collagenase (Worthington, LS004177), 0.01 mg / ml elastase (Sigma, E1250), and 0.6 mg / ml protease (Sigma, P5147). The heart was digested for 20-25 minutes, and the perfusion solution was maintained at 37°C and oxygenated with 100% O2. The enzyme concentration and digestion time were adjusted according to individual circumstances. After heart digestion, the AVN tissue was cut into small pieces and transferred to solution D, which contained (in mM): 100 mg / mL K-glutamate, 10 mg / mL K-aspartate, 25 mg / mL KCl, 10 mg / mL KH2PO4, 2 mg / mL MgSO4, 20 mg / mL taurine, 5 mg / mL creatine, 0.5 mg / mL EGTA, 20 mg / mL glucose, 5 mg / mL Hepes, and 1 mg / mL BSA. The pH was adjusted to 7.2 with KOH. The AVN tissue was gently pipetted in solution D at 37°C for 2-5 minutes to obtain individual AVNPCs. Then, individual AVNPCs cells were progressively recalcified for further patch-clamp recording. Alternatively, RNA was extracted from individual AVNPCs cells for subsequent single-cell PCR analysis to determine the expression levels of genes related to the GABA neurotransmitter system.

[0125] Method 2: Whole-cell patch-clamp recording

[0126] Whole-cell patch-clamp recordings of individual rat AVNPCs were performed using an EPC-10 amplifier (HEKA, Germany) and Clampfit 10.7 software (HEKA, Germany) to detect membrane potentials and currents. Membrane potentials and currents were recorded at 22–26 °C. Two types of microelectrodes (borosilicate glass, 2-6 MΩ) were fabricated using a horizontal puller (Sutter Instrument): one for electrical stimulation and recording, filled with intracellular fluid containing (in mM): KCl (140), EGTA (10), HEPES (10), glucose (5), Na2ATP (3), and pH adjusted to 7.2 with KOH; the other was a 10 μm inner diameter microelectrode used to rapidly and concentratedly apply different drugs to the cell membrane of AVNPCs using a PL1-100 pixel injector (Harvard Apparatus, USA), filled with extracellular fluid including (in mM): NaCl (140), KCl (5.4), CaCl2 (1.8), KH2PO4 (1.2), taurine (50), D-glucose (18.5), HEPES (5), BSA 1 mg / mL. Tool drugs targeting key components of the GABA neurotransmitter system (such as the agonist Afloqualone) are dissolved in extracellular fluid. Stimulating pulses are applied via an EPC-10 amplifier (HEKA, Germany) synchronized with the picoinjector to release the drug. Action potentials are recorded in current-clamp mode, and currents are recorded in voltage-clamp mode.

[0127] Method 3: Electrocardiogram recording of an isolated heart

[0128] At 37°C, the heart was rapidly removed from adult rats and suspended on a modified Langendorff system, and perfused via the aorta with an oxygenated perfusion solution containing (mM): NaCl 140, glucose 5.5, KCl 5.4, CaCl2 1.8, K2HPO4 1.2, HEPES 5, and MgCl2 1 (pH 7.4, adjusted with NaOH). For electrocardiogram (ECG) recording, one electrode was placed at the base of the heart near the right atrium, and the other at the apex. The heart was paced via a pacing electrode (Powerlab, ADInstruments, USA) placed in the right atrium. The stimulation protocol was set as follows: stimulation thresholds were determined during the stabilization period and during pacing stimulation, typically 1.5–2 times the threshold, with a pulse duration of 2 ms and a frequency of 6 Hz. The heart was stabilized for 30 minutes before ECG recording, and then different pharmacological agents were added to the perfusion solution. ECG recordings were continuously obtained from Langendorff perfused hearts using a Powerlab amplifier (Powerlab, ADInstruments, USA).

[0129] The AVB model was constructed by adding verapamil (250 nM) to the perfusion solution after equilibration perfusion in an isolated heart for 30 minutes. An AVB model was considered successfully constructed when the isolated heart developed second-degree or high-degree AVB lasting longer than 15 minutes.

[0130] Method 4: Telemetry ECG recording

[0131] Adult rats were anesthetized with 2% isoflurane. A telemetry electrocardiogram transmitter was implanted into the abdominal cavity of the rats.

[0132] (DataSciences International, St. Paul, Minnesota) Paired wire electrodes were placed in the chest cavity (lead II configuration). Post-surgery, the rats were placed in individual cages with free access to food and water. Seven days after implantation surgery, ECGs were recorded for 24 hours using LabChart software (v8.1.9, ADInstruments Inc., Colorado) and a telemetry receiver. ECG parameters, including P wave, PR interval, QRS interval, and QT interval, were measured as previously described.

