Use of drug targeting γ-aminobutyric acid transmitter system in treatment of cardiac diseases

Abstract

The present invention provides use of a drug targeting the γ-aminobutyric acid transmitter system in the treatment of cardiac diseases. Specifically, the present invention provides use of a drug targeting the γ-aminobutyric acid transmitter system (GABA-TS) in the preparation of a medicament for treating or preventing cardiac diseases. Specifically, the present invention identifies the endogenous γ-aminobutyric acid transmitter system and the regulatory functions contained in atrioventricular node pacemaker cells, providing a novel intervention strategy for arrhythmias.

Description

Use of a drug targeting gamma-aminobutyric acid neurotransmitter system in the treatment of heart diseases TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to use of a drug targeting gamma-aminobutyric acid neurotransmitter system in the treatment of heart diseases. BACKGROUND

[0002] Under physiological conditions, electrical stimulation starts from the sinoatrial node, i.e. the source of electrical activity, and electrical excitation first reaches the atrium, and then reaches the ventricle through the atrioventricular node (AVN) and His-Purkinje fiber network, and finally triggers ventricular contraction. The conduction velocity of electrical excitation is slowest in the AVN, and this feature ensures the sequential contraction of the atrium and the ventricle and effective cardiac pumping. Clinically, AVN conduction defects are one of the most common causes of arrhythmia, which can cause different degrees of atrioventricular block (AVB) or more serious cardiac arrest and sudden cardiac death. Severe AVB is the main clinical indication for cardiac pacemaker implantation. Unfortunately, people know little about the mechanism of the slow conduction of electrical excitation in the AVN. Therefore, there is a lack of effective treatment for heart diseases such as the slow conduction of electrical excitation in the AVN or AVN conduction defect-related arrhythmia.

[0003] In view of the above, there is an urgent need in the art to develop a new method for treating or preventing AVN conduction defect-related arrhythmia. SUMMARY

[0004] The purpose of the present application is to provide a new method for treating or preventing AVN conduction defect-related heart diseases such as arrhythmia.

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

[0006] In another preferred embodiment, the gamma-aminobutyric acid neurotransmitter system is the gamma-aminobutyric acid neurotransmitter system in atrioventricular node pacemaker cells (AVNPC).

[0007] In another preferred embodiment, the targeting of the gamma-aminobutyric acid neurotransmitter system refers to targeting one or more targets in the gamma-aminobutyric acid neurotransmitter system.

[0008] In another preferred embodiment, the gamma-aminobutyric acid neurotransmitter system comprises one or more targets selected from the group consisting of 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 alpha 3 (GABRA3), GABA receptor GABA A R subunit beta 2 (GABRB2), GABA receptor GABA A R subunit gamma 2 (GABRG2), vesicular GABA transporter (vGAT), GABA transporter 1 (GAT-1), GABA transaminase (GABA-T), and GABA-degrading enzyme (SSADH).

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

[0018] In another preferred embodiment, the drug targeting the GABA-TS comprises one or more of: a small molecule compound, an antibody, and an shRNA; preferably, a small molecule compound.

[0019] In another preferred embodiment, the drug targeting the gamma-aminobutyric acid neurotransmitter system comprises: a GABA A R modulator, a vGAT modulator, a GABA reuptake modulator, a GAT-1 modulator, a GABA transaminase modulator, a SSADH modulator.

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

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

[0022] inhibiting GABA A R;

[0023] inhibiting vGAT;

[0024] promoting GABA reuptake;

[0025] activating GAT-1;

[0026] activating GABA transaminase.

[0027] In another preferred embodiment, the drug targeting the gamma-aminobutyric acid neurotransmitter system is selected from the group consisting of: a GABA A R antagonist, a vGAT inhibitor, a GABA reuptake promoter, a GAT-1 activator, a GABA transaminase activator, or a combination thereof.

[0028] In another preferred embodiment, the GABA AR antagonists include: Gabazine, Picrotoxinin, Bicuculline, Etbicyphat, Oroxylin A, Songorine, Thiocolchicoside, (-)-Securinine, 6,2'-Dihydroxyflavone, and the like.

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

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

[0031] activating GABA A R;

[0032] activating vGAT;

[0033] inhibiting GABA reuptake;

[0034] inhibiting GAT-1;

[0035] inhibiting GABA transaminase.

[0036] In another preferred embodiment, the drug targeting the gamma-aminobutyric acid neurotransmitter system or the GABA-TS agonist is selected from the group consisting of a GABA A R agonist, a vGAT activator, a GABA reuptake inhibitor, a GAT-1 inhibitor, a GABA transaminase inhibitor, or a combination thereof.

[0037] In another preferred embodiment, the GABA reuptake inhibitor includes: Tiagabine or a salt thereof, and the like.

[0038] In another preferred embodiment, the GAT-1 inhibitor includes: SKF 89976A or a salt thereof, LU-32-176B,

[0039] Guvacine or a salt thereof, CI 966 or a salt thereof, NO-711 or a salt thereof, and the like.

[0040] In another preferred embodiment, the cardiac disease is a cardiac disease characterized by or caused by a PR interval abnormality.

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

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

[0045] In another preferred embodiment, the cardiac disease is an atrioventricular node (AVN) conduction defect related cardiac disease.

[0046] In another preferred embodiment, the cardiac disease is an atrioventricular node (AVN) conduction slow related cardiac disease.

[0047] In another preferred embodiment, the cardiac disease is selected from the group consisting of: arrhythmia (including tachyarrhythmia and bradyarrhythmia), atrioventricular block (AVB), cardiac arrest, sudden cardiac death, atrial fibrillation, atrial flutter, atrial premature beat, Wolff-Parkinson-White syndrome, short PR syndrome, or a combination thereof.

[0048] In another preferred embodiment, the cardiac disease characterized by or caused by a prolonged PR interval comprises: atrioventricular block (AVB), bradyarrhythmia.

[0049] In another preferred embodiment, the cardiac disease characterized by or caused by a shortened PR interval comprises: Wolff-Parkinson-White syndrome, short PR syndrome.

