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.

Biological Relativity Requires Circular Causality but Not Symmetry of Causation: So, Where, What and When Are the Boundaries?

Since the Principle of Biological Relativity was formulated and developed there have been many implementations in a wide range of biological fields. The purpose of this article is to assess the status of the applications of the principle and to clarify some misunderstandings. The principle requires circular causality between levels of organization. But the forms of causality are also necessarily different. They contribute in asymmetric ways. Upward causation can be represented by the differential or similar equations describing the mechanics of lower level processes. Downward causation is then best represented as determining initial and boundary conditions. The questions tackled in this article are: (1) where and when do these boundaries exist? and (2) how do they convey the influences between levels? We show that not all boundary conditions arise from higher-level organization. It is important to distinguish those that do from those that don't. Both forms play functional roles in organisms, particularly in their responses to novel challenges. The forms of causation also change according to the levels concerned. These principles are illustrated with specific examples.

Electrotonic suppression of early afterdepolarizations in the neonatal rat ventricular myocyte monolayer.

Pathologies that result in early afterdepolarizations (EADs) are a known trigger for tachyarrhythmias, but the conditions that cause surrounding tissue to conduct or suppress EADs are poorly understood. Here we introduce a cell culture model of EAD propagation consisting of monolayers of cultured neonatal rat ventricular myocytes treated with anthopleurin-A (AP-A). AP-A-treated monolayers display a cycle length dependent prolongation of action potential duration (245 ms untreated, vs. 610 ms at 1 Hz and 1200 ms at 0.5 Hz for AP-A-treated monolayers). In contrast, isolated single cells treated with AP-A develop prominent irregular oscillations with a frequency of 2.5 Hz, and a variable prolongation of the action potential duration of up to several seconds. To investigate whether electrotonic interactions between coupled cells modulates EAD formation, cell connectivity was reduced by RNA silencing gap junction Cx43. In contrast to well-connected monolayers, gap junction silenced monolayers display bradycardia-dependent plateau oscillations consistent with EADs. Further, simulations of a cell displaying EADs electrically connected to a cell with normal action potentials show a coupling strength-dependent suppression of EADs consistent with the experimental results. These results suggest that electrotonic effects may play a critical role in EAD-mediated arrhythmogenesis.

How the Hodgkin-Huxley equations inspired the Cardiac Physiome Project.

Early modelling of cardiac cells (1960-1980) was based on extensions of the Hodgkin-Huxley nerve axon equations with additional channels incorporated, but after 1980 it became clear that processes other than ion channel gating were also critical in generating electrical activity. This article reviews the development of models representing almost all cell types in the heart, many different species, and the software tools that have been created to facilitate the cardiac Physiome Project.

Ca²âº-induced delayed afterdepolarizations are triggered by dyadic subspace Ca2²âº affirming that increasing SERCA reduces aftercontractions.

Ca(2+)-induced delayed afterdepolarizations (DADs) are depolarizations that occur after full repolarization. They have been observed across multiple species and cell types. Experimental results have indicated that the main cause of DADs is Ca(2+) overload. The main hypothesis as to their initiation has been Ca(2+) overflow from the overloaded sarcoplasmic reticulum (SR). Our results using 37 previously published mathematical models provide evidence that Ca(2+)-induced DADs are initiated by the same mechanism as Ca(2+)-induced Ca(2+) release, i.e., the modulation of the opening of ryanodine receptors (RyR) by Ca(2+) in the dyadic subspace; an SR overflow mechanism was not necessary for the induction of DADs in any of the models. The SR Ca(2+) level is better viewed as a modulator of the appearance of DADs and the magnitude of Ca(2+) release. The threshold for the total Ca(2+) level within the cell (not only the SR) at which Ca(2+) oscillations arise in the models is close to their baseline level (∼1- to 3-fold). It is most sensitive to changes in the maximum sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump rate (directly proportional), the opening probability of RyRs, and the Ca(2+) diffusion rate from the dyadic subspace into the cytosol (both indirectly proportional), indicating that the appearance of DADs is multifactorial. This shift in emphasis away from SR overload as the trigger for DADs toward a multifactorial analysis could explain why SERCA overexpression has been shown to suppress DADs (while increasing contractility) and why DADs appear during heart failure (at low SR Ca(2+) levels).

Competing oscillators in cardiac pacemaking: historical background.