[0133] Method 5: Optical mapping

[0134] Optical mapping was performed on isolated rat AVN tissue. Briefly, rats were anesthetized with sodium pentobarbital (30 mg / kg). The rat heart was rapidly removed and placed in a warm, oxygenated Tyrode solution. The right atrial-AVN tissue, comprising the atrium and AVN including the Koch triangle, was then dissected. The AVN tissue was stained with Di-4-ANEPPS (15 μM, AAT, 90134-00-2) in an oxygenated Tyrode solution at 37°C for 40–60 min. Blebbistatin (10 μM, MCE, HY-13813) was perfused for 10 min to eliminate motion artifacts during optical mapping recording. Optical fluorescence signals were recorded at 1000 frames / second in the Koch triangle under right atrial pacing (5 Hz pacing rate, 2x pulse amplitude threshold, 2 ms pulse duration) using a high-speed 10,000-pixel camera (SciMedia, MiCAMULTIMA, USA). Prior to optical mapping analysis, the AVN formulation was perfused with DMSO, Afloqualone, Tiagabine, or SKF89976A for 30–60 minutes. AVN conduction time was calculated based on the gradient of the action potential activation map.

[0135] Method 6: Immunofluorescence staining

[0136] For immunofluorescence staining, isolated AVNPC cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes and washed twice with PBS. Cells were then permeabilized with 0.5% Triton X-100 solution for 10 minutes, washed twice with PBS, and blocked with 4% goat serum at room temperature for 1 hour. For immunostaining, cells were incubated overnight with primary antibody at 4°C. The next day, after washing twice with PBST, cells were incubated with the corresponding secondary antibody at room temperature for 1 hour, followed by DAPI (4,6-diamidinyl-2-phenylindole dihydrochloride) staining for 30 minutes. Representative images were captured using a Leica confocal microscope.

[0137] For immunohistochemical staining, the hearts of adult rats were rapidly excised, fixed overnight at 4°C with 4% PFA, and embedded in paraffin. The hearts were then longitudinally sectioned into 6 μm sections for immunostaining. Heart sections were dewaxed with xylene, then rehydrated in progressively decreasing concentrations of ethanol, and then antigen-repaired in citrate buffer. Sections were blocked with 5% goat serum at room temperature for 1 h, followed by incubation overnight at 4°C with a primary antibody diluted in 5% goat serum. After washing three times in 0.1% PBST, sections were stained with fluorescent secondary antibody at room temperature for 1 h, followed by 10 min DAPI staining to label cell nuclei. Representative images were captured using a Leica confocal microscope.

[0138] Method 7, Electron Microscopy

[0139] Fresh heart tissue (less than 1 mm) 3 Fixation was performed overnight at 4°C in 5 ml of fixation buffer (0.1 M sodium phosphate buffer of 2.5% glutaraldehyde and 2.0% PFA, pH 7.4). Images were acquired using a TEM (JOEL TEM1230, Japan).

[0140] Method 8, Statistical Analysis

[0141] Statistical analysis was performed using GraphPad Prism 9 software. All statistics are presented as mean ± standard deviation (SD). One-way ANOVA and Dunnett's multiple comparison test were used for statistical analysis of two or more groups. Fisher's exact test was used to compare the differences in the proportion of second-degree or high-degree AVB among the AAV2 / 9-Control, AAV2 / 9-Gabrb2, and AAV2 / 9-Slc32a1 viral injection groups. For concentration-response curves, logarithmic inhibitors (agonists) were fitted to the normalized response-variable slope. P < 0.05 was considered statistically significant. The exact P-values ​​are shown in the corresponding graphs.

[0142] Under physiological conditions, electrical stimulation begins in the sinoatrial node, the source of electrical activity. The electrical excitation first reaches the atria, then travels through the atrioventricular node (AVN) and the His-Purkinje fiber network to the ventricles, ultimately triggering ventricular contraction. The conduction velocity of electrical excitation is slowest in the AVN, a characteristic that ensures sequential contraction of the atria and ventricles and efficient cardiac pumping. Clinically, AVN conduction defects are one of the most common causes of arrhythmias, leading to varying degrees of atrioventricular block (AVB) or even more serious cardiac arrest and sudden cardiac death. Severe AVB is a major clinical indication for pacemaker implantation. Unfortunately, little is known about the mechanisms underlying the slow conduction of electrical excitation within the AVN. Therefore, effective treatments are lacking for cardiac diseases such as slow conduction of electrical excitation within the AVN or arrhythmias related to AVN conduction defects.