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

[0051] In another preferred embodiment, the cardiac disease is an atrioventricular block caused by AVN conduction defect.

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

[0053] In another preferred embodiment, the drug targeting gamma-aminobutyric acid neurotransmitter system is a GABA-TS inhibitor, and the cardiac disease is an atrioventricular block or bradyarrhythmia.

[0054] In another preferred embodiment, the drug targeting gamma-aminobutyric acid neurotransmitter system is a GABA A R antagonist and / or vGAT inhibitor, and the cardiac disease is an atrioventricular block or bradyarrhythmia.

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

[0056] In a second aspect of the present application, there is provided a pharmaceutical combination comprising: (1) a drug targeting the gamma-aminobutyric acid neurotransmitter system (GABA-TS), and (2) a further drug.

[0057] In another preferred embodiment, the further drug is a drug selected from the group consisting of a drug for treating or preventing a cardiac disease, a drug for treating or preventing a disease caused by a cardiac disease, a drug for alleviating a condition caused by a cardiac disease.

[0058] In another preferred embodiment, the further drug is not a drug targeting the gamma-aminobutyric acid neurotransmitter system (GABA-TS).

[0059] In a third aspect of the present application, there is provided a kit comprising: (1) a drug targeting the gamma-aminobutyric acid neurotransmitter system (GABA-TS), and (2) a further drug.

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

[0061] In a fourth aspect of the present application, there is provided a pharmaceutical composition comprising: (1) a first active agent which is a drug targeting the gamma-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 a cardiac disease.

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

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

[0065] In a fifth aspect of the present application, there is provided a method of modulating the PR interval of a subject, comprising the step of contacting the subject with a drug targeting the GABA-TS, thereby modulating the PR interval.

[0066] In another preferred embodiment, the modulating the PR interval is a shortening of the PR interval, and the drug targeting the GABA-TS is a GABA-TS inhibitor (as defined above).

[0067] In another preferred embodiment, said modulating the PR period means lengthening the PR period, and said drug targeting GABA-TS means a GABA-TS agonist (as defined above).

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

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

[0070] In a sixth aspect of the application, there is provided a method of modulating the electrical excitability of an atrioventricular node pacemaker cell (AVNPC), comprising the step of contacting the atrioventricular node pacemaker cell with a drug targeting GABA-TS, thereby modulating the electrical excitability of the atrioventricular node pacemaker cell.

[0071] In another preferred embodiment, said modulating the electrical excitability of the atrioventricular node pacemaker cell is increasing the electrical excitability of the atrioventricular node pacemaker cell, and said drug targeting GABA-TS means a GABA-TS inhibitor (as defined above).

[0072] In another preferred embodiment, said modulating the electrical excitability of the atrioventricular node pacemaker cell is decreasing the electrical excitability of the atrioventricular node pacemaker cell, and said drug targeting GABA-TS means a GABA-TS agonist (as defined above).

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

[0074] In a seventh aspect of the application, there is provided a method of modulating AVN electrical signalling, comprising the step of contacting an atrioventricular node pacemaker subject with a drug targeting GABA-TS, thereby modulating the AVN electrical signalling.

[0075] In another preferred embodiment, said modulating the AVN electrical signalling is speeding up the AVN electrical signalling, and said drug targeting GABA-TS means a GABA-TS inhibitor (as defined above).

[0076] In another preferred embodiment, said modulating the AVN electrical signalling is slowing down the AVN electrical signalling, and said drug targeting GABA-TS means a GABA-TS agonist (as defined above).

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

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

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

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

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

[0082] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 shows the presence of GABA vesicles in rat atrioventricular nodal pacemaker cells and GABA-induced ligand-gated currents. a: Transmission electron microscopy (TEM) images showing the presence of a large number of vesicles under the cell membrane of rat atrioventricular nodal pacemaker cells (AVNPCs). The right panel is a magnified version of the white dashed box in the left panel. White arrows indicate vesicles. Scale bar, 500 nm. b: Immunofluorescence images showing the colocalization of GABA with the vesicle marker CAST in single rat AVNPCs. Scale bar, 10 μm. c: Patch-clamp recordings of currents in rat AVNPCs evoked by different concentrations of GABA (0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM). Holding potential was set at -60 mV. Horizontal bars, 500 ms, vertical bars, 10 pA.

[0084] Figure 2 is a heat map showing the expression of GABA transmitter system genes in single atrioventricular nodal pacemaker cells of mice (Hcn4CreERT2(+); Rosa26 TomRed+ mice. Rows represent samples of atrioventricular nodal pacemaker cells (AVNPCs), and columns represent Ct values of GABA transmitter system genes. Color scale shows the relative expression of genes (measured as Ct values). Score -1 (red) indicates high expression level, and score 1 (blue) indicates low expression level.

[0085] Figure 3 shows the expression of key elements of the GABA transmitter system in rat atrioventricular nodal tissue. a: Immunofluorescence staining of GABA metabolic enzymes (GAD2, GABA-T, and SSADH) in rat atrioventricular nodal (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 transporters (vGAT and GAT-1) in rat AVN tissue. Scale bar, 25 μm.

[0086] Figure 4 shows expression of key elements of the GABAergic system in rat atrioventricular nodal pacemaker cells. a: Immunofluorescence staining showing expression and localisation of GABA metabolising enzymes (GAD2, GABA-T and SSADH) in rat atrioventricular nodal pacemaker cells (AVNPCs). Scale bar, 10 μm. b: Immunofluorescence staining showing expression and localisation of GABAAreceptors (GABRA3, GABRB2 and GABRG2) in rat AVNPCs. Scale bar, 10 μm. c: Immunofluorescence staining showing expression and localisation of GABA transporters (vGAT and GAT-1) in rat AVNPCs. GABA A R, GABA A R receptors. Scale bar, 10 μm.

[0087] Figure 5 shows that GABA and GABA A receptor activation reduces the excitability of atrioventricular nodal pacemaker cells. Summary data of the maximum diastolic potential (MDP) of spontaneous action potentials (APs) recorded using current-clamp patch-clamp recordings in adult rat atrioventricular nodal pacemaker cells (AVNPCs). n = 7. Data shown as mean ± s.d. P values were calculated by one-way ANOVA and Dunnett's multiple comparison test.

