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Investigation of the Cellular Pharmacological Mechanism and Clinical Evidence of the Multi-Herbal Antiarrhythmic Chinese Medicine Xin Su Ning.
Xin Su Ning (XSN), a China patented and certified multi-herbal medicine, has been available in China since 2005 for treating cardiac ventricular arrhythmia including arrhythmia induced by ischemic heart diseases and viral myocarditis, without adverse reactions being reported. It is vitally important to discover pharmacologically how XSN as a multicomponent medicine exerts its clinical efficacy, and whether the therapeutic effect of XSN can be verified by standard clinical trial studies. In this paper we report our discoveries in a cellular electrophysiological study and in a three-armed, randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. Conventional electrophysiological techniques were used to study the cellular antiarrhythmic mechanism of XSN. Data was then modeled with computational simulation of human action potential (AP) of the cardiac ventricular myocytes. The clinical trial was conducted with a total of 861 eligible participants randomly assigned in a ratio of 2:2:1 to receive XSN, mexiletine, or the placebo for 4 weeks. The primary and secondary endpoint was the change of premature ventricular contraction (PVC) counts and PVC-related symptoms, respectively. This trial was registered in the Chinese Clinical Trial Register Center (ChiCTR-TRC-14004180). We found that XSN prolonged AP duration of the ventricular myocytes in a dose-dependent, reversible manner and blocked potassium channels. Patients in XSN group exhibited significant total effective responses in the reduction of PVCs compared to those in the placebo group (65.85% vs. 27.27%, P < 0.0001). No severe adverse effects attributable to XSN were observed. In conclusion, XSN is an effective multicomponent antiarrhythmic medicine to treat PVC without adverse effect in patients, which is convincingly supported by its class I & III pharmacological antiarrhythmic mechanism of blocking hERG potassium channels and hNaV1.5 sodium channel reported in our earlier publication and prolongs AP duration both in ventricular myocytes and with computational simulation of human AP presented in this report.
A role for GABAergic interneuron diversity in circuit development and plasticity of the neonatal cerebral cortex.
GABAergic interneurons are a highly heterogeneous group of cells that are critical for the mature function and development of the neocortex. In terms of the latter, much attention has focused on the well-established role of parvalbumin (PV+)-expressing, fast spiking, basket cells in determining the critical period plasticity. However recent endeavours have started to shed the light on the contribution of other interneuron subtypes to early circuit formation and plasticity. Data suggests that there are significant interactions between PV+ cells and other interneuron subtypes that regulate circuit development in rodents in the first postnatal week. Moreover, a number of these early interactions are transient which points to an important, distinct role for interneuron diversity in setting up emergent neocortical processing.
Fast, quantitative, murine cardiac 19F MRI/MRS of PFCE-labeled progenitor stem cells and macrophages at 9.4T.
PURPOSE: To a) achieve cardiac 19F-Magnetic Resonance Imaging (MRI) of perfluoro-crown-ether (PFCE) labeled cardiac progenitor stem cells (CPCs) and bone-derived bone marrow macrophages, b) determine label concentration and cellular load limits, and c) achieve spectroscopic and image-based quantification. METHODS: Theoretical simulations and experimental comparisons of spoiled-gradient echo (SPGR), rapid acquisition with relaxation enhancement (RARE), and steady state at free precession (SSFP) pulse sequences, and phantom validations, were conducted using 19F MRI/Magnetic Resonance Spectroscopy (MRS) at 9.4 T. Successful cell labeling was confirmed using flow cytometry and confocal microscopy. For CPC and macrophage concentration quantification, in vitro and post-mortem cardiac validations were pursued with the use of the transfection agent FuGENE. Feasibility of fast imaging is demonstrated in murine cardiac acquisitions in vivo, and in post-mortem murine skeletal and cardiac applications. RESULTS: SPGR/SSFP proved favorable imaging sequences yielding good signal-to-noise ratio values. Confocal microscopy confirmed heterogeneity of cellular label uptake in CPCs. 19F MRI indicated lack of additional benefits upon label concentrations above 7.5-10 mg/ml/million cells. The minimum detectable CPC load was ~500k (~10k/voxel) in two-dimensional (2D) acquisitions (3-5 min) using the butterfly coil. Additionally, absolute 19F based concentration and intensity estimates (trifluoroacetic-acid solutions, macrophages, and labeled CPCs in vitro and post-CPC injections in the post-mortem state) scaled linearly with fluorine concentrations. Fast, quantitative cardiac 19F-MRI was demonstrated with SPGR/SSFP and MRS acquisitions spanning 3-5 min, using a butterfly coil. CONCLUSION: The developed methodologies achieved in vivo cardiac 19F of exogenously injected labeled CPCs for the first time, accelerating imaging to a total acquisition of a few minutes, providing evidence for their potential for possible translational work.