[0143] AVN pacemaker cells (AVNPCs) are the main functional cells in the AVN. In this study, the inventors identified key components of the endogenous gamma-aminobutyric acid neurotransmitter system (GABA-TS) in AVNPCs, including numerous GABA neurotransmitter vesicles beneath the AVNPC cell membrane and key molecular elements of GABA-TS, such as GABA metabolic enzymes, GABA receptors, and GABA transporters. Electrophysiological studies confirmed that GABA-TS significantly regulates the conduction of electrical signals in the AVN and between the atria and ventricles. Importantly, intervention targeting GABA-TS significantly prevented the occurrence and development of severe atrial fibrillary block (AVBs). The inventors' evidence reveals a novel bioelectrical control system in the heart.

[0144] Example 1. Identification of intact endogenous GABA-TS in AVNPC.

[0145] First, using ultrastructural images of AVN tissue obtained by transmission electron microscopy (see Method 7), the inventors detected the presence of numerous neurotransmitter vesicles in AVNPCs. Neurotransmitter vesicles are essential ultrastructures for neuronal function, mediating the storage, transport, and synaptic release of neurotransmitters. The inventors discovered through transmission electron microscopy (TEM) that these vesicles were located beneath the cell membrane of rat AVNPCs and in the gaps between two adjacent AVNPCs. Figure 1 a). As a neurotransmitter of GABAergic neurons, GABA can inhibit excitatory conduction between GABAergic neurons. Next, the inventors performed immunofluorescence staining (see Method 6) to confirm the co-localization of GABA and the vesicle marker CAST in rat AVNPCs, indicating that the vesicles discovered by the inventors are GABA neurotransmitter vesicles. Figure 1 b).

[0146] Given the abundance of GABA neurotransmitter vesicles in AVNPCs, the inventors analyzed whether GABA could induce GABA ligand-gated currents in AVNPCs. Whole-cell patch-clamp recordings (see Method 2) showed that spraying different concentrations (0.01 to 100 μM) of GABA onto the surface membrane of isolated rat AVNPCs after setting the potential to –60 mV induced transient inward currents in AVNPCs in a concentration-dependent manner. These results indicate that GABA possesses electrophysiological functions in AVNPCs similar to those of GABAergic neurons. Figure 1 c).

[0147] In the mammalian central nervous system, GABA is synthesized by glutamate decarboxylase (GAD) and transported to the postsynaptic cleft via vesicular GABA transporter protein (vGAT), ultimately acting on GABA ionotropic or metabolotropic receptors. These components constitute a complete GABAergic neurotransmitter system. The inventors used single-cell PCR technology to detect the expression of GABA neurotransmitter system genes in a single mouse atrioventricular node pacemaker cell. Figure 2 Genes with high expression abundance in the GABA neurotransmitter system were identified (Gad2, Gabra3, Gabrb2, Gabrg2, Slc16a1, Slc32a1, Abat, Aldh5a1).

[0148] In addition, immunofluorescence staining (Method 6) also confirmed the presence of GABA synthase GAD2 and GABA receptor GABA. A R subunit α3 (GABRA3), GABA A R subunit β2 (GABRB2), GABA A High expression of R subunit γ2 (GABRG2), vesicle GABA transporter (vGAT), GABA transporter 1 (GAT-1), GABA transferase (GABA-T), and GABA degrading enzyme (SSADH) in AVN tissues and AVNPCs. Figure 3 , Figure 4 It is worth noting that GABA A The R subtypes, including GABRA3, GABRB2, and GABRG2, are enriched on the cell membrane of AVNPC. Figure 4 In summary, the inventors discovered a complete endogenous GABA-TS in AVNPC.

[0149] Example 2. GABA-TS regulation of AVN electrical signal conduction.

[0150] Electrical excitability is a prerequisite for the electrical conductivity of AVNPCs. The inventors first investigated the effect of GABA on the spontaneous action potentials (APs) of individual rat AVNPCs using method 2. Spontaneous APs of AVNPCs were recorded in current-clamp mode. The results showed that GABA (100 μM) reduced the maximum diastolic membrane potential (MDP) of AVNPCs, suggesting that GABA increases the electrical excitability threshold of AVNPCs, thus reducing their excitability. Figure 5 ). Given GABA A R mediates the fastest inhibitory activity in mammalian GABAergic neurons, combined with observed GABA... A Given the high expression and membrane localization characteristics of R in AVNPC, the inventors utilized GABA... A Detection of GABA by the specific agonist Afloqualone (100 μM) A The effect of R activation on spontaneous active techniques (APs) in AVNPC. Experiments revealed that Afloqualone has a similar effect to GABA, indicating that GABA... A Activation of R reduces the excitability of AVNPCs. Figure 5 The inventors also studied GABA. A Can an antagonist of R compensate for GABA? A R activation inhibits the excitability of AVNPC. Experiments have shown that GABA... A The specific antagonist of R, Gabazine (30 μM), can effectively block the reduction of MDP induced by Afloqualone. Figure 5 These data indicate that GABA and GABA A R is a key factor in determining the arousal level of AVNPCs.