[0088] Figure 6 shows that the GABAergic system controls electrical signalling in the atrioventricular node. a: Representative activation map showing the activation of the atrioventricular node in response to a single extracellular electrical stimulus in the presence of solvent control DMSO, GABA AElectrical activation and conduction in perfused rat atrioventricular node (AVN) tissue after superfusion with the R-specific agonist Afloqualone (320 μM), the GABA reuptake inhibitor Tiagabine (64 μM) or the GAT-1 inhibitor SKF89976A (128 μM). The magnified activation map shows electrical activation and conduction in the central region of the AVN. Electrical activity in the rat AVN tissue was recorded by optical mapping using the fluorescent dye Di-4-ANBDQBS. Activation times, conduction velocities and vector maps were obtained during continuous pacing at the stimulation electrode located at the crux (5 Hz, 2 V). b: Statistical analysis of electrical conduction times in the AVN for different treatment groups (n = 6 samples for control group, n = 7 samples for Afloqualone treatment group, n = 9 samples for Tiagabine treatment group, n = 8 samples for SKF89976A treatment group). Data are shown as mean ± s.d. P values were calculated by one-way ANOVA and Dunnett's multiple comparison test. c, f and i: Representative electrocardiogram (ECG) recordings from 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 horizontal 100 ms, vertical 2 mV. d, g and j, Dose response of Afloqualone (d), Tiagabine (g) and SKF89976A (j) on PR interval in perfused rat hearts. n = 5 for Afloqualone and SKF89976A treatment groups, n = 6 for Tiagabine treatment group. Data are shown as mean ± s.d. P values were calculated using one-way ANOVA and Dunnett's multiple comparison test. e, h and k, Concentration-effect curves for PR interval changes induced by Afloqualone (EC50, 165.70 μM) (e), Tiagabine (IC50, 20.22 μM) (h) and SKF89976A (IC50, 37.57 μM) (k). Concentration-effect curves were fitted to the Hill equation using non-linear regression and normalized to the maximum changed PR interval. Data are shown as mean ± s.d. n = 5 for Afloqualone and SKF89976A treatment groups, n = 6 for Tiagabine treatment group. l, Representative ECGs from conscious rats recorded by telemetry recording system after injection of AAV2 / 9-Control, AAV2 / 9-Gabrb2, AAV2 / 9-Slc32a1 or AAV2 / 9-Abat viruses. Scale bar horizontal 100 ms, vertical 0.5 mV.m and n, histogram of the mean PR interval (m) and P-wave duration (n) of the rats in the group. n = 7 for the AAV2 / 9-Control group, n = 6 for the AAV2 / 9-Gabrb2 group, n = 6 for the AAV2 / 9-Slc32al group, n = 6 for the AAV2 / 9-Abat group. Data are shown as mean ± s.d. P values were calculated by one-way ANOVA and Dunnett's multiple comparison test.

[0089] Figure 7 shows that intervention on the GABAergic system results in severe atrioventricular block in isolated rat hearts. a: Representative electrocardiogram (ECG) recordings showing that when the heart is perfused with 640 μΜ GABA A receptor agonist (Afloqualone) (a), GABA reuptake inhibitor (Tiagabine) (b) and GABA transporter inhibitor (SKF89976A) (c). n = 5 for Afloqualone and SKF89976A treated groups, n = 6 for Tiagabine treated group. Data are expressed as mean ± s.d. P values were calculated by one-way ANOVA and Dunnett's multiple comparison test.

[0090] Figure 8 shows that GABAergic system agonists or inhibitors do not change the QRS and QT intervals in isolated rat hearts. a-c, Different concentrations of GABA A receptor agonist (Afloqualone) (a), GABA reuptake inhibitor (Tiagabine) (b) and GABA transporter inhibitor (SKF89976A) (c). n = 5 for Afloqualone and SKF89976A treated groups, n = 6 for Tiagabine treated group. Data are expressed as mean ± s.d. P values were calculated by one-way ANOVA and Dunnett's multiple comparison test.

[0091] Figure 9 shows that intervention of GABAergic system does not affect QRS interval, QT interval and heart rate in vivo. a-c, Histograms showing the statistical graphs of mean QRS interval (a), QT interval (b) and heart rate (beats per minute, BPM) (c) of AAV2 / 9-Control, AAV2 / 9-Gabrb2, AAV2 / 9-Slc32al and AAV2 / 9-Abat virus infected rat hearts, respectively. n=7 for AAV2 / 9-Control group, n=6 for AAV2 / 9-Gabrb2 group, n=6 for AAV2 / 9-Slc32al group, n=6 for AAV2 / 9-Abat group. Data are shown as mean ± s.d. P values were calculated by one-way ANOVA and Dunnett's multiple comparison test.

[0092] Figure 10 shows that intervention targeting GABAergic system can prevent the occurrence and development of atrioventricular block. Left, Representative electrocardiogram (ECG) recordings showing that severe atrioventricular block (AVB) was successfully constructed by perfusion of verapamil (250 nM) in isolated hearts of AAV2 / 9-Control virus injected rats. Arrows indicate representative second degree AVB (2: 1). AAV2 / 9-Gabrb2 and AAV2 / 9-Slc32al virus injected hearts did not develop to second or high degree AVB. Scale bar, 100 ms horizontally and 2 mV vertically. Right, Proportion of second or high degree AVB occurrence in AAV2 / 9-Control, AAV2 / 9-Gabrb2 or AAV2 / 9-Slc32al virus injected hearts. AAV2 / 9-Control: n=10, AAV2 / 9-Gabrb2: n=6, AAV2 / 9-Slc32al: n=8. P values were calculated by Fisher's exact test. DETAILED DESCRIPTION

[0093] After long-term and in-depth research, the inventors unexpectedly found that the main functional cells in atrioventricular node (AVN), i.e. atrioventricular node pacemaker cells (AVNPCs), exist endogenous γ-aminobutyric acid neurotransmitter system (GABA-TS) and its key elements, for example, GABA metabolic enzyme, GABA receptor (GAB A R) and GABA transporter such as vesicular GABA transporter (vGAT), GABA transporter 1 (GAT-1); in addition, the inventors also found that intervention of GABA-TS, for example, targeting the key elements therein, can significantly regulate electrical signal conduction in AVN, thereby preventing the occurrence and development of severe AVB. Based on this, the inventors completed the present application.

