One of the main focuses in Chinese Medicine research is the identification of efficacious components in Chinese herbal medicine (CHM). Studies in such area are difficult due to the complexity and the synergistic characteristics of CHM. Current methods to track and separate active components are not adequate to meet the needs of revealing effects and identify substances and pharmacological mechanisms, which directly restrict the modernization and globalization of CHM. In this paper, a new methodology to deplete a single active component via immunoassay was introduced. The specific active component in a CHM mixture can then be identified and studied through comparative analyses of the pharmacological effects before and after immune depletion. With this new methodology, degree of contribution of a particular component to the whole complex herbal mixture can be elucidated, and its synergistic property with other components can be determined. The new method can reflect not only the overall combined pharmacological effects of CHM but also the effect of individual component. It is an effective way to explain the degree of contribution of one specific component to the overall activity of a CHM prescription. Complexities are the basic character of Chinese Herbal Medicine . The composition of CHM is complex in terms of its chemical structure and therapeutic effects. The pharmacodynamics of CHM is generated from the combined effects of different herbal components in a complex CHM mixture. And such combination is a result of a linear or nonlinear accumulation of synergistic or antagonistic medicinal interactions in vivo. In order to properly and comprehensively study and understand the pharmacological mechanism and synergistic effects in CHM and to establish a standard CHM quality control (QC), it is vital to identify the most active component in any given CHM prescription, the “principal component” (PC). And how to accurately explain the relationship between the therapeutic effects of a CHM preparation and the variation in concentration of principal compound is one of the key questions that needed answers in CHM research. This will be a key to develop CHM with factual bases.When it comes to establishing connections between a principal component and a primary medicinal property of a herb, simply focusing on one specific chemical compound is insufficient. The pharmacological action of a herb is far more sophisticated and every active component should be taken into consideration and analysed as a whole. Even though the dominant component may be the major contributor to the herb's pharmacological function, but its synergistic relationship with other components is just as important. Current existing methods are unsubstantial to satisfy the needs of material-based CHM research. New ideas and perspectives are needed to explore new techniques and applications.The primary objective in the CHM research is to identify the principal component and determine its main contributions to the pharmacodynamics of component herbs. In recent years, with the advancements in detection method development and the application of “fingerprint” technology, researchers have successfully isolated and analysed the primary active components in herbs and herbal mixtures, but study on CHM trace elements has just begun. It was necessary to study herbal medicine in vivo and define the absorption, metabolism, and distribution in different organs, tissues and cells, but to reveal the whole medicinal component system was not easy. Moreover, after the absorption and metabolism, it was exceedingly difficult to identify the pharmacological mechanism in vivo because of the content descend and conformational change of active components. Due to the complexity of CHM, it has been difficult to examine its pharmacodynamics and pharmacokinetics in vivo. However, scholars in the field of CHM research continuously come up with better strategies and have made some breakthrough in the area , 2. One Progress has been made in principal component identification –7, whichThe unique nature of CHM is created from the combined actions of different components. In order to achieve standardized QC, establish material-based pharmacodynamics, and analyse the metabolism of any of the CHM prescriptions, it is necessary to correlate the medicinal property of the principal component identified and the prescription's known therapeutic effects. And it is also necessary to correlate the amount of principal component present and the overall CHM therapeutic performance. A “quantity-effect” (QE) relationship of the principal component can then be generated and studied. This QE relationship can then be further applied into other different research aspects, such as the metabolism, the in vivo distribution, and the medicinal behaviors of CHM. Then, the accurate functional consistency of herbs and the main active component will be described. How to accurately and precisely establish such relationship is a critical issue being faced in basic CHM research. Such relationship can also affect the planting, harvesting, processing, and quality control processes of CHM. It is also a significant foundation for future CHM researches in areas such as herbal compatibility, CHM pharmacokinetics, and CHM pharmacodynamics, which can ultimately lead to a more effective, more stable, and safer application of CHM principal component.Defining the corresponding mechanisms of CHMs is enormously challenging due to the complexity and unique nature of each herb. A single herb possesses a complex system that consists of a broad spectrum of compounds. When a herb is introduced into an animal or a human, each of the compound's pharmacodynamic characteristics will be altered after they are metabolized or interacted with other compounds, producing a different therapeutic effect. The interactions between various components in herbal medicine can be synergistic and antagonistic. Due to technical limitations and sample acquisition difficulties, the pharmacokinetic study on a single active component has become a great challenge. On top of that, synergistic nature of CHM compounds makes the study even more complicated.There has long been an interest in CHM innovation and pressing demand for drug development in the field of material basis of CHM research. Researchers who hold “Reductionist” opinions worldwide have launched a research campaign focusing on herbal medicinal chemical compositions and their role in CHM efficacy. Some studies have tried to isolate the primary compound that is directly associated with the pharmacodynamics of a CHM prescription through direct separation analysis of herbal chemical compositions , 11. ThiIn the interpretation of the material basis of CHM, one approach for multicomponent analysis in herbal medicine is to remove one component at a time and eventually identify the target component of interest according to the changes inefficacy. This methodology is derived from the philosophy of reductionism. It highlights the individual component and elicits its pharmacological effect. However, such approach may not be the best option in analysing CHM on the material basis due to the following reasons. (1) Reductionistic thinking is contradictory to the basic philosophy of Chinese medicine, which emphasizes the “on a whole” approach to every matter. (2) The research object is centred on the effect and mechanism of one single component, which can result in a loss of focus of the overall performance of a CHM mixture. (3) The reduction process of ridding one compound at a time eliminates any synergistic or antagonistic activities between compounds and is not suitable for studies that desire multicomponent interactions. Therefore, the steps of “track” then “separation” are not fully applicable to the material basis of CHM.The goal of pharmacodynamic analysis of whole compound system of CHM material-based research is difficult to realize in the near future. And the approach to track and analyse single active component still possesses a certain degree of technical difficulties and the results obtained are still questionable. New strategies and methodologies are needed. Protein specific immunodepletion assay may be a valuable solution to the technical obstacles mentioned above.The basic principle of immunodepletion assay is to utilize antigen-antibody reaction to remove specific proteins in a complex CHM mixture. The objective is to establish and study the correlation between one specific protein and the overall efficacy of a CHM prescription. Comparing the pharmacological properties of a CHM mixture before and after protein deletion can then reveal the specific function of that particular protein. The advantage of adopting this method is the specific nature of antibody, which can selectively target the protein of interest and eliminate all other undesirable factors. The method not only discloses the pharmacodynamics and pharmacokinetics of a single active component but also reveals properties of other untargeted components and their synergistic behaviors. This method allows us to analyse the target component within the herbs and can accurately describe the degree of contribution of the target component to the overall efficacy without destroying the