A Network Pharmacology Study of the Multi-Targeting Profile of an Antiarrhythmic Chinese Medicine Xin Su Ning.
Xin Su Ning (XSN) is a China patented and certified traditional Chinese herbal medicine used to treat premature ventricular contractions (PVCs) since 2005. XSN is formulated with 11 herbs, designed to treat arrhythmia with phlegm-heat heart-disturbed syndrome (PHHD) according to Chinese medicine theory. The rational compatibility of the 11 herbs decides the therapeutic outcome of XSN. Due to the multicomponent nature of traditional Chinese medicine, it is difficult to use conventional pharmacology to interpret the therapeutic mechanism of XSN in terms of clear-cut drug molecule and target interactions. Network pharmacology/systematic pharmacology usually consider all the components in a formula with the same weight; therefore, the proportion of the weight of the components has been ignored. In the present study, we introduced a novel coefficient to mimic the relative amount of all the components in relation with the weight of the corresponding herb in the formula. The coefficient is also used to weigh the pharmacological effect of XSN on all relative biological pathways. We also used the cellular electrophysiological data generated in our lab, such as the effect of liensinine and isoliquiritigenin on NaV1.5 channels; we therefore set sodium channel as one of the targets of these two components, which would support the clinical efficacy of XSN in treating tachyarrhythmia. Combining the collected data and our discovery, a panoramagram of the pharmacological mechanism of XSN was established. Pathway enrichment and analysis showed that XSN treated PHHD arrhythmia through multiple ion channels regulation, protecting the heart from I/R injury, inhibiting the apoptosis of cardiomyocyte, and improving glucose and lipid metabolism.
Decoding the transcriptional basis for GABAergic interneuron diversity in the mouse neocortex.
The locally projecting GABAergic interneurons of the mammalian cerebral cortex are a highly heterogeneous population, whose malfunction or deficit has been implicated in a wide range of neurological disorders. However, the low incidence of the various distinct interneuron populations within the neocortex, combined with the lack of molecular or physiological markers specific to these subtypes, have hampered investigations into their function in the normal and dysfunctional brain. A number of research groups have begun to elucidate the developmental genetic mechanism that underpins this diversity in the mouse neocortex, spurred on by the knowledge that the temporal and spatial origin of an interneuron in the embryonic brain is predictive of its eventual intrinsic properties in the mature cortex. In this review we highlight a number of recent findings that strengthen our understanding of the transcription factor code that is at the heart of generating this diversity. Further understanding of this code will enable selective observation, targeting and manipulation of interneuron subtypes across both in vitro and in vivo systems.
Developmental mechanisms for the generation of telencephalic interneurons.
Interneurons, which release the neurotransmitter γ-aminobutyric acid (GABA), are the major inhibitory cells of the central nervous system (CNS). Despite comprising only 20-30% of the cerebral cortical neuronal population, these cells play an essential and powerful role in modulating the electrical activity of the excitatory pyramidal cells onto which they synapse. Although interneurons are present in all regions of the mature telencephalon, during embryogenesis these cells are generated in specific compartments of the ventral (subpallial) telencephalon known as ganglionic eminences. To reach their final destinations in the mature brain, immature interneurons migrate from the ganglionic eminences to developing telencephalic structures that are both near and far from their site of origin. The specification and migration of these cells is a complex but precisely orchestrated process that is regulated by a combination of intrinsic and extrinsic signals. The final outcome of which is the wiring together of excitatory and inhibitory neurons that were born in separate regions of the developing telencephalon. Disruption of any aspect of this sequence of events during development, either from an environmental insult or due to genetic mutations, can have devastating consequences on normal brain function.
Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.
By combining an inducible genetic fate mapping strategy with electrophysiological analysis, we have systematically characterized the populations of cortical GABAergic interneurons that originate from the caudal ganglionic eminence (CGE). Interestingly, compared with medial ganglionic eminence (MGE)-derived cortical interneuron populations, the initiation [embryonic day 12.5 (E12.5)] and peak production (E16.5) of interneurons from this embryonic structure occurs 3 d later in development. Moreover, unlike either pyramidal cells or MGE-derived cortical interneurons, CGE-derived interneurons do not integrate into the cortex in an inside-out manner but preferentially (75%) occupy superficial cortical layers independent of birthdate. In contrast to previous estimates, CGE-derived interneurons are both considerably greater in number (approximately 30% of all cortical interneurons) and diversity (comprised by at least nine distinct subtypes). Furthermore, we found that a large proportion of CGE-derived interneurons, including the neurogliaform subtype, express the glycoprotein Reelin. In fact, most CGE-derived cortical interneurons express either Reelin or vasoactive intestinal polypeptide. Thus, in conjunction with previous studies, we have now determined the spatial and temporal origins of the vast majority of cortical interneuron subtypes.
The requirement of Nkx2-1 in the temporal specification of cortical interneuron subtypes.