[0094] TERMS

[0095] Unless otherwise indicated, each abbreviation herein has the meaning ascribed to it by the person of ordinary skill in the art.

[0096] As used herein, the term "PR interval" refers to the process in an electrocardiogram from the beginning of the P wave to the beginning of the QRS wave.

[0097] Atrioventricular nodal pacemaker cell (AV NPC)

[0098] Atrioventricular node (AVN, Atrio-ventricular node)

[0099] The atrioventricular node is located in the subendocardium of the lower part of the interatrial septum on the right atrial side, in front of the ostium of the coronary sinus, in the shape of a flat ellipse, smaller than the sinoatrial node, and the lower front end of the atrioventricular node continues as the atrioventricular bundle. Its function is to transmit impulses from the sinoatrial node to the ventricle. Electrical impulses in the atrioventricular node will experience a brief delay to ensure sequential contraction of atrial and ventricular muscle, i.e. the atrium contracts first, and the ventricle contracts later.

[0100] Pacemaker cell (PC)

[0101] A pacemaker cell is capable of spontaneous excitation and can transmit the current it generates to all or part of the non-autonomic myocardial cells or cell groups. The pacemaker cell is the smallest unit of myocardium with automaticity and is the organizational basis of the pacemaker. The characteristic of the pacemaker cell is that the phase 4 of its action potential has a certain slope, and it can spontaneously and slowly perform diastolic automatic depolarization, thus having automaticity. In a physiological state, pacemaker cells are mainly distributed in the cardiac conduction system, and a small number are scattered in ordinary myocardium, especially in atrial muscle.

[0102] Gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid transmitter system (GABA-TS)

[0103] Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. The GABAergic system refers to a series of molecules and cellular structures, including GABA, GABA receptors, and GABA transporters, that work together to maintain balance and information transmission between neurons. Among them, 1) GABA: GABA is the main inhibitory neurotransmitter, widely distributed in the brain. It promotes inhibitory signal transmission of neurons by binding to GABA receptors; 2) GABA receptors: GABA receptors are the core elements of GABA's action. They are divided into three types: 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 that, when GABA binds, allow Cl ions to pass through, increasing the degree of intracellular hyperpolarization and leading to inhibitory effects on neurons; GABA-B receptors are G protein-coupled receptors that, when GABA binds, activate related signal transduction pathways, thereby producing inhibitory effects. 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 into neurons or remove excess GABA from the synaptic cleft to maintain appropriate neurotransmitter levels.

[0104] The function of the GABAergic system is crucial for maintaining the stability of the brain and proper signal transmission between neurons. It is involved in emotional regulation, motor control, cognitive function, and other physiological and behavioral processes. Abnormal GABAergic system function may be related to various neurological diseases, such as anxiety, depression, epilepsy, etc. Drugs targeting the GABAergic system are widely used in clinical practice to treat various neurological and psychiatric diseases. These drugs are mainly divided into two categories: drugs that increase GABA levels and drugs that modulate GABA receptor function.

[0105] Drugs that increase GABA levels: benzodiazepines Benzodiazepines: such as Lorazepam, Alprazolam, etc., which increase the activity of GABA-A receptors to produce sedative, anxiolytic, and antidepressant effects. Baclofen: a GABA-B receptor agonist used to treat muscle spasms and movement disorders.

[0106] Drugs that modulate GABA receptor function: Anti-epileptic drugs: such as Sodium valproate, Gabapentin, etc. They inhibit abnormal nerve excitation by modulating GABA receptors or increasing GABA levels, thus preventing and controlling seizures. Pregabalin: Similar to Gabapentin, also used to treat epilepsy and neuropathic pain. Ketamine: It is an NMDA receptor antagonist, but it can also affect the GABAergic system, and is used to treat depression and other mental illnesses.

[0107] The gamma-aminobutyric acid transmitter system (GABA-TS) in the atrioventricular nodal pacemaker cells (AVNPCs) and its role

[0108] Here, the core components of GABA-TS in AVNPCs were identified by single-cell qPCR and immunofluorescence staining, such as GAD2, vGAT, ionotropic GABA A Importantly, by TEM and confocal microscopy, the inventors found that AVNPCs not only express the molecular elements of GABA-TS, but also are rich in GABA vesicles under the cell membrane and in the interstitial space of AVNPCs. The inventors' research first showed that AVNPCs have a complete endogenous GABA-TS.

[0109] The in vitro and in vivo data provided herein demonstrate that the endogenous GABA-TS of AVNPCs plays an important role in the electrical signaling of AVN. The slow conduction property of AVN and the resulting physiological "conduction delay" phenomenon between atrium and ventricle have been a difficult problem in the field of cardiac rhythmology. The presence of unique gap junction proteins in AVNPCs is considered an important factor leading to the slow conduction of AVN. Here, the inventors found that intervention on multiple molecules of the endogenous GABA-TS in AVNPCs can significantly change the electrical signaling speed of AVN, which means that GABA-TS is another functional bioelectricity regulation system that controls the electrical signaling of AVN. In addition, the findings of this study also provide a unique mechanism for regulating the sequential contraction of atrium and ventricle.

[0110] AVB can develop into fatal arrhythmia. Currently, the effect of drugs for treating AVB is very small, and severe AVB is mainly treated by cardiac pacemaker. Due to the extremely limited number of AVNPCs and the difficulty of sampling, the research on AVN at the cellular level has been relatively lacking, and the research progress of AVB has been relatively lagging behind. Therefore, these factors greatly hinder the progress of the clinical prevention and treatment of AVB. The present study found that the use of tool drugs to intervene GABA AR or GAT-1 can significantly affect the conduction of electrical excitation within the AVN. Knocking out the GABA-TS-encoding genes Gabrb2, Slc32al or Abat can significantly alter PR interval in rats. Importantly, the inventors demonstrated that inhibiting GABA A R or vGAT can prevent the occurrence and development of severe AVB. These data suggest that GABA-TS can be a promising intervention target for AVB.