relations of other components.After the removal of a target protein in a herbal extract, the protein can then be reintroduced into the solution at variable concentration. And a quantity-efficacy (QE) correlation of that protein can then be generated, which demonstrates that the variation in concentration of a principal component can affect the overall therapeutic performance of a CHM preparation. Obtaining the QE correlation of the principal component can provide valuable support for new drug development. Mixing different monoclonal antibodies to create a cocktail can remove several different target proteins at one time. And through orthogonal design and titration method, the synergistic relationships between different active components can then be revealed. Illustrating such synergistic interactions between CHM compounds can provide better scientific information for future CHM formulation and compatibility studies. The immunodepletion of specific CHM proteins allows the analysis of a single component independent of others. It can also be used to study the combined effects of certain different components or all the components altogether and other various related issues. It is a more reasonable way to carry out future CHM researches.Monoclonal antibody is currently one of the most important research tools in life science research. And monoclonal antibody against CHM compound, if can be widely accepted, will be a very well-prospected research application. CHM researchers are now learning antibody technologies to study the complexities of herbal medicine. In 2005, Zhang et al.  reportedThere is no essential difference between the path of making monoclonal antibodies of TCM components and other small molecules. Scholars around the globe have established several hapten synthesizing methods, such as the carbodiimide method  and the In recent years, a variety of monoclonal antibodies against active components in herbal medicine have been successfully prepared against liquorice acid , paeonifIn contrast, limited by outdated research strategies, the usage of specific protein immunodepletion in the field of CHM basic research has not truly begun. It is also due to the lack of high titer monoclonal antibodies and lack of suitable instruments. Another reason for the setback is that more than 90% of the small molecules (MW < 2500) isolated from CHM are haptens, which are incapable of direct stimulation of antibody production in vivo. Another reason is that there are compounds that have similar structures with the active components in a CHM mixture  and may cause undesired cross-reaction with the monoclonal antibody, which further complicates the process of polyclonal antibody preparation and antigen synthesis and clone selection. Furthermore, the structures of the active components in CHM are diverse and can create obstacles during the coupling process with macromolecules. Therefore, preparation of high potent monoclonal antibodies for small CHM molecules and establishing a CHM small molecule monoclonal antibody library are the foundations for promoting protein specific immunodepletion technology.Pueraria root (Radix puerariae) is a sweet, acrid, cool, and nontoxic herbal remedy. It benefits the spleen and stomach. It is commonly used in the treatment for diarrhoea, fever, thirst, exogenous fever, headache, neck and shoulder pain, chest pain, measles, and so forth. Clinical application of R. puerariae is widespread in both classic and modern Chinese medicine prescriptions, such as GuiZhiJiaGeGen decoction, GeGenJiaBanXia decoction, GeGenHuanQinHuangLian decoction, Puerarin injection, GanMao QingRe granules, XinKeShu tablets, YuFengNingXin Tablets, TianBaoNingYinXing preparations, and XiaoKe capsules.Radix puerariae contains more than 30 types of flavonoid and isoflavonoid compounds, including daidzein and puerarin. It also contains triterpenoids, steroids, coumarin, GE phenolic glycosides, amino acids, and starch [ Pueraria. A typical Pueraria usually contains from 3% to 5% of puerarin, which is the primary therapeutic component of Pueraria [ Pueraria root and also as a QC standard for any medications that use Pueraria.d starch . PuerariPueraria . Testing Pueraria is associated with its antisympathetic, calcium antagonistic, and extensive β receptor blocking effects which result in vasodilation, lowering blood sugar and lipoproteins, and lowering blood pressure and subsequently protects the heart, liver, and kidneys [ Pueraria root affecting Pueraria's efficacy?” and “should puerarin be taken as the QC of any Pueraria's preparations?” need to be explicitly delineated in order to guide the real-world usage to Pueraria.Puerarin's pharmacological activity has been a primary research focus in the field of medicine. Literatures suggest that the pharmacological action of kidneys –40. The Series of research in the production of monoclonal antibody against the active CHM component were launched since 2008. The study involves the synthesis of artificial antigens by conjugating CHM micromolecules with macromolecules, such as bovine serum albumin. The conjugate obtained was then used to immunize lab animals and generate corresponding cell fusions, which were then screened for positive clones. Researchers have successfully generated monoclonal antibodies against the hormone geniposide, Radix puerariae-baicalin, berberine, and many other CHM components. Mastering and widely adopting monoclonal antibody technology have made significant advancements in the CHM industry. Geniposide and puerarin immunoassay kits were introduced, allowing the completion of study in allergens in Qingkailing injections. The immunoassay chip used in the study of allergen, chlorogenic acid, in QingKaiLing Injection resulted in a number of patents –47, and  Pueraria water extract. The puerarin immunoaffinity column prepared had strong specificity to puerarin present in the water extract and was able to delete the puerarin without affecting the remaining components in the solution. The bound puerarin was then eluted and a high purity sample of puerarin was obtained. These tools allow researchers to have a clear understanding on how to conduct a more in-depth study in CHM research. Radix puerariae was used here as an example for demonstrating monoclonal antibody technology using mAb deletion. There are animal and cellular models created for the purpose of studying the classic and modern medicinal property of Pueraria root, which can be used to collect pharmacological data of Pueraria under many different experimental criteria. By comparing the pharmacological properties and pharmacodynamics of Pueraria extract before and after undergoing puerarin immunodepletion, the main contribution of puerarin to the overall medicinal property of Pueraria can then be easily evaluated, in vivo and in vitro, static and dynamic. As a result, puerarin can then be better utilized in QC, compatibility studies and pharmacokinetic studies of Pueraria roots, which expands the usage of Pueraria roots in clinical medicine in the future. And also as a result, the experimental foundation for any future CHM studies using target specific deletion techniques was established. However, the methodology introduced in this paper still has its limitations, for instance (1) it is not suitable for identifying unknown CHM components and (2) it is also incapable of complete separation of compounds that have already interacted with others. Taking everything into account, the new research strategy still holds many advantages and has proven to provide more effective and convenient ways of pursuing CHM research and help the modernisation of Chinese medicine.By using puerarin monoclonal antibody and immunoaffinity chromatography technology, the puerarin was deleted from


Clusters of ryanodine receptors within atrial myocytes are confined to spatially separated layers. In many species, these layers are not juxtaposed by invaginations of the plasma membrane , so that calcium-induced-calcium signals rely on centripetal propagation rather than voltage-synchronized channel openings to invade the interior of the cell and trigger contraction. The combination of this specific cellular geometry and dynamics of calcium release can lead to novel autonomous spatio-temporal calcium waves, and in particular ping waves. These are waves of calcium release activity that spread as counter-rotating sectors of elevated calcium within a single layer of ryanodine receptors, and can seed further longitudinal calcium waves. Here we show, using a computational model, that these calcium waves can dominate the response of a cell to electrical pacing and hence are pro-arrhythmic. This highlights the importance of modeling internal cellular structures when investigating mechanisms of cardiac dysfunction such as atrial arrhythmia. Cardiac excitation-contraction coupling (EC-coupling) is initiated by an action potential that sweeps from the sinoatrial node, the pacemaking region of the heart. When the depolarizing action potential reaches individual myocytes it causes depolarization of their sarcolemma leading to activation of L-type voltage-operated Ca2+ channels (VOCs), and consequently a brief influx of Ca2+. The Ca2+ influx through VOCs provides a trigger signal to provoke