[0151] Next, the inventors conducted optical mapping experiments in rat AVN tissue to analyze the effect of GABA-TS on the conduction of electrical signals within the AVN. First, GABA was detected... A The effect of R activation on the electrical signal conduction velocity in AVN. AVN tissue was loaded with the voltage-sensitive dye Di-4-ANEPPS (15 μM) to track the propagation of electrical signals. Compared with the control group, GABA was used... A The conduction time of AVN tissues treated with the R agonist Afloqualone was significantly increased, indicating that GABA A Activation of R reduces the electrical conduction velocity of AVN. Figure 6(a, 6b). Furthermore, the effect of GABA transporters on AVN electrical signal transduction was investigated. In the brain, GAT mediates the reuptake of GABA from the synaptic cleft. The inventors found that administration of GABA reuptake inhibitors (Tiagabine) or GAT-1 inhibitors (SKF89976A) reduced the electrical signal transduction velocity in AVN tissues. Figure 6 (a, 6b) This indicates that GABA transporter protein, as one of the key factors of GABA-TS, plays an important regulatory role in the conduction of electrical excitation in AVN, which is similar to its mechanism of action in the nervous system.

[0152] This study further validated the role of GABA-TS in AVN conduction in perfused rat hearts using electrocardiogram recordings of isolated hearts (Method 3). The PR interval of surface electrocardiograms (ECG) was used to assess the role of GABA-TS. A The effects of R and GATs on AVN electrical signal conduction. During the experiment, right atrial pacing was used to control the heart rate of perfused rats, thereby eliminating the effect of heart rate on the PR interval. The results showed that GABA... A The R agonist afloqualone caused a concentration-dependent prolongation of the PR interval. After treatment with afloqualone, the PR interval increased from 35.00 ± 3.54 ms to 66.20 ± 7.19 ms (0 to 640 μM). The half-maximal effective concentration (EC50) was 165.70 μM (95% confidence interval (CI): 140.90 to 194.90 μM). Figure 6 It is noteworthy that, due to severe atrioventricular block (AVB), cardiac arrest even occurred when 640 μM of Afloqualone was infused. Figure 7 a). This study also evaluated the effects of GABA reuptake inhibitors (Tiagabine) and GAT-1 inhibitors (SKF89976A) on the PR interval, finding that Tiagabine or SKF89976A induced PR interval prolongation in isolated rat hearts in a concentration-dependent manner. After perfusion with Tiagabine, the PR interval increased from 38.17 ± 2.23 ms to 79.67 ± 6.83 ms (0 to 64 μM). Figure 6 fh). SKF89976A extended the PR interval from 39.40±5.68ms to 70.80±11.08ms (0 to 128μM) ( Figure 6 The half-maximal inhibitory concentrations (IC50) were 20.22 μM (95% CI: 18.10 to 22.59 μM, Tiagabine) Figure 6 fh) and 37.57 μM (95% CI: 31.05 to 45.45 μM, SKF89976A) Figure 6 In the experiment, it was also observed that high concentrations of Tiagabine (64 μM) or SKF89976A (128 μM) may lead to second- or even third-degree AVB. Figure 7 b, 7c). Furthermore, Afloqualone, Tiagabine, and SKF89976A did not alter the QRS and QT intervals (b, 7c). Figure 8 ).

[0153] Finally, this paper explores the effect of GABA-TS on AVN electroconduction in vivo. AAV2 / 9 virus was constructed to knock down the key gene in GABA-TS: GABA. A R (Gabrb2, encoding GABRB2), GABA transporter (Slc32a1, encoding vGAT), or GABA transferase (Abat, encoding GABA-T). AAV2 / 9 virus was then locally injected into rat AVN tissue using an insulin injector. Atrioventricular conduction was investigated using telemetry electrocardiogram recordings. Eight weeks post-injection, Abat knockdown prolonged the PR interval in rat hearts, while Gabrb2 or Slc32a1 knockdown shortened the PR interval, consistent with the effects of the corresponding GABA-TS agonists or inhibitors on the PR interval. Figure 6 Furthermore, knocking down Gabrb2, Slc32a1, or Abat does not affect P wave duration, QRS interval, QT interval, or heart rate. Figure 9 These data indicate that endogenous GABA-TS in AVNPC effectively control the electrical signal conduction in AVN.