[0111] In summary, the inventors found that endogenous GABA-TS in the AVNPC modulates the excitability and electrical signaling of the AVN, which is an important basis for the electrical conduction between atria and ventricles. The inventors' study reveals a new electrophysiological mechanism of AVN conduction based on the intrinsic transmitter system, providing a new intervention strategy for arrhythmia.

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

[0113] As used herein, the term "drug targeting the gamma-aminobutyric acid transmitter system" refers to a substance (such as a small molecule compound, an antibody, etc.) that targets the gamma-aminobutyric acid transmitter system and has the ability to intervene or modulate the function of the gamma-aminobutyric acid transmitter system. Preferably, the compound targeting the gamma-aminobutyric acid transmitter system is capable of targeting one or more key elements in the gamma-aminobutyric acid transmitter system (such as GABA A R, vGAT, GAT-1, SSADH). In some alternatives, the compound targeting the gamma-aminobutyric acid transmitter system is specific or non-specific. In some embodiments, the "drug targeting the gamma-aminobutyric acid transmitter system" is as defined in the first aspect.

[0114] As used herein, the term "GABA-TS inhibitor" refers to a drug or substance that is capable of modulating the gamma-aminobutyric acid transmitter system by targeting one or more targets in the gamma-aminobutyric acid transmitter system, reducing the inhibitory effect of GABA, and thus being capable of, for example, promoting / accelerating electrical signaling, increasing atrioventricular node excitability, shortening PR interval. According to the present text, a GABA-TS inhibitor can be a GABA A R antagonist, a vGAT inhibitor, a GABA reuptake promoter, a GAT-1 activator, a GABA transferase activator, etc. Similarly, the term "GABA-TS agonist" refers to a drug or substance that is capable of modulating the gamma-aminobutyric acid transmitter system by targeting one or more targets in the gamma-aminobutyric acid transmitter system, enhancing the inhibitory effect of GABA, and thus being capable of, for example, slowing down electrical signaling, reducing atrioventricular node excitability, prolonging PR interval. According to the present text, a GABA-TS inhibitor can be a GABA AR agonists, vGAT activators, GABA reuptake inhibitors, GAT-1 inhibitors, GABA transaminase inhibitors, and the like.

[0115] Since GABA-TS exists in AVN and intervention of GABA-TS in AVN has excellent effects on modulating the excitability and electrical signaling of AVN, and further mediates the electrical conduction between atrium and ventricle, the drugs or compounds having the ability to intervene / modulate GABA-TS or targeting GABA-TS (especially the drugs or compounds having the effects of inhibiting GABA A R, vGAT, GABA reuptake, GAT-1, GABA transaminase) can be used for treating or preventing heart diseases. According to the prior art, these heart diseases include, but are not limited to, arrhythmia, atrioventricular block (AVB), cardiac arrest, sudden cardiac death, heart failure, cardiomyopathy, congenital heart disease, myocardial infarction, and the like. In particular, the drugs or compounds having the effects of inhibiting GABA A R, inhibiting vGAT, promoting GABA reuptake, activating GAT-1, activating GABA transaminase are particularly suitable for treating or preventing the related heart diseases such as slow arrhythmia which are the functional defects of atrioventricular node (AVN) (e.g. slow conduction of atrioventricular node (AVN)).

[0116] In some embodiments, the drugs or compounds having the ability to intervene GABA-TS or targeting GABA-TS can be the drugs or compounds having the ability to intervene GABA-TS in cardiovascular system, nervous system, immune system or targeting GABA-TS in cardiovascular system, nervous system, and / or immune system. Therefore, the anti-epileptic drugs, sedative drugs, benzodiazepine drugs, anti-insomnia drugs, muscle relaxants, and the like can be used for treating or preventing heart diseases. drugs, anti-insomnia drugs, muscle relaxants, and the like can be used for treating or preventing heart diseases.

[0117] In this context, the pharmaceutical composition of the present application comprises a safe and effective amount of the drugs targeting γ-aminobutyric acid neurotransmitter system. Wherein "safe and effective amount" refers to the amount of drugs sufficient to significantly improve the condition without causing serious side effects.

[0118] The drugs targeting γ-aminobutyric acid neurotransmitter system in the present application can be administered alone or in combination with other drugs (such as other drugs for treating or preventing heart diseases or diseases or conditions related to or caused by heart diseases).

[0119] The main advantages of the present application include:

[0120] The technical scheme of the present application provides a new and effective intervention strategy for heart diseases, especially arrhythmia.

[0121] The present application is further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0122] Experimental methods

[0123] Method 1, AVNPCs isolation

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

[0125] Method 2, whole-cell patch-clamp recording

[0126] Whole-cell patch-clamp recordings of membrane potential and currents were performed on single rat AVNPCs using an EPC-10 amplifier (HEKA, Germany) and Clampfit 10.7 software (HEKA, Germany). Membrane potential and currents were recorded at 22-26 °C. Two types of microelectrodes (borosilicate glass, 2-6 MΩ) were used, one for electrical stimulation and recording, filled with intracellular solution 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 for rapid and localized application of different drugs onto the cell membrane of AVNPCs by using a PL1-100 picoinjector (Harvard Apparatus, USA), filled with extracellular solution containing (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 elements of the GABAergic system (e.g. the agonist Afloqualone) were dissolved in the extracellular solution. Stimulus pulses to release the drugs were applied by the EPC-10 amplifier (HEKA, Germany) synchronized with the picoinjector. Action potentials were recorded in current-clamp mode and currents in voltage-clamp mode.