more substantial Ca2+ release from closely apposed ryanodine receptor (RyR) clusters on the SR, by a process known as Ca2+-induced Ca2+ release  is governed byWe employ a recently developed three-dimensional model of an atrial myocyte  = σH(t)H(trel − t). The Ca2+ release strength is measured by σ, and the Heaviside function H, which is zero for negative arguments and 1 otherwise, ensures that release occurs over a period of trel. We solve equation (1) in a cylinder of length h = 100.4 μm and radius of a = 6 μm, consistent with the actual shape and size of an atrial myocyte . The initiation of the unpaced Ca2+ waves is due to the supercritical value of the residual Ca2+ concentration when the RyRs emerge from being refractory. Keeping the same value of the refractory period as in Figure 2+ waves by lowering the release strength as illustrated in Figure 2+ waves occur. The larger the release strength, the longer is the time window for initiating stimulus-independent Ca2+ waves.Following each systolic Ca2+ waves that derives from the specific geometry of atrial myocytes occurs within a single z-plane under appropriate conditions of refractory period and Ca2+ release strength. Specifically, the limited recruitment of a few RyR clusters triggers a perpetual, rotating Ca2+ ping wave within a z-plane .Another manifestation of self-sustaining, spontaneous Ca Figures B–G. The 2+ waves that pervade the entire cellular volume. Figure 2+ waves. The ping wave is clearly visible as the light bluish ribbon running along the center of the Figure. Emanating from the ping wave activity are diagonal rays of elevated Ca2+ activity that correspond to longitudinal Ca2+ waves. The only stimulus required for all the Ca2+ activity evident in Figure tref, longitudinal Ca2+ waves also exhibit periods with duration tref. The perpetual, ping wave Ca2+ release activity only occurs in the central z-plane. In all other z-planes, there is a clear quiescence phase between consecutive longitudinal waves. In the remainder of this paper, we will address the robustness of ping waves, especially with respect to physiologically relevant pacing conditions.Although ping waves occur within a single z-plane, their influence is not restricted to that domain of the cell. Rather, they have the capacity to trigger longitudinal Ca2+ waves. With this aim, we increased the refractory period at all z-planes except the one where we triggered the ping wave. Figure 2+ waves. As soon as the RyRs outside the central z-plane exit the refractory period, the ping wave triggers the longitudinal Ca2+ wave. Increasing the refractory period further . At the time indicated by the asterisk, peripheral release sites are stimulated as would be the case for electrical pacing. At this point, the longitudinal Ca2+ wave has reached the z-planes marked by the two green dots. The RyRs that lie between the central z-plane and the currently responding z-planes (red solid line) cannot liberate Ca2+ because they are still refractory due to the actual longitudinal Ca2+ wave (dashed red lines). The RyRs that are located above and below the green dots have not yet recovered from refractoriness induced by the previous longitudinal Ca2+ wave (dashed black lines). The only channels that are ready to respond are those in the two z-planes indicated by the green dotes. However, such localized activity does not significantly perturb the Ca2+ flow of the longitudinal Ca2+ wave, hence rendering the external stimulation ineffective.The mechanism by which electrical pacing fails to elicit a response once a ping wave has given rise to a fully developed longitudinal Ca2+ waves. If the refractory period is too long, then ping waves will die out. The simple reason is that the Ca2+ concentration will decline substantially once a ping wave has traversed its z-plane so that there is an insufficient trigger to initiate another event. An example of the significance of the refractory period in altering ping wave activity is depicted in Figure 2+ waves are evident. Since the longitudinal Ca2+ waves have reached the top and bottom of the cell, electrical pacing is ineffective. At the time indicated by the white arrow, the refractory period of the RyRs within the whole cell is immediately doubled. This provokes a rapid cessation of Ca2+ release and a quiescent phase whilst the RyRs recover. Since all triggers for autonomous Ca2+ release are halted, once the RyRs recover from being refractory the cell has to start responding to subsequent pacing events, and remain in-phase for the duration of stimulation. Some localized Ca2+ release activity is observed in the central z-plane, but is not sufficient to trigger a ping wave or longitudinal Ca2+ wave. These simulated data show that ping waves are critically dependent on refractory period and can be reversed once initiated.The results presented above indicate the robustness of ping waves in the face of electrical pacing. A key question is how such pro-arrhythmic activity could be quashed once initiated. Once again, the refractory period plays a critical role. Altering the refractory period can affect the sustainability of ping waves and their ability to trigger longitudinal Ca42+ signals. Any condition that allows Ca2+ dynamics to operate independently of electrical pacing has the potential to be arrhythmogenic. In particular, it is widely accepted that the activation of spontaneous Ca2+ signals can lead to arrhythmias by activating electrogenic ion transporters, or altering the responsiveness of cells to pacing. However, the triggers for spontaneous Ca2+ signaling, and the spatial pattern of such events are not well known.Fidelity of EC-coupling requires the appropriate translation of electrical stimulation into temporally- and spatially-coordinated Ca2+ signals in atrial myocytes. These cells have a well-defined geometry, and the relative positions of Ca2+ release sites (RyR clusters) are known  and RyR activity  will impact on the potential triggering of successive ping waves. In particular, SERCA activity will serve to diminish the residual Ca2+ concentration that remains in the wake of a ping wave, so that a successive ping wave will not occur. In the present study, we altered the refractory period as a way of changing the time available for Ca2+ to be diminished by SERCA. As we demonstrate, short refractory periods favor ping wave activity, while they are eradicated by increasing refractory period. However, it is not the refractoriness of RyRs per se that controls ping wave firing, but rather the processes that occur after Ca2+ release termination. Long refractory periods quash ping wave activity because Ca2+ diffusion and SERCA activity remove the trigger.When exploring the movement of Cae Figure . Ping wa2+ signal that then propagates in a saltatory manner between the RyR clusters within the z-disk via successive rounds of diffusion and CICR. In the example shown in Figure 2+ release sites to be involved (data not shown) – there simply needs to be a subcellular triggering event of sufficient magnitude to cause CICR in a localized spreading fashion.Ping waves depend on the activation of just a few RyR clusters within a z-plane. This is necessary to generate a local Ca2+ signal (Figure 2+ to trigger another ping wave after the previous one has propagated and declined. We therefore consider that ping waves are most likely to occur under conditions such as those found in heart failure where the RyRs are believed to have increased leakiness, SR Ca2+ load is reduced, diastolic Ca2+ levels may be elevated and Ca2+ pumps are less effective at removing cytosolic Ca2+. These conditions would favor the scenario where RyRs emerge from being refractory in the presence of elevated Ca2+ concentration, with the consequent triggering of spontaneous Ca2+ release. There is no need for back flux of Ca2+ from adjacent z-planes to maintain ping wave activity, as the results for long refractory periods illustrate (Figure 2+ waves in the presence of long refractory periods outside the central z-plane (Figure 2+ signal generated by the ping wave is the main mechanism that triggers longitudinal Ca2+ waves under these conditions. For short refractory periods, ping waves are responsible for seeding the first longitudinal Ca2+ wave. All subsequent longitudinal Ca2+ waves occur autonomously due to the short refractory period similar to the results shown in Figure A key aspect of ping wave survival is that the refractory period of the RyRs must be shorter than the recovery time of the cytosolic Cal Figure B. Otherwe Figure . Moreovee Figure  demonstr2+, intrinsic channel gating properties and/or decreased SR Ca2+ loading. Current evidence suggests that SR Ca2+ load may be a principle determinant of RyR refractoriness, but is not the sole factor. Indeed, the SR can refill with Ca2+, but RyRs can still be refractory (Ramay et al., 2+ release by cytosolic Ca2+, intrinsic gating or SR Ca2+ reduction. Essentially, with each mechanism, there would be a period of Ca2+ release followed by RyR closure, during which cytosolic Ca2+ can decline. Experimental data show that local Ca2+ release events recover within 200–250 ms, which is well within the period of ping waves described in this study (Figure 2+ to replenish, and RyR refractoriness