[0154] Example 3. Key components of GABA-TS can serve as potential intervention targets for AVB.

[0155] As a common arrhythmia associated with AVN conduction defects, AVB can be classified into grade I, II, and III AVB according to the severity of the block. Slow AVN electrical signal conduction can ultimately lead to severe AVB or cardiac arrest. Data from this study show that knockdown of Gabrb2 or Slc32a1 significantly shortened the PR interval in rat hearts. Therefore, Gabrb2 and Slc32a1 may be potential interventional targets for preventing or terminating AVB development.

[0156] This paper further establishes an AVB model in isolated rat hearts by perfusion with verapamil (see steps in Method 3). Verapamil at 250 nM can induce second-degree or high-degree AVB. Figure 10Compared with the control group, the incidence of second-degree or high-degree atrioventricular block (AVB) was significantly reduced in AAV2 / 9-Gabrb2 and AAV2 / 9-Slc32a1 knockdown rats. These data indicate that GABA A Knockdown of R or vGAT prevented the occurrence and development of AVB, suggesting that GABA-TS may serve as a systemic intervention target for this disease.

[0157] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a drug targeting the γ-aminobutyric acid neurotransmitter system (GABA-TS) in the preparation of a medicament for the treatment or prevention of heart disease.

2. The use as described in claim 1, characterized in that, The drugs targeting the γ-aminobutyric acid (GABA) neurotransmitter system include: GABA A One or more of the following: R regulator, vGAT regulator, GABA reuptake regulator, GAT-1 regulator, and GABA transferase regulator.

3. The use as described in claim 1, characterized in that, (a) The drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS inhibitor, and the GABA-TS inhibitor is selected from the group consisting of GABA... A R antagonists, vGAT inhibitors, GABA reuptake promoters, GAT-1 activators, GABA transferase activators, or combinations thereof; or (b) The drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS agonist, and the GABA-TS agonist is selected from the group consisting of GABA... A R agonists, vGAT activators, GABA reuptake inhibitors, GAT-1 inhibitors, GABA transferase inhibitors, or combinations thereof.

4. The use as described in claim 3, characterized in that, GABA A R-antagonists are selected from the group consisting of: gabazine, picrotoxinin, bicuculline, etbicyphat, oroxylin A, songorine, thiocolchicoside, (-)-securinine, 6,2'-dihydroxyflavone, or combinations thereof; and / or GABA reuptake inhibitors are selected from the following group: tiagabine or its salts; and / or, GAT-1 inhibitors include those selected from the following group: SKF89976A or its salts, LU-32-176B, Guvacine or its salts, CI 966 or its salts, NO-711 or its salts, or combinations thereof.

5. The use as described in claim 1, characterized in that, The heart disease is selected from the following group: arrhythmia, atrioventricular block (AVB), cardiac arrest, sudden cardiac death, atrial fibrillation, atrial flutter, premature atrial contractions, Wolff-Parkinson-White syndrome, short PR syndrome, or a combination thereof.

6. The use as described in claim 1, characterized in that, (a) The drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS inhibitor, and the cardiac disease is characterized by cardiac disease including PR interval prolongation or cardiac disease caused by PR interval prolongation; or, (b) The drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS agonist, and the heart disease is characterized by a shortened PR interval or a heart disease caused by a shortened PR interval.

7. The use as described in claim 1, characterized in that, The drug targeting the γ-aminobutyric acid neurotransmitter system is a GABA-TS inhibitor, and the heart disease is atrioventricular block or bradycardia.

8. The use as described in claim 1, characterized in that, The drug targeting the γ-aminobutyric acid neurotransmitter system is GABA. A R antagonists and / or vGAT inhibitors, and the cardiac disease is atrioventricular block or bradycardia.

9. A reagent kit, characterized in that, include: (1) a drug targeting the γ-aminobutyric acid neurotransmitter system (GABA-TS), and (2) another drug; wherein the other drug is selected from the group consisting of: drugs for treating or preventing heart disease, drugs for treating or preventing diseases caused by heart disease, and drugs for relieving symptoms caused by heart disease.

10. A method for modulating the PR interval of a subject, comprising the step of contacting the subject with a drug targeting GABA-TS to modulate the PR interval.

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