[0127] Method 3, electrocardiogram recordings from isolated hearts

[0128] At 37 °C, hearts were rapidly removed from adult rats and mounted on a modified Langendorff system, perfused through the aorta with oxygenated perfusate containing (in 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 recordings, one electrode was placed at the bottom of the heart close to the right atrium and the other at the apex. The heart was paced by a pacing electrode (Powerlab, ADInstruments, USA) placed in the right atrium. The stimulation protocol was set as follows: the stimulation threshold was determined during a stabilization period and during pacing stimulation, usually 1.5-2 times the threshold, pulse duration 2 ms, frequency 6 Hz. Before recording the electrocardiogram, the heart was stabilized for 30 min, then different pharmacological drugs were added to the perfusate solution. ECG recordings were continuously obtained from the Langendorff perfused heart using a Powerlab amplifier (Powerlab, ADInstruments, USA).

[0129] AVB models were constructed by adding verapamil (250 nM) to the perfusate solution after 30 minutes of equilibration perfusion in isolated hearts. An AVB model was considered to be successfully constructed when the isolated heart developed a second degree or high grade AVB that lasted more than 15 minutes.

[0130] Method 4, Telemetry ECG recordings

[0131] Adult rats were anesthetized with 2% isoflurane. Rats were implanted intraperitoneally with a telemetry ECG transmitter (Data Sciences International, St. Paul, MN) and paired wire electrodes were placed on the chest (II lead configuration). After surgery, rats were placed in individual cages with free access to food and water. ECGs were recorded for 24 hours using LabChart software (v8.1.9, ADInstruments Inc., Colorado) and a telemetry receiver after 7 days of recovery from the implant surgery. ECG parameters, including P-wave, PR interval, QRS interval, and QT interval, were measured as previously described.

[0132] Method 5, Optical mapping

[0133] Isolated rat AVN tissue was subjected to optical mapping. Briefly, rats were anesthetized with sodium pentobarbital (30 mg / kg). The rat heart was rapidly removed and placed in a warm oxygenated Tyrode’s solution. Then, the right atrium-AVN tissue, which refers to the atrial and AVN tissue including the Koch triangle, was dissected. The AVN tissue was stained with Di-4-ANEPPS (15 mM, AAT, 90134-00-2) for 40-60 minutes in 37 °C oxygenated Tyrode’s solution. Blebbistatin (10 mM, MCE, HY-13813) was perfused for 10 minutes to eliminate motion artifacts during optical mapping recordings. Optical fluorescence signals were recorded at the Koch triangle at a speed of 1000 frames / second using a high-speed 10,000 pixel camera (SciMedia, MiCAM ULTIMA, USA) under right atrial pacing (5 Hz pacing rate, 2-fold pulse amplitude threshold, 2 ms pulse duration). Prior to optical mapping analysis, AVN preparations were perfused with DMSO, Afloqualone, Tiagabine, or SKF89976A for 30-60 minutes. AVN conduction times were calculated from the gradient of the action potential activation map.

[0134] Method 6, Immunofluorescence staining

[0135] For immunofluorescence staining, isolated AVNPCs were fixed in 4% paraformaldehyde (PFA) for 15 min and washed twice with PBS. Cells were then permeabilized with a solution of 0.5% Triton X-100 for 10 min, washed twice in PBS and blocked with 4% goat serum for 1 h at room temperature. For immunostaining, cells were incubated with primary antibodies overnight at 4°C. The next day, after washing twice with PBST, cells were incubated with the corresponding secondary antibodies for 1 h at room temperature, followed by DAPI (4,6-diamidino-2-phenylindole dihydrochloride) staining for 30 min. Representative images were taken using a Leica confocal microscope.

[0136] For immunohistochemistry staining, adult rat hearts were rapidly excised, fixed with 4% PFA overnight at 4°C and embedded in paraffin. Hearts were then cut longitudinally into 6 pm sections for immunostaining. Heart sections were deparaffinized with xylene and then rehydrated in stepwise decreasing concentrations of ethanol and then antigen-repaired in citrate buffer. Sections were blocked with 5% goat serum for 1 h at room temperature and then incubated with primary antibodies diluted in 5% goat serum overnight at 4°C. After washing 3 times in 0.1% PBST, sections were stained with fluorescent secondary antibodies for 1 h at room temperature, followed by 10 min DAPI staining to label nuclei. Representative images were taken using a Leica confocal microscope.

[0137] Method 7, Electron Microscopy

[0138] Fresh heart tissue (less than 1 mm 3 ) was fixed in 5 ml of fixation buffer (2.5% glutaraldehyde, 2.0% PFA in 0.1 M sodium phosphate buffer, pH 7.4) overnight at 4°C. Images were acquired using TEM (JOEL TEM 1230, Japan).

[0139] Method 8, Statistical Analysis

[0140] Statistical analysis of data was performed using GraphPad Prism 9 statistical software. All statistical data are shown as mean ± standard deviation (s.d.). Statistical analysis was performed on more than two groups using one-way ANOVA and Dunnett’s multiple comparison test. Fisher’s exact test was used to compare the difference in the proportion of second or high degree AVB between AAV2 / 9-Control, AAV2 / 9-Gabrb2 and AAV2 / 9-Slc32a1 virus injected groups. For concentration-effect curves, a log-inhibitor (agonist) versus normalized response-variable slope fit was used. P < 0.05 was considered statistically significant. Exact P values are shown in the corresponding figures.

[0141] Under physiological conditions, electrical stimulation starts from the sinoatrial node, the source of electrical activity, and the electrical excitation first reaches the atrium, then reaches the ventricle through the atrioventricular node (AVN) and the His-Purkinje fiber network, and finally triggers ventricular contraction. The conduction velocity of electrical excitation is the slowest in the AVN, and this feature ensures the sequential contraction of the atrium and the ventricle and effective cardiac pumping. Clinically, AVN conduction defects are one of the most common causes of arrhythmia, which can cause different degrees of atrioventricular block (AVB) or more severe cardiac arrest and sudden cardiac death. Severe AVB is the main clinical indication for cardiac pacemaker implantation. Unfortunately, people know very little about the mechanism of the slow conduction of electrical excitation in the AVN. Therefore, there is a lack of effective treatment for cardiac diseases such as slow conduction of electrical excitation in the AVN and arrhythmia related to AVN conduction defects.