to reverse.The physiological processes underlying RyR refractoriness are not fully established, but are believed to involve increased cytosolic Cay Figure . Hence, 2+ that is independent of pacing. More serious, however, is their ability to cause longitudinal Ca2+ waves by triggering CICR from neighboring z-planes. As demonstrated in Figure 2+ waves will have an increasingly dominant effect on the cell. This is because ping waves can trigger longitudinal Ca2+ waves as soon as the RyRs emerge from being refractory. Whereas, the centripetal Ca2+ waves that are caused by pacing occur at longer intervals, thereby giving the longitudinal Ca2+ waves the chance to propagate. Once the longitudinal Ca2+ waves have reached the top and bottom of the cell centripetal Ca2+ waves cannot occur. Under these conditions, pacing an atrial myocyte would have no effect in terms of contraction. It is worth noting that persistent Ca2+ wave activity that is independent of electrical pacing has been observed in atrial myocytes when the cells were stimulated with endothelin-1 (Bootman et al., An important feature of ping waves is their robustness, and their ability to make cells insensitive to electrical pacing. By themselves, ping waves would compromise atrial myocyte function because they occupy a fraction of the RyR clusters in a futile cycling of Ca2+ dynamics: cytosolic Ca2+ diffusion, CICR trigger by cytosolic Ca2+, and Ca2+ extrusion by SERCA pumps. As such, the model is minimal, but it allows us to study in detail the Ca2+ dynamics in an atrial myocyte at the subcellular and cellular level at the same time. The low computational demand of the model comes at the price of some limitations. At the moment, we assume a static SR, which prevents us from incorporating SR depletion and refilling. Undoubtedly, SR Ca2+ dynamics is vital in shaping cardiac Ca2+ patterns (Thul et al., 2+ concentration. We here focused on deterministic Ca2+ release, where Ca2+ liberation starts at a fixed value of the cytosolic Ca2+ concentration and lasts for a given amount of time comparable to the duration of a Ca2+ spark. The main motivation for neglecting stochastic channel openings was our goal to better understand autonomous Ca2+ release in general and ping waves in particular. As we demonstrate, autonomous Ca2+ release can be achieved without spontaneous Ca2+ liberation by balancing release strength, SERCA pump activity and the refractory period. Introducing random openings of RyR cluster would certainly modify the shape of ping waves, but would not completely abolish them. As we showed previously (Thul et al., 2+ release activity can be easily incorporated by randomizing the threshold for Ca2+ liberation. By construction, parameters such as the release strength σ or the release duration trel subsume a considerable number of mechanistic processes. Instead of modeling detailed molecular interactions, we chose a coarse-grained approach to investigate the consequences of changes at the micro scale – irrespective of their origin – for physiological and pathological Ca2+ signals.Our simulation framework is based on the core components of intracellular Ca2+ release that is favored by conditions of low SR Ca2+ load. Once acquired, these ping waves are difficult to stop and can persist indefinitely. Furthermore, although the simulations presented in this study have employed a ping wave occurring in a single z-plane, it is plausible that multiple ping waves could be present within a single cell. Analogous to cells acquiring multiple mutations of their DNA, several z-planes within an atrial myocyte could initiate ping wave phenomena and thereby trigger complex patterns of longitudinal Ca2+ wave formation. If the conditions described above are encountered then the progressive development of ping waves is inevitable.Arrhythmias induced by overloading the SR are well documented. Our computational studies have highlighted another form of arrhythmogenic CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The physical association between the endoplasmic reticulum (ER) and mitochondria, known as the mitochondria-associated ER membrane (MAM), enables highly efficient transmission of Ca2+ from the ER to mitochondria under both physiological and pathological conditions. Molecular chaperones are well known for their protective effects during cerebral ischemia. It has been demonstrated recently that many molecular chaperones coexist with MAM and regulate the MAM and thus Ca2+ concentration inside mitochondria. Here, we review recent research on cerebral ischemia and MAM, with a focus on molecular chaperones and ER-mitochondrial calcium transfer.It is commonly believed that sustained elevations in the mitochondrial matrix Ca Stroke is one of the leading causes of death worldwide and a major cause of long-term disability . Althoug2+ channels and exchangers located on the inner mitochondrial membrane. Recently ER stress was found to be one of the effects of excitotoxicity, that is, exposure to toxic levels of excitatory neurotransmitters, with release of Ca2+ from the ER via both ryanodine receptors and IP3R, with release from inositol trisphosphate receptors (IP3Rs) leading to mitochondrial Ca2+ overload and activation of apoptosis [2+ alter the permeability of mitochondria and finally open the MPT pore [A generally accepted cell death pathway after cerebral ischemia is mitochondrial permeability transition (MPT) pore opening . Ischemipoptosis . ExcessiMPT pore , causingMPT pore  and otheMPT pore .2+-selective channel known as the mitochondrial Ca2+ uniporter (MCU) [2+ affinity [2+ transients not exceeding concentrations of 1–3 μM, mitochondrial Ca2+ concentrations rise almost simultaneously to values above 10 μM [2+ at MAM leads to the formation of microdomains of high Ca2+ concentration that is crucial for efficient Ca2+ uptake by mitochondria [Mitochondria can accumulate large amounts of calcium through a Caer (MCU) , 13. Howaffinity . It is ive 10 μM . The exichondria , 17.Molecular chaperones are a functionally related group of proteins that assist protein folding in cells and protect cells from injury after cerebral ischemia or other stress. It has been demonstrated recently that MAM coexists with many molecular chaperones . The relAnimal models of ischemic stroke are used to study the basic pathophysiological processes and potential therapeutic interventions in this disease; the extension of knowledge gained from these animal models will lead to improvement of medical treatment for human ischemic stroke in the future. Focal cerebral ischemia by middle cerebral artery occlusion (MCAO) in rats or mice is the rodent model most immediately relevant to human stroke. Using this method, transient ischemia is achieved by inserting a suture into the left middle cerebral artery, temporarily blocking blood flow to the middle cerebral artery territory, and removing the suture to allow reperfusion after a duration of minutes to hours depending on the specific study , 19, 20.in vitro models of brain ischemia. Either cell cultures or slice cultures are subjected to medium lacking glucose, and in the case of OGD, also placed in a chamber with very low oxygen levels for a fixed period of time [ Glucose deprivation (GD) and combined oxygen-glucose deprivation (OGD) are common  of time , 21–26, in vivo and in vitro models of stroke [Grp78 mRNA was induced following MCAO both in the core and outside the infarcted area, GRP78 protein was only induced in the penumbra, not within the area of infarction.Molecular chaperones were originally defined as a functionally related group of proteins that assist protein folding in bacterial, plant, and animal cells. The heat shock proteins of the 70 kDa molecular weight family (HSP70), including HSP72 (cytosol), GRP75/mortalin (mitochondria), and GRP78/BIP , are highly evolutionarily conserved and have been extensively studied. Studies, including those from our laboratory, show that all three of these HSP70 family members are protective in both f stroke , 27–32. f stroke –35 and Gf stroke , that cef stroke . AlthougRecently a more complex, integrating role of these proteins has been recognized, that of stabilizing intracellular morphological and functional networks through protein-protein interactions with numerous client proteins –39. ThisIn addition to the new concept of the chaperone network, each individual chaperone has been found to have additional functions beyond that of functioning as a molecular chaperone. For example, GRP78 is traditionally considered to be a major endoplasmic reticulum chaperone as well as a master regulator of the unfolded protein response. Due to recent findings that significant amounts of GRP78 are present on the surface of cancer cells, it has emerged as an important regulator of tumor cell viability signaling, and cell surface GRP78 is now being used for therapeutic targeting . In addiAlthough the association of endoplasmic reticulum (ER) with mitochondria was first observed in the 1960s by several independent groups , 50, mor2+-binding ER resident chaperones have been found in the MAM fraction, for example, calnexin (CNX), calreticulin, and ERp44 [2+ signals [Numerous proteins have recently been proposed to participate in the interaction