Previous work has demonstrated that the character of mouse cortical interneuron subtypes can be directly related to their embryonic temporal and spatial origins. The relationship between embryonic origin and the character of mature interneurons is likely reflected by the developmental expression of genes that direct cell fate. However, a thorough understanding of the early genetic events that specify subtype identity has been hampered by the perinatal lethality resulting from the loss of genes implicated in the determination of cortical interneurons. Here, we employ a conditional loss-of-function approach to demonstrate that the transcription factor Nkx2-1 is required for the proper specification of specific interneuron subtypes. Removal of this gene at distinct neurogenic time points results in a switch in the subtypes of neurons observed at more mature ages. Our strategy reveals a causal link between the embryonic genetic specification by Nkx2-1 in progenitors and the functional attributes of their neuronal progeny in the mature nervous system.
Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors.
Inhibitory GABAergic interneurons of the mouse neocortex are a highly heterogeneous population of neurons that originate from the ventral telencephalon and migrate tangentially up into the developing cortical plate. The majority of cortical interneurons arise from a transient embryonic structure known as the medial ganglionic eminence (MGE), but how the remarkable diversity is specified in this region is not known. We have taken a genetic fate mapping strategy to elucidate the temporal origins of cortical interneuron subtypes within the MGE. We used an inducible form of Cre under the regulation of Olig2, a basic helix-loop-helix transcription factor highly expressed in neural progenitors of the MGE. We observe that the physiological subtypes of cortical interneurons are, to a large degree, unique to their time point of generation.
Phenotype of V2-derived interneurons and their relationship to the axon guidance molecule EphA4 in the developing mouse spinal cord.
The ventral spinal cord consists of interneuron groups arising from distinct, genetically defined, progenitor domains along the dorsoventral axis. Many of these interneuron groups settle in the ventral spinal cord which, in mammals, contains the central pattern generator for locomotion. In order to better understand the locomotor networks, we have used different transgenic mice for anatomical characterization of one of these interneuron groups, called V2 interneurons. Neurons in this group are either V2a interneurons marked by the postmitotic expression of the transcription factor Chx10, or V2b interneurons which express the transcription factors Gata2 and Gata3. We found that all V2a and most V2b interneurons were ipsilaterally projecting in embryos as well as in newborns. V2a interneurons were for the most part glutamatergic while V2b interneurons were mainly GABAergic or glycinergic. Furthermore, we demonstrated that a large proportion of V2 interneurons expressed the axon guidance molecule EphA4, a molecule previously shown to be important for correct organization of locomotor networks. We also showed that V2 interneurons and motor neurons alone did not account for all EphA4-expressing neurons in the spinal cord. Together, these findings enable a better interpretation of neural networks underlying locomotion, and open up the search for as yet unknown components of the mammalian central pattern generator.
V1 spinal neurons regulate the speed of vertebrate locomotor outputs.
The neuronal networks that generate vertebrate movements such as walking and swimming are embedded in the spinal cord. These networks, which are referred to as central pattern generators (CPGs), are ideal systems for determining how ensembles of neurons generate simple behavioural outputs. In spite of efforts to address the organization of the locomotor CPG in walking animals, little is known about the identity and function of the spinal interneuron cell types that contribute to these locomotor networks. Here we use four complementary genetic approaches to directly address the function of mouse V1 neurons, a class of local circuit inhibitory interneurons that selectively express the transcription factor Engrailed1. Our results show that V1 neurons shape motor outputs during locomotion and are required for generating 'fast' motor bursting. These findings outline an important role for inhibition in regulating the frequency of the locomotor CPG rhythm, and also suggest that V1 neurons may have an evolutionarily conserved role in controlling the speed of vertebrate locomotor movements.
Indirect phosphorylation-dependent modulation of postsynaptic nicotinic acetylcholine responses by 5-hydroxytryptamine.
Ionotropic nicotinic acetylcholine (ACh) receptors have been shown to be modulated by protein kinase-mediated phosphorylation in vitro. Here we demonstrate that 5-hydroxytryptamine (5-HT) can downregulate postsynaptic nicotinic ACh responses, elicited in an identified arthropod motoneuron in situ, by a mechanism dependent on protein kinase activity. Serotonergic modulation can be mimicked by perfusion with membrane-permeable analogues of either adenine (cAMP) or guanine (cGMP) cyclic nucleotides, and is prolonged in the presence of phosphodiesterase inhibitors. Furthermore, suppression of the ACh response by 5-HT is blocked by specific competitive inhibitors of protein kinase A and G, as well as the broad specificity protein kinase inhibitor staurosporine. The protein phosphatase inhibitor cantharidin similarly blocks recovery of the ACh response from suppression mediated by 5-HT. Thus, it appears that the nicotinic ACh response is modulated by a cAMP-mediated phosphorylation-dependent intracellular signalling pathway that is distinct from the direct block of mammalian nicotinic ACh receptors by 5-HT previously reported in vitro.