[0142] AVN pacemaker cells (AVNPCs) are the main functional cells in the AVN. In this study, the inventors identified the presence of key elements of the endogenous gamma-aminobutyric acid transmitter system (GABA-TS) in AVNPCs, including a large number of GABA transmitter vesicles under the cell membrane of AVNPCs and key molecular elements of GABA-TS, such as GABA metabolic enzymes, GABA receptors and GABA transporters. Electrophysiological examination confirmed that GABA-TS significantly regulates the conduction of electrical signals in the AVN and between the atrium and the ventricle. Importantly, intervention against GABA-TS significantly prevents the occurrence and development of severe AVB. The inventors' evidence reveals a new bioelectric control system in the heart.

[0143] Example 1. Identification of complete endogenous GABA-TS in AVNPCs.

[0144] Firstly, by acquiring the ultrastructure images of AVN tissues by transmission electron microscopy (see Method 7), the inventors detected the presence of a large number of transmitter vesicles in AVNPCs. Transmitter vesicles are essential ultrastructures for neurons to function, mediating neurotransmitter storage, transport and synaptic release. The inventors found these vesicles under the cell membrane of rat AVNPCs and in the gap between two adjacent AVNPCs by transmission electron microscopy (TEM) (Figure 1a). As a transmitter of GABAergic neurons, GABA can inhibit the conduction of excitation between GABAergic neurons. Next, the inventors performed immunofluorescence staining (see Method 6) to confirm the colocalization of GABA and the vesicle marker CAST in rat AVNPCs, indicating that the vesicles discovered by the inventors are GABA transmitter vesicles (Figure 1b).

[0145] In view of the abundant GABAergic vesicles in AVNPCs, the inventors analyzed whether GABA can induce GABA ligand-gated currents in AVNPCs. Whole-cell patch clamp recordings (see Methods 2) showed that upon perfusion of different concentrations (0.01 to 100 mM) of GABA on the surface membrane of isolated rat AVNPCs after setting the potential to -60 mV, GABA induced transient inward currents in AVNPCs in a concentration-dependent manner. These results suggest that GABA has electrophysiological functions similar to GABAergic neurons in AVNPCs (Fig. lc).

[0146] In the central nervous system of mammals, GABA is synthesized by glutamate decarboxylase (GAD) and transported to the postsynaptic cleft by vesicular GABA transporters (vGAT), and finally acts on GABA ionotropic or metabotropic receptors. These elements constitute a complete GABAergic neurotransmitter system. The inventors detected the expression of GABAergic system genes in single mouse AVN pacemaker cells by single-cell PCR technique (Fig. 2), and identified genes with higher expression abundance in the GABAergic system (Gad2, Gabra3, Gabrb2, Gabrg2, Slcl6al, Slc32al, Abat, Aldh5al).

[0147] In addition, immunofluorescence staining (Methods 6) also confirmed the high expression of GABA synthesis enzyme GAD2, GABA receptor GABA A R subunit a3 (GABRA3), GABA A R subunit b2 (GABRB2), GABA A R subunit g2 (GABRG2), vesicular GABA transporter (vGAT), GABA transporter 1 (GAT-1), GABA transaminase (GABA-T), and GABA degrading enzyme (SSADH) in AVN tissue and AVNPCs (Figs. 3, 4). Notably, GABA A R subtypes, including GABRA3, GABRB2, and GABRG2, are enriched on the cell membrane of AVNPCs (Fig. 4). Overall, the inventors found a complete endogenous GABA-TS in AVNPCs.

[0148] Example 2. GABA-TS regulates electrical signaling of AVN.

[0149] Electroexcitability 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 electroexcitability 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 AVNPC (Figure 5). The inventors also investigated 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.

[0150] 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 AActivation of R decreased the electrical conduction velocity of AVN (Fig. 6a, 6b). In addition, the effect of GABA transporters on the electrical conduction of AVN was examined. In the brain, GATs mediate the re-uptake of GABA from the synaptic cleft. The inventors found that administration of GABA re-uptake inhibitor (Tiagabine) or GAT-1 inhibitor (SKF89976A) decreased the electrical conduction velocity of AVN tissue (Fig. 6a, 6b), suggesting that GABA transporters, as one of the key factors of GABA-TS, play an important regulatory role in the electrical excitation conduction of AVN, similar to the mechanism of action in the nervous system.

[0151] The role of GABA-TS in the conduction of AVN of perfused rat heart was further verified by electrocardiogram recording of isolated heart (Method 3) herein. The PR interval of electrocardiogram (ECG) was used to evaluate the effect of GABA A The effect of R and GATs on the electrical conduction of AVN. During the experiment, right atrial pacing was used to control the beating frequency of perfused rat heart, so as to eliminate the effect of heart rate on PR interval. The results showed that GABA AThe R agonist Afloqualone caused prolongation of PR interval in a concentration-dependent manner. After Afloqualone treatment, PR interval was prolonged from 35.00 ± 3.54 ms to 66.20 ± 7.19 ms (0 to 640 μΜ). The half maximal effective concentration (EC50) was 165.70 μΜ (95% confidence interval (CI): 140.90 to 194.90 μΜ) (Fig. 6c-e). Notably, due to severe atrioventricular block (AVB), the heart even experienced cardiac arrest when perfused with 640 μΜ of Afloqualone (Fig. 7a). The effects of GABA reuptake inhibitor (Tiagabine) and GAT-1 inhibitor (SKF89976A) on PR interval were also evaluated, and it was found that Tiagabine or SKF89976A caused prolongation of PR interval in isolated rat hearts in a concentration-dependent manner. After perfusion of Tiagabine, PR interval was prolonged from 38.17 ± 2.23 ms to 79.67 ± 6.83 ms (0 to 64 μΜ) (Fig. 6f-h). SKF89976A prolonged PR interval from 39.40 ± 5.68 ms to 70.80 ± 11.08 ms (0 to 128 μΜ) (Fig. 6i-k). The half maximal inhibitory concentration (IC50) was 20.22 μΜ (95% CI: 18.10 to 22.59 μΜ, Tiagabine) (Fig. 6f-h) and 37.57 μΜ (95% CI: 31.05 to 45.45 μΜ, SKF89976A) (Fig. 6i-k), respectively. High concentrations of Tiagabine (64 μΜ) or SKF89976A (128 μΜ) were also observed to potentially cause second or even third degree AVB (Fig. 7b, 7c) in the experiments. In addition, Afloqualone, Tiagabine and SKF89976A did not change QRS and QT intervals (Fig. 8).