and communication between the mitochondria and the ER, highlighting the emerging role of this region in bioenergetics, cell survival, and cell death , 59. Onend ERp44 –67. The nd ERp44 . This fr signals . Recentl signals .MAM can be isolated from tissues and cells to investigate the mechanisms and functions involved , 71. Wie2+ released upon activation of the IP3R at the ER is taken up into mitochondria via VDAC and then MCU [It is commonly accepted that the main structure responsible for ER-mitochondrial calcium transfer at the MAM is composed of the IP3R on the ER, VDAC on the OMM and MCU on the IMM . Ca2+ rethen MCU , 73.2+ signaling between ER and mitochondria, a central topic of major interest both in normal physiology and pathophysiology. This second messenger has been proposed to have multiple roles in modulating intracellular events including bioenergetics and autophagy. Constitutive calcium release via the IP3R was found to be essential for maintaining normal bioenergetics and suppressing autophagy in conditions of ready nutrient availability [2+ increase seems to be required for triggering autophagy [2+ release via the IP3R can also induce autophagy [2+ dependent and independent pathways . Under  cascade . Some st2+ imposed upon the mitochondrion. Previous studies have shown that the reduction in the Ca2+ amount that can be released from ER to mitochondria decreases the probability of Ca2+-dependent apoptosis. On the other hand, conditions that increase ER Ca2+ storage have the opposite effect on Ca2+-dependent apoptosis [2+ within the ER/mitochondrial complex. Knockout of the proapoptotic proteins BAX and BAK reduced the resting concentration of ER Ca2+ decreasing the uptake of Ca2+ by mitochondria after Ca2+ release from the ER [2+ release, and diminished cellular sensitivity to Ca2+-mediated apoptotic stimuli [2+ leak from the ER, while AKT hyperactivation induces a decrease in ER Ca2+ release, probably through phosphorylation of the IP3R [The ER can play an important role in regulating apoptosis by adjusting the load of Capoptosis –82. It hm the ER . The act stimuli , 82. Antthe IP3R , 80.2+ signaling between ER and mitochondria. It was found that the mitochondrial chaperone GRP75 regulates IP3R-mediated mitochondrial Ca2+ signaling [2+-release channel IP3R through GRP75, highlighting chaperone-mediated conformational coupling between the IP3R and the mitochondrial Ca2+ uptake machinery (in vivo and in vitro cerebral ischemia [Some important chaperones are enriched in MAM and may play a key role in regulating Caignaling . It was achinery . We haveischemia , 83.2+-sensitive and ligand-operated receptor chaperone at the MAM [2+ depletion or after ligand stimulation, SIG1R can dissociate from GRP78 and begin to chaperone conformationally unstable IP3R (2+ signaling from the ER into mitochondria to increase the production of ATP in the cell through the tricarboxylic acid cycle in the mitochondria [ER protein SIG1R, implicated in neuroprotection, carcinogenesis, and neuroplasticity, is a Ca the MAM . Normallble IP3R  to enhanchondria . If stimchondria , 84–86. 2+ overload, and increases Ca2+ uptake capacity in isolated mitochondria after stress [Recent evidence  indicater stress . In order stress . As in tr stress , the mol2+ uniporter is the primary influx pathway for Ca2+ into respiring mitochondria, and hence is a key regulator of mitochondrial Ca2+. Although the uniporter's biophysical properties have been studied extensively, its molecular composition remained elusive for more than 50 years. A very recent report has identified a 40-kDa protein which fulfills the requirements for being the long sought mitochondrial calcium uniporter [2+ uptake in HeLa cells indicating that additional components or chaperones may be limiting in some settings [2+ with overexpression [2+ overload in intact cells, but also increases Ca2+ uptake capacity in isolated mitochondria [2+. Future validation of the hypothesis depends on further development of molecular approaches to confirm this property of MCU and its relationship with GRP78.The mitochondrial Caniporter . Overexpsettings , though pression . The MCUpression . Considechondria , it is p2+ signaling between these two organelles and control bioenergetics, cell survival, and cell death decisions. In the brain, ER calcium release has been found to directly contribute to excitotoxicity, a neuronal death mechanism important both in acute and chronic neurodegenerative diseases. Better understanding the roles of chaperones and Ca2+ handling in vivo should in the future provide new therapeutic strategies to protect brain cells during ischemia.In summary these findings together support a new emerging picture: chaperone machineries at both the ER and mitochondrion orchestrate the regulation of Ca


V1.2 channel. Calfacilitin binds to this channel and is co-expressed with it in the embryo. Regulation of intracellular calcium by Calfacilitin is required for expression of the neural plate specifiers Geminin and Sox2 and for neural plate formation. Loss-of-function of Calfacilitin can be rescued by ionomycin, which increases intracellular calcium. Our results elucidate the role of calcium fluxes in early neural development and uncover a new factor in the modulation of calcium signalling.Calcium fluxes have been implicated in the specification of the vertebrate embryonic nervous system for some time, but how these fluxes are regulated and how they relate to the rest of the neural induction cascade is unknown. Here we describe Calfacilitin, a transmembrane calcium channel facilitator that increases calcium flux by generating a larger window current and slowing inactivation of the L-type Ca Calcium signalling has been implicated in neural induction in the embryo. The authors identify Calfacilitin as a regulator of CaV1.2 calcium channels, which is required for induction of the pre-neural specifiers Geminin and Sox2 in the early chick embryo. A graft of a special region of the embryo, known as Spemann’s organizer1245678110111213Sox2, requires about 12 h exposure of competent ectoderm to neural inducing signals from a grafted organizer and a morphologically recognizable neural plate only appears several hours later1516171920212223252728293031323334353619However, development of the neural plate takes a relatively long time; for example, in the chick embryo expression of the earliest definitive neural plate marker, Timed organizer transplantation and removal experiments suggested that a minimum of 5 h signalling from a grafted Hensen’s node is required for the responding epiblast to become sensitive to BMP inhibition39404121434417V1.2 L-type channels and therefore named it Calfacilitin. We further show that Calfacilitin is required for neural induction . Calfacilitin is induced by FGF and is required for induction of Geminin and Sox2, two key effectors of neural induction. Our findings uncover a new regulator of calcium signalling as well as a new link between calcium and other signals during neural induction.Here we show that C3 encodes a multi-pass transmembrane protein, which facilitates calcium signalling through CaIn situ hybridization reveals that expression of C3 mRNA starts weakly in the epiblast before gastrulation  and the steady-state inactivation towards a positive potential , therefore evoking a larger window current. Furthermore, C3 slows inactivation in the presence of either Ba2+ or Ca2+ as the charge carrier  experiments in HEK293T cells. Two tagged versions . Whole embryos present particular challenges to Ca2+-imaging, which are further complicated by combining in experiments using fluorescein-labelled morpholinos and GFP-labelled reporter constructs (see below). The main problem is imaging large areas of the embryo with enough sensitivity and discrimination of Ca2+ levels over a long period of time (hours) without loss of dye from the cells. We explored Fura2 (ratio-imaged with IR excitation in a multi-photon microscope or with UV excitation by standard fluorescence microscopy), the ratio between Fura-Red+Fluo4 in a confocal or conventional fluorescence microscope, and Rhod-2 . The latter was most stable and allowed combining with FITC-labelled morpholino or GFP-labelled constructs. Although Rhod-2 tends to accumulate in mitochondria, the emitted signal is still sensitive to cytosolic Ca2+ levels; for example, this can be seen in ionomycin control experiments (see below). With the microscopy methods used, in whole embryos, we were unable to detect fast Ca2+ transients either in the normal embryo or in experimentally manipulated regions. However, we were able to detect slow changes over several hours.To test this further we used various Ca2+ signal, starting after about 3–4 h in the adjacent host epiblast . Calfacilitin-transfected cells display elevated Ca2+ as compared with neighbouring non-transfected cells or to control GFP- or DS-Red-transfected cells . These effects resemble the activity of BMP antagonists, which can only expand the neural plate and its border into the adjacent non-neural ectoderm when misexpressed along a continuous line of cells extending from the neural plate40422+ is sufficient for neural induction, which involves additional signals.To determine whether Calfacilitin is