Interaction between a membrane oscillator generated by voltage-dependent ion channels and an intracellular calcium signal oscillator was present in the earliest models (1984 to 1985) using representations of the sarcoplasmic reticulum. Oscillatory release of calcium is inherent in the calcium-induced calcium release process. Those historical results fully support the synthesis proposed in the articles in this review series. The oscillator mechanisms do not primarily compete with each; they entrain each other. However, there is some asymmetry: the membrane oscillator can continue indefinitely in the absence of the calcium oscillator. The reverse seems to be true only in pathological conditions. Studies from tissue-level work and on the development of the heart also provide valuable insights into the integrative action of the cardiac pacemaker.

Mathematical models of the electrical action potential of Purkinje fibre cells.

Early development of ionic models for cardiac myocytes, from the pioneering modification of the Hodgkin-Huxley giant squid axon model by Noble to the iconic DiFrancesco-Noble model integrating voltage-gated ionic currents, ion pumps and exchangers, Ca(2+) sequestration and Ca(2+)-induced Ca(2+) release, provided a general description for a mammalian Purkinje fibre (PF) and the framework for modern cardiac models. In the past two decades, development has focused on tissue-specific models with an emphasis on the sino-atrial (SA) node, atria and ventricles, while the PFs have largely been neglected. However, achieving the ultimate goal of creating a virtual human heart will require detailed models of all distinctive regions of the cardiac conduction system, including the PFs, which play an important role in conducting cardiac excitation and ensuring the synchronized timing and sequencing of ventricular contraction. In this paper, we present details of our newly developed model for the human PF cell including validation against experimental data. Ionic mechanisms underlying the heterogeneity between the PF and ventricular action potentials in humans and other species are analysed. The newly developed PF cell model adds a new member to the family of human cardiac cell models developed previously for the SA node, atrial and ventricular cells, which can be incorporated into an anatomical model of the human heart with details of its electrophysiological heterogeneity and anatomical complexity.

Role of pacemaking current in cardiac nodes: insights from a comparative study of sinoatrial node and atrioventricular node.

Cardiac pacemaking in the sinoatrial (SA) node and atrioventricular (AV) node is generated by an interplay of many ionic currents, one of which is the funny pacemaker current (If). To understand the functional role of If in two different pacemakers, comparative studies of spontaneous activity and expression of the HCN channel in mouse SA node and AV node were performed. The intrinsic cycle length (CL) is 179+/-2.7 ms (n=5) in SA node and 258+/-18.7 ms (n=5) in AV node. Blocking of If current by 1 micromol/L ZD7288 increased the CL to 258+/-18.7 ms (n=5) and 447+/-92.4 ms (n=5) in SA node and AV node, respectively. However, the major HCN channel, HCN4 expressed at low level in the AV node compared to the SA node. To clarify the discrepancy between the functional importance of If and expression level of HCN4 channel, a SA node cell model was used. Increasing the If conductance resulted in decreasing in the CL in the model, which explains the high pacemaking rate and high expression of HCN channel in the SA node. Resistance to the blocking of If in the SA node might result from compensating effects from other currents (especially voltage sensitive currents) involved in pacemaking. The computer simulation shows that the difference in the intrinsic CL could explain the difference in response to If blocking in these two cardiac nodes.

The role of the Na+/Ca2+ exchangers in Ca2+ dynamics in ventricular myocytes.

The role of the Na+/Ca2+ exchanger (NCX) as the main pathway for Ca2+ extrusion from ventricular myocytes is well established. However, both the role of the Ca2+ entry mode of NCX in regulating local Ca2+ dynamics and the role of the Ca2+ exit mode during the majority of the physiological action potential (AP) are subjects of controversy. The functional significance of NCXs location in T-tubules and potential co-localization with ryanodine receptors was examined using a local Ca2+ control model of low computational cost. Our simulations demonstrate that under physiological conditions local Ca2+ and Na+ gradients are critical in calculating the driving force for NCX and hence in predicting the effect of NCX on AP. Under physiological conditions when 60% of NCXs are located on T-tubules, NCX may be transiently inward within the first 100 ms of an AP and then transiently outward during the AP plateau phase. Thus, during an AP NCX current (INCX) has three reversal points rather than just one. This provides a resolution to experimental observations where Ca2+ entry via NCX during an AP is inconsistent with the time at which INCX is thought to become inward. A more complex than previously believed dynamic regulation of INCX during AP under physiological conditions allows us to interpret apparently contradictory experimental data in a consistent conceptual framework. Our modelling results support the claim that NCX regulates the local control of Ca2+ and provide a powerful tool for future investigations of the control of sarcoplasmic reticulum (SR) Ca2+ release under pathological conditions.