[0152] Finally, the effect of GABA-TS on electrical conduction of AVN was explored in vivo. AAV2 / 9 virus was constructed to knock down the key gene in GABA-TS: GABA AR (Gabrb2, encoding GABRB2), GABA transporter (Slc32al, encoding vGAT), or GABA transaminase (Abat, encoding GABA-T). The AAV2 / 9 virus was then injected locally into the AVN tissue of the rats using an insulin syringe. The atrioventricular electrical conduction was studied by telemetry electrocardiogram recording. At 8 weeks after virus injection, knockdown of Abat prolonged the PR interval of the rat heart, while knockdown of Gabrb2 or Slc32al shortened the PR interval, which was consistent with the effects of the corresponding GABA-TS agonists or inhibitors on the PR interval (Fig. 61- n). In addition, knockdown of Gabrb2, Slc32al, or Abat did not affect the P-wave duration, QRS interval, QT interval, and heart rate (Fig. 9). These data suggest that the endogenous GABA-TS in the AVN PC effectively controls the electrical signal conduction of the AVN.

[0153] Example 3. Key elements of GABA-TS can serve as potential intervention targets for AVB.

[0154] As a common arrhythmia associated with conduction defects of the AVN, AVB can be classified into first-degree, second-degree, and third-degree AVB according to the severity of the block. The over-slow electrical signal conduction of the AVN eventually leads to severe AVB or cardiac arrest. The data herein show that knockdown of Gabrb2 or Slc32al significantly shortened the PR interval of the rat heart. Therefore, it can be speculated that Gabrb2 and Slc32al can be potential intervention targets to prevent the occurrence or terminate the development of AVB.

[0155] The AVB model was further constructed in the isolated rat heart by perfusion of verapamil (see Step of Method 3), which induced second- or high-degree AVB at 250 nM (Fig. 10). Compared with the control group, the incidence of second- or high-degree atrioventricular block (AVB) was significantly reduced in the AAV2 / 9-Gabrb2 and AAV2 / 9-Slc32al knockdown rats. These data suggest that GABA A Knockdown of R or vGAT prevented the occurrence and development of AVB, indicating that GABA-TS can be a potential systemic intervention target for this disease.

[0156] All documents referred to in this disclosure are incorporated by reference herein as if each were individually incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the disclosure herein, and it is intended that the same be considered as falling within the scope of the application as defined by the appended claims.

Claims

1. Use of a drug targeting the γ-aminobutyric acid transmitter system (GABA-TS) for the manufacture of a medicament for the treatment or prevention of a cardiac disorder.

2. Use according to claim 1, characterized in that, The drugs targeting the gamma-aminobutyric acid neurotransmitter system include: GABA A one or more of a GABA modulator, a GABA transaminase modulator, a GABA reuptake modulator, a GAT-1 modulator, a vGAT modulator.

3. The use according to claim 1, wherein the cardiac disorder is selected from the group consisting of: arrhythmia, atrioventricular block (AVB), cardiac arrest, sudden cardiac death, atrial fibrillation, atrial flutter, atrial premature beats, Wolff-Parkinson-White syndrome, short PR syndrome, or a combination thereof. (a) the drug targeting the gamma-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 that targets the gamma-aminobutyric acid neurotransmitter system is a GABA-TS agonist, and the GABA-TS agonist is selected from the group consisting of GABA A a R agonist, a vGAT activator, a GABA reuptake inhibitor, a GAT-1 inhibitor, a GABA transferase inhibitor, or a combination thereof.

4. The use according to claim 3, wherein the cardiac disorder is selected from the group consisting of: arrhythmia, atrioventricular block (AVB), cardiac arrest, sudden cardiac death, atrial fibrillation, atrial flutter, atrial premature beats, Wolff-Parkinson-White syndrome, short PR syndrome, or a combination thereof. GABA A the R antagonist is selected from the group consisting of Gabazine, Picrotoxinin, Bicuculline, Etbicyphat, Oroxylin A, Songorine, Thiocolchicoside, (-)-Securinine, 6,2'-Dihydroxyflavone, or a combination thereof; and / or GABA reuptake inhibitor is selected from the group consisting of: Tiagabine or a salt thereof; and / or, GAT-1 inhibitor comprises an agent selected from the group consisting of: SKF 89976A or a salt thereof, LU-32-176B, Guvacine or a salt thereof, CI 966 or a salt thereof, NO-711 or a salt thereof, or a combination thereof.

5. The use according to claim 1, characterized in that, The cardiac disorder is selected from the group consisting of: arrhythmia, atrioventricular block (AVB), cardiac arrest, sudden cardiac death, atrial fibrillation, atrial flutter, atrial premature beats, Wolff-Parkinson-White syndrome, short PR syndrome, or a combination thereof.

6. The use according to claim 1, wherein: (a) the drug targeting the γ-aminobutyric acid transmitter system is a GABA-TS inhibitor and the cardiac disorder is a cardiac disorder characterized by or resulting from a prolonged PR interval; or (b) the drug targeting the γ-aminobutyric acid transmitter system is a GABA-TS agonist and the cardiac disorder is a cardiac disorder characterized by or resulting from a shortened PR interval.

7. The use according to claim 1, characterized in that, The drug targeting the γ-aminobutyric acid transmitter system is a GABA-TS inhibitor and the cardiac disorder is atrioventricular block or bradyarrhythmia.

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

9. A kit characterized in that, comprises: (1) a drug targeting the γ-aminobutyric acid transmitter system (GABA-TS), and (2) an additional drug; wherein the additional drug is a drug selected from the group consisting of: a drug for the treatment or prevention of a cardiac disorder, a drug for the treatment or prevention of a disease resulting from a cardiac disorder, a drug for the alleviation of a condition resulting from a cardiac disorder.

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

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