sufficient to induce t 26/32; ; arrow, t 26/32; , expandsCalfacilitin into the prospective neural plate of early primitive streak stage embryos. When electroporated into the embryo, this causes exon skipping, generating a truncated Calfacilitin protein , whereas Calfacilitin-MO significantly lowers Ca2+ with respect to neighbouring cells . Next, we determined whether this treatment affects expression of the neural plate marker Sox2. Indeed it does  targeting internal splice sites of  protein . First, her 6/31 , as compher 6/31 . CalfaciCalfacilitin cDNA (lacking the targeted splice site). This is the case . This predicts that the MO lowers Ca2+ in cells adjacent to the grafted node; to test this, we imaged Ca2+ using Rhod-2. The MO prevented the increase in Ca2+ caused by node grafts , Calfacilitin-MO  were used for most experiments. Quail eggs were obtained from Potter Poultry Farm, UK. All animal experiments conform to UK Home office regulations and were restricted to embryos during the first 2 days of incubation. Embryos were staged according to Hamburger and Hamilton17212+ levels, embryos were incubated in Rhod-2 , or a mixture of Fura-Red and Fluo4  or Fura2  at 37 °C for 30 min, rinsed in saline and left to recover for 30 min–1 h in new culture before grafting a node and/or electroporation of either Control-MO or Calfacilitin-MO. Time-lapse imaging was performed . Fura2 was imaged in a conventional fluorescence microscope as the ratio between emission at 510 nm generated by excitations at 340 and 380 nm or in a confocal microscope by excitation at 351 and 364 nm. Fluo4 and Fura-Red were excited at 488 nm and the ratio between green and red fluorescence generated was imaged by conventional microscopy. Rhod-2 was imaged by conventional fluorescence, using TRITC filters.To visualize Ca2+ levels  were measured in fixed areas (a rectangle or circle) of electroporated and contralateral control tissue, measuring the total intensity of red pixels; Student’s t-tests were used for analysis. In Ca2+-imaging experiments, implantation of a AG1X2 bead soaked in 2 μM ionomycin (Sigma I9657) in DMSO caused an explosive increase in the calcium signal with all dyes—this was performed as a control in time-lapse calcium imaging experiments. For inhibition of calcium channels, embryos were incubated in nicardipine  at 37 °C for 30 min and rinsed in saline before incubation of the embryo. To drive Ca2+ into cells, AG1X2 beads were incubated in 2 μM ionomycin in DMSO, rinsed in Pannett–Compton and implanted into embryos.For quantification in Rhod-2 experiments with electroporation of a morpholino or GFP construct, CaCalfacilitin was identified from a previously described screenCalfacilitin ORF was cloned into pCAβ-IRES-GFP and electroporated from a stock at 0.5 mg ml−1. To facilitate visualization, GFP cloned in the same vector was co-electroporated. For loss-of-function, fluorescein-labelled morpholinos (Gene Tools)  targeting intron–exon splice sites of Calfacilitin were electroporated either singly or together, from a stock containing 0.5 mM of each. The efficiency of the MO was tested by RT–PCR using primers: CalFac-F 5′-GAACCTCCTCGTTTCCTTCG-3′ and CalFac-R 5′-ACGAGGCCAACAAGTACGTC-3′. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, with primers: GAPDH-F 5′-GTGGGGGAGACAGAAGGGAAC-3′ and GAPDH-R  and cloned into pcDNA3.1. The constructs were transfected into COS cells using Lipofectamine-2000V1.2 was a gift from Dr Emmanuel Bourinet to TWS and contains the HA epitope in the extracellular S5-H5 loop of domain IIHaemagglutinin (HA) tagged CaV1.2 α1, β2a-GFP, α2δ-subunits, TAG and C3-myc using lipofectamine 2000(Invitrogen). Control cells were transfected with an equal amount of pcDNA3 vector. 24 h later, the cells were harvested and lysed in 100 μl Hepes lysis buffer containing: 20 mM Hepes, 137 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2 and 1 mM EGTA. After incubation for 1 h at 4 °C, 50 μl of protein lysate were taken as input and the remaining for IP. Eight microgram anti-CaV1.2 antibody (Alomone ACC-003) were incubated with the protein lysate and protein A beads (Roche) for 3 h. After washing three times with lysis buffer containing 0.1% Triton X-100, the protein was separated by SDS–PAGE. Calfacilitin-myc was detected with anti-myc antibody (Sigma C3956) and CaV1.2 protein by anti-CaV1.2 antibody.The electrophysiological properties of Calfacilitin were studied by patch clamp techniques after transfection into HEK293 using calcium phosphateC.P. and I.D.A. performed all the embryo experiments, some of them with participation of E.K., L.Z. and A.Sh. P.L. performed the electrophysiological experiments in T.W.S.’s laboratory with the assistance of S.-Q.L. and D.Y. G.S. and A.S. performed the initial screen that led to isolation of Calfacilitin. N.M.M.O. performed transfection experiments in COS cells. The project was conceived and directed by C.D.S. and largely performed in his laboratory. The paper was written by C.D.S. with contributions from C.P., I.D.A., P.L. and T.W.S.Accession codes: Sequence data have been deposited in Genbank/EMBL/DDBJ under accession number GQ504719.How to cite this article: Papanayotou, C. et al. Calfacilitin is a calcium channel modulator essential for initiation of neural plate development. Nat. Commun. 4:1837 doi: 10.1038/ncomms2864 (2013).Supplementary Figures S1-S4


Development of modern proteomic methods in recent years has opened also new perspectives in the identification of new biomarkers which ensure more effective diagnosis, treatment monitoring and prediction of therapeutic outcome. We evaluated usefulness of comparative proteomics (MALDI-TOF) in two subtypes of acute myeloid leukemia (AML), M1 and M2, according to FAB classification. The bone marrow or blood cell proteomes were examined in 33 newly diagnosed patients before “3 + 7” induction therapy, after treatment and when the disease relapsed. We found that bone marrow and peripheral mononuclear cells from healthy volunteers revealed a number of quantitative and qualitative differences between the two proteomes, reflecting differences in the maturational status of normal cells. Such differences were not detected in our AML M1/M2 patients. Additionally, we found 9 proteins, which are potential biomarkers differentiating between the AML patients and healthy volunteers. Using comparative proteomics, we found that annexin I, glutathione transferase omega, esterase D and gamma 1 actin had prognostic significance. Applying statistical methods, we detected two proteins which might allow to predict results of induction therapy in AML M1/M2. One of them was esterase D, the higher concentration of which was associated with higher complete remission rate, and the other was gamma 1 actin, the higher concentration of which was related to resistance. In the article, we also discussed the role of these two proteins in the biology of AML, and we suggested potential usefulness of modification in induction therapy reflecting the presence of proteins. FLT3, NPM1, CEBPA) improved prognostic allocation especially in the group of patients with the, so-called, normal cytogenetics. During the last decade, DNA microarrays were extensively used to identify genes involved in pathogenesis of human myeloid malignancies  and FLT3-ITD in mononuclear cells was determined by RT-PCR. Cytogenetic analysis was performed on metaphases from samples of bone marrow obtained prior to induction therapy (T0) by using standard banding techniques. Karyotypes (GTG) were determined according to the International System for Cytogenetic Nomenclature . The. The9]. p-value was < 0.05 in the univariate analysis were included into a multivariate logistic regression model. Adjusted odds ratios (OR) and 95 % confidence intervals were calculated.The relationship between clinical parameters and results of induction chemotherapy (CR or RES) was analyzed by Fisher’s exact test. Univariate analysis of the major outcome variables and the protein concentrations  was performed using logistic regression. Factors for which the p < 0.05. Statistical analyses were performed using Statistica 10  software and MedCalc version 10.3.2 .Receiver operating characteristics (ROC) curves were calculated to determine the potential of analyzed factors to discriminate the result of induction therapy. An optimal cut-off point was calculated according to the highest accuracy . The area under the ROC curve (AUC) was used to check the prognostic value of a particular variable. The tests were considered statistically significant at The comparison between AML-BMMC and AML-PBMC proteomes at T0, T1 and T2 did not show any statistically significant differences between them, and therefore, data for these samples were calculated together. On the other hand, a comparative analysis of BMMC and PBMC from HV revealed numerous quantitative and qualitative differences between the two proteomes. The set of proteins differentiating them was finally limited to 28, which matched the established criteria (we excluded extracellular proteins that were found to be contaminants or proteins for which the relative accumulation levels were below 0.004).Comparative analysis of AML M1/M2 and control BM/PB cells revealed 25 differentially accumulating proteins. They