Functional significance of Na+/Ca2+ exchangers co-localization with ryanodine receptors.

Co-localization of Na+/Ca2+ exchangers (NCX) with ryanodine receptors (RyRs) is debated. We incorporate local NCX current in a biophysically detailed model of L-type Ca2+ channels (LCCs) and RyRs and study the effect of NCX on the regulation of Ca2+-induced Ca2+ release and the shape of the action potential. In canine ventricular cells, under pathological conditions, e.g., impaired LCCs, local NCXs become an enhancer of sarcoplasmic reticulum release. Under such conditions incorporation of local NCXs is critical to accurately capture mechanisms of excitation-contraction coupling.

Resistance of cardiac cells to NCX knockout: a model study.

The effects of NCX knockout were determined in a variety of cardiac cell models. Those of the mouse and rat ventricles, and of atrial cells in other species behave similarly to the experiments on mouse ventricle showing only small effects, and considerable tolerance of NCX knockout. Models of ventricular cells with high action potential plateaus, however, are more sensitive and require compensatory mechanisms to adjust other conductance parameters to enable the cells to resist NCX knockout. The effects can therefore be expected to be species-specific, and it is not possible to extrapolate the mouse results to those that may occur in the Guinea pig or human.

Modelling of calcium handling in airway myocytes.

Airway myocytes are the primary effectors of airway reactivity which modulates airway resistance and hence ventilation. Stimulation of airway myocytes results in an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and the subsequent activation of the contractile apparatus. Many contractile agonists, including acetylcholine, induce [Ca(2+)](i) increase via Ca(2+) release from the sarcoplasmic reticulum through InsP(3) receptors. Several models have been developed to explain the characteristics of InsP(3)-induced [Ca(2+)](i) responses, in particular Ca(2+) oscillations. The article reviews the modelling of the major structures implicated in intracellular Ca(2+) handling, i.e., InsP(3) receptors, SERCAs, mitochondria and Ca(2+)-binding cytosolic proteins. We developed theoretical models specifically dedicated to the airway myocyte which include the major mechanisms responsible for intracellular Ca(2+) handling identified in these cells. These biocomputations pointed out the importance of the relative proportion of InsP(3) receptor isoforms and the respective role of the different mechanisms responsible for cytosolic Ca(2+) clearance in the pattern of [Ca(2+)](i) variations. We have developed a theoretical model of membrane conductances that predicts the variations in membrane potential and extracellular Ca(2+) influx. Stimulation of this model by simulated increase in [Ca(2+)](i) predicts membrane depolarisation, but not great enough to trigger a significant opening of voltage-dependant Ca(2+) channels. This may explain why airway contraction induced by cholinergic stimulation does not greatly depend on extracellular calcium. The development of such models of airway myocytes is important for the understanding of the cellular mechanisms of airway reactivity and their possible modulation by pharmacological agents.

Ascending aortic stenosis selectively increases action potential-induced Ca2+ influx in epicardial myocytes of the rat left ventricle.

A decrease of the transient outward potassium current (Ito) has been observed in cardiac hypertrophy and contributes to the altered shape of the action potential (AP) of hypertrophied ventricular myocytes. Since the shape and duration of the ventricular AP are important determinants of the Ca2+ influx during the AP (QCa), we investigated the effect of ascending aortic stenosis (AS) on QCa in endo- and epicardial myocytes of the left ventricular free wall using the AP voltage-clamp technique. In sham-operated animals, QCa was significantly larger in endocardial compared to epicardial myocytes (803 +/- 65 fC pF(-1), n = 27 vs. 167 +/- 32 fC pF(-1), n = 38, P < 0.001). Ascending aortic stenosis significantly increased QCa in epicardial myocytes (368 +/- 54 fC pF(-1), n = 42, P < 0.05), but did not alter QCa in endocardial myocytes (696 +/- 65 fC pF(-1), n = 26). Peak and current-voltage relation of the AP-induced Ca2+ current were unaffected by AS. However, the time course of the current-voltage relation was significantly prolonged in epicardial myocytes of AS animals. Model calculations revealed that the increase in QCa can be ascribed to a prolonged opening of the activation gate, whereas an increase in inactivation prevents an excessive increase in QCa. In conclusion, AS significantly increased AP-induced Ca2+ influx in epicardial but not in endocardial myocytes of the rat left ventricle.