included 8 proteins , which simultaneously differentiated healthy bone marrow/blood mononuclear cells from AML M1/M2 mononuclear cells and healthy blood cells from healthy bone marrow. Additionally, we found 9 proteins , which represented potential biomarkers differentiating between the AML M1/M2 patients and healthy volunteers.t  were in the same subgroup, defined as AML M2. There were no differences in protein accumulation between patients with or without this translocation in the group of AML M2 patients. Next, we performed a comparative proteomic analysis within the AML M1-T0 and AML M2-T0 subgroups. As a result, we identified 5 proteins differentially accumulating in two subgroups. Three from those proteins—annexin III, L-plastin isoform and 6-phosphogluconate dehydrogenase—were detected only in the AML M2-T0 group. Catalase and peroxiredoxin 6, on the other hand, were manifested in both analyzed groups; however, they showed a higher level of concentration in the AML M2-T0 group.We used a function of the Image Master 2D Platinum 6.0 program to distinguish between two subtypes of AML according to FAB classification–M1 and M2 at proteome level. We found that AML M1/M2-T0 group can be divided into 2 subgroups. The important fact was that samples from the same patients (BMMCs and PBMCs) were always in the same subgroup. Moreover, all samples from patients with the diagnosed We also found that such proteomic-based classification was in 88 % consistent with the clinical diagnosis, based on cytomorphology, immunophenotyping and peroxidase reaction.We compared the proteomes of individual patients before treatment (T0) and after treatment, when CR was obtained (T1), and we did not succeed in identifying any proteins, which would differentiate both groups. The results obtained showed that in case of samples collected at T1, we dealt with a highly diversified pool of cells. Their proteome exhibited very high individual variability.In addition, we attempted to determine whether at the time of relapse (T2), the proteome of mononuclear leukemic cells is similar to or different from the proteome of the cells collected at T0. The analyses did not reveal any statistically significant differences. Therefore, we suggested that the proteome of cells before treatment and at relapse was similar, at least at the level of highly abundant proteins.No dependence between results of treatment and traditional clinical characteristics—age, sex, subtype of AML, performance status, history of prior malignancy and chemotherapy, dysplasia, infection status, cardiac status, white blood cell count, hemoglobin, platelet count, percentage of blood and bone marrow blasts, percentage cells expressing CD 7, 11b, 14, 34, 56, HLA-DR, creatinine and bilirubin concentration in blood, cytogenetics, molecular findings was observed (data not presented).p < 0.001) in the subgroup with CR (a threefold difference). Gamma 1 actin accumulated to a significantly higher level (a 2.85-fold difference) in mononuclear cells in AML M1/M2-T0 subgroup with resistance to induction therapy and also in patients with short-term remission (below 12 months). Additionally, results obtained for annexin I and gamma 1 actin were verified using the Western blot method.On the basis of proteomic analysis of blood and bone marrow samples from patients with AML before induction therapy (T0) who achieved complete remission or were resistant, we found four proteins, the presence of which significantly correlated with results of treatment. The prognostic significance was manifested by annexin I, glutathione transferase omega, esterase D/formylglutathione hydrolase/and gamma 1 actin Table . Two prop = 0.0032). An increase in esterase D concentration by 1 unit augmented about 1015-fold the probability of CR. On the other hand, an increase in gamma1 actin concentration by 1 unit multiplied the probability of RES by about 103. This statistical model gave correct prediction in 92 % of cases. Cross-validation gave 94.4 % accuracy for CR results and 85.7 % accuracy for RES results.Glutathione transferase omega manifested the best prognostic value; but on the basis of logistic regression analysis, we identified two other proteins, esterase D and gamma 1 actin, which provided the best index pointing to a complete remission . Moreover, all samples from patients with diagnosed t translocation have fitted the same subgroup defined as AML M2. There exist no differences in protein accumulation between patients with or without this translocation in group of AML M2 patients . Some auIn adult patients with acute myeloid leukemia (without acute promyelocytic leukemia), the use of intense induction chemotherapy “3 + 7” is the worldwide standard of care. Its administration allowed to obtain CR in approximately 70–80 % of patients under 60 years of age. However, most of them relapse, and therefore, overall survival in this group is only 40–45 % at 5 years. The results of therapy are much worse in the group of older patients (over 60 years old) . Due to In this paper, authors have focused on the pretreatment prognostic factors. On the one hand, we have attempted to evaluate influence of clinical, cytogenetic, molecular data on results of chemotherapy. On the other hand, we have correlated all the mentioned above data with results of comparative proteomics to establish its usefulness to foresee the results of induction therapy (complete remission or resistance).p = 0.048) and consolidation therapy based on alloHSCT (p = 0.019) prolonged overall survival (OS). Meanwhile, occurrence of disease relapse (p = 0.043) and cardiologic side effects (0.024) has shortened OS.The studied group of patients with AML M1 and M2 has consisted mostly of younger people (below 60 years old) in good performance status. The statistical analysis of traditional clinical characteristics—age, sex, subtype of AML, performance status, history of prior malignancy and chemotherapy, dysplasia, infection status, cardiac status, white blood cell count, hemoglobin level, platelet count, percent blood and marrow blasts, percent cells expressing CD 7, 11b, 14, 34, 56, HLADR, creatinine and bilirubin concentration, cytogenetics, molecular findings and results of induction chemotherapy (“3 + 7”) has demonstrated no significant correlations. We are aware that our study group was too small to draw final conclusions in this subject. However, we have performed also very initial survival analyses, and we have found that favorable cytogenetics  while Kornblau et al.  assayed The first proteomic biomarker proposed by us is esterase D, also known as S-formylglutathione hydrolase. The enzyme is involved in detoxification of formaldehyde . HoweverIn our opinion, the second protein which potentially carries a prognostic value in AML M1 and M2 patients is gamma 1 actin. Actins are an essential component of the cytoskeleton, with critical roles in a wide range of cellular processes, including cell migration, cell division and the regulation of gene expression . An overIn conclusion, there are no doubts that esterase D and gamma 1 actin, suggested by the authors to represent prognostic factors involving results of induction therapy in acute myeloid leukemia M1 and M2, should provide targets of further more detailed investigations. We want to establish not only the utility of these proteins in prognosis of a complete remission or resistance in other subtypes of AML, but also in prognosis of the effects of combined induction therapy with or without arsenic trioxide in a group of patients with high gamma 1 actin level. We suggest that ATO represents a drug which could improve results of treatment in AML, but we think that also other drugs which influence Rho-ROCK-JNK axis deserve further investigation.


Background: Initial studies on cardiac NAADP signaling were published, but no role for NAADP in cardiac arrhythmias has been reported.Results: NAADP affects spontaneous diastolic Ca2+ transients in cardiac myocytes and arrhythmias in awake mice.Conclusion: Results indicate a pivotal role for NAADP in fine-tuning of cardiac excitation-contraction coupling.Significance: First evidence is reported for involvement of NAADP in cardiac arrhythmias evoked by β-adrenergic stimulation. 2+-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca2+ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca2+ signals sensitive to inhibitors of both acidic Ca2+ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca2+ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca2+ transients were recorded both as increases of the free cytosolic Ca2+ concentration and as decreases of the sarcoplasmic luminal Ca2+ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca2+ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy.Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca Similare family –9, ryanoe family –16. A une family ; the cenchannels , 19.2+ release from heart microsomes  NAADP evoked Cacrosomes ; (ii) NAcrosomes ; (iii) ecrosomes , 21, andcrosomes . ADP-ribcrosomes , is prescrosomes , 25. Fur+ sparks .2+ signaling, we hypothesized that it might also play a significant role in aspects of myocyte function. We therefore analyzed activation of Ca2+ signaling upon NAADP infusion in quiescent adult mouse cardiac myocytes, and we studied both cell-based (in vitro) and animal (in vivo) models of ventricular arrhythmic events to evaluate an involvement of NAADP signaling. Our findings indicate a hitherto unappreciated pivotal role for NAADP in fine tuning of cardiac excitation-contraction coupling and open the way for novel therapeutic treatment of cardiac arrhythmias.In view of the strong evidence for an involvement of NAADP in cardiac CaO-methyladenosine-3′,5′-cyclic monophosphate (8-pCPT) and N6-benzoyladenosine-3′,5′-cyclic monophosphate (6-Bnz-cAMP) were obtained from Biolog . Creatine kinase and creatine phosphate were purchased from Roche Applied Science. Saponin was obtained from Fluka/Sigma. Fura-2/free acid was purchased from Calbiochem. This investigation conforms to the Guide for the Care and Use of Laboratory Animals .BZ194 was synthesized as described previously , purifieTM research grade (Roche Applied Science) and 12.5 μm CaCl2 for 6–9 min. A total of 150,000–250,000 rod-shaped cells were obtained per heart. Cells were plated onto laminin 111-coated  μ-Slide 8-well chambers  at a density of 20,000 rod-shaped myocytes/ml in the plating medium containing minimum Eagle's medium with Hanks' salts and l-glutamine (Invitrogen), 5% (v/v) NCS or FCS, 10 mm 2,3-butanedione monoxime and incubated 30 min at 37 °C in an incubator at 2% (v/v) CO2 in air.Cardiac myocyte isolation from wild-type Black Swiss, FVB, or C57BL/6J mice (6–15 weeks old) and cell culture was performed as described previously . Hearts 2+ transients (SCT) was studied in cardiac myocytes from FVB mice  in plating medium in a reaction tube for 30 min at room temperature. Myocytes were washed twice with plating medium. For some experiments, cells were incubated with 0.5 μm bafilomycin A1 or 0.4% (v/v) DMSO for 20 min after loading with Fura-2/AM. All experiments were performed with myocytes in buffer A  attached to 35-mm glass bottom culture dishes  at room temperature. The patch electrodes were made from 1.5-mm diameter borosilicate glass capillaries and filled with intracellular solution. The pipette solution contained (in mm) the following: 140 KCl, 2 MgCl2, 10 HEPES adjusted to pH 7.4 with KOH. Fresh solution containing Fura-2 free acid  and NAADP  were prepared each day. For co-infusion experiments, pipette solution with Fura-2, 37.5 nm NAADP, and BZ194 , 0.1% (v/v) DMSO, ruthenium red , or ryanodine (1 mm) were prepared.For the whole-cell infusion experiments, an EPC10 patch clamp amplifier was used in conjunction with the PATCHMASTER software . Cardiac myocytes were loaded with Fura-2/AM (4 μ2+ imaging was performed as described before using an imaging system (PerkinElmer Life Sciences) built around a Leica microscope (type DM IRBE) (Ratiometric CaDM IRBE) , 14. To DM IRBE) . Data prg for 10 min at 4 °C). The supernatant was divided into two identical halves (twin samples). Authentic NAADP (15 pmol) was added to one of the twin samples to calculate recovery. After extraction of trichloroacetic acid using water-saturated diethyl ether, the samples were freeze-dried overnight. The cell extracts were purified by gravity-fed anion-exchange chromatography, and NAADP content was determined using the NAADP cycling assay as described previously ) for 20 min at room temperature, and afterward lysosomes were labeled with 75 nrecently , 30. Them final concentration) for 45 min at room temperature in plating medium (composition see above). Then the medium was removed by gentle aspiration and replaced by buffer A. This step was repeated twice to completely remove indo-1/AM as well as rounded or nonattached myocytes. Ratiometric Ca2+ imaging was performed as described previously  set to 30 V and custom-made platinum electrodes. Iso or 8-pCPT was added about 5 min and 6-Bnz-cAMP about 20 min before the start of electrical stimulation. Myocytes were incubated with BZ194 or vehicle DMSO  for 1 h before the start of electrical stimulation. Myocytes were incubated with bafilomycin A1 or vehicle DMSO  for 20 min before the start of electrical stimulation.Cardiac myocytes, attached to laminin 111-coated chamber slides, were loaded with indo-1/AM . The experiments were either performed without incubation (control), after incubation of the myocytes in 0.5% (v/v) DMSO , or after incubation with 2 mm BZ194 . The holding potential for the measurements of L-type Ca2+ channels was −80 mV. A prepulse to −40 mV for 100 ms was applied to inactivate Na+ channels. IV relationships were recorded from −40 to +40 mV. Series resistance was compensated by 70–90%. Data were low pass filtered at 1 kHz and stored on a personal computer. Analysis was performed with PulseFit software (HEKA) and Excel (Microsoft). All experiments were performed at room temperature.For whole-cell patch clamp experiments, an EPC9 patch clamp amplifier was used in conjunction with the PULSE stimulation and data acquisition software . The patch electrodes were pulled from 1.5 mm diameter borosilicate glass capillaries with horizontal puller (Sutter) and filled with intracellular solution. The pipette solution contained 156 m3H]ryanodine binding was carried out with 50 μg of heavy SR, which was incubated for 3 h at 30 °C in a buffer containing (in mm) 20 HEPES, pH 7.4, 140 KCl, 50 NaCl, and 20 nm [3H]ryanodine supplemented by protease inhibitors benzenesulfonyl fluoride hydrochloride, Pefabloc SC). The free [Ca2+] was adjusted by the ratio of CaCl2 and EGTA (m ryanodine.Cardiac heavy sarcoplasmic reticulum was prepared from rabbit hearts according to a previous protocol . High afand EGTA . The bin7) were rinsed twice in intracellular buffer I ). Afterward, cells were resuspended in permeabilization buffer (intracellular buffer I with 60–80 μg/ml saponin) and incubated for 5 min at 37 °C. After incubation, cells were rinsed with intracellular buffer II  and resuspended in intracellular buffer II). To measure free [Ca2+], 0.5 μg/ml Fura-2/free acid, 20 units/ml creatine kinase, and 20 mm creatine phosphate were added to the cells in a quartz cuvette. Fura-2 fluorescence at 495 ± 10 nm was determined in a Hitachi F-2000 fluorescence spectrophotometer  with alternating excitation at 340 ± 10 and 380 ± 10 nm every 5 s. After 100 s, 1 mm ATP was added to activate Ca2+ uptake. After 800 s, 1 μm ionomycin was added to release Ca2+ from the luminal space. Each measurement was calibrated according to Ref. m CaCl2 for maximal ratio and 8 mm EGTA, 60 mm Tris for minimal ratio. Curves after addition of ATP were fitted to three-parameter exponential decay using SigmaPlot (10.0).HEK293 cells (1·10m) 140 KCl, 2 MgCl2, 1 CaCl2, 2.5 EGTA, 10 HEPES, titrated to pH 7.40 using KOH. The extracellular solution consisted of (mm) 135 NaCl, 4.7 KCl, 0.6 KH2PO4, 1.2 MgSO4, 0.6 NaH2PO4, 1.25 CaCl2, 20 glucose, 10 HEPES, titrated to pH 7.4 with NaOH. Before beginning the experiments, myocytes were incubated for 1 h with BZ194  or vehicle DMSO (0.5%). Action potentials were elicited at room temperature at a rate of 2 Hz by depolarizing current pulses of 5 ms duration throughout the experiment. The current injection was adapted to match the excitation threshold of each individual myocyte. Data were low pass filtered at 1 kHz and analyzed using the FitMaster software  and custom-made procedures in IgorPro .Action potentials of cardiac myocytes were recorded using the ruptured patch whole-cell configuration in combination with an EPC-10 amplifier controlled by the PATCHMASTER software. Patch pipettes were pulled from 1.5-mm diameter borosilicate glass capillaries with a horizontal puller (Sutter Instruments). The pipette solution contained  in plating medium (composition see above) in a reaction tube and attached to laminin 111-coated chamber slides for 30 min at 37 °C in an incubator at 2% (v/v) CO2 in air. To remove mag-Fura-2/AM and nonattached myocytes, the chamber slides were washed with buffer A (composition see above). The myocytes were then incubated with 1 mm BZ194 or vehicle DMSO  for 60 min at 37 °C and 2% (v/v) CO2 in air. Iso  was added 5 min before imaging.Cardiac myocytes were loaded with mag-Fura-2/AM (5 μ2+ imaging was performed as described above using a PerkinElmer Life Sciences imaging system built around a Leica microscope (type DM IRBE). Illumination at 380 nm was carried out using a Sutter DG-4 ultra high speed wavelength device (PerkinElmer Life Sciences). Images of emission light at 510 nm were acquired using a gray scale EM-CCD camera . The spatial resolution was 1000 × 1000 pixels at 40-fold magnification. The acquisition rate was ∼30 frames/s. Raw data images were stored on hard disk. Data processing was performed using Openlab software version 5.5.2 (PerkinElmer Life Sciences).Ca−1) and subsequent addition of caffeine (10 mm) using a field stimulator  set to 30 V and custom-made platinum electrodes.Myocytes were monitored under repeated electrical stimulation  were implanted subcutaneously in male BALB/c mice  with the leads approximating an Eindhoven II configuration  and Prism .Amplitudes obtained from Ca2+ mobilizing activity of NAADP in ventricular cardiac myocytes, cells were infused with 37.5 nm NAADP resulting in an increase in [Ca2+]i, whereas infusion of nominally Ca2+-free intracellular buffer did not . A slow wave of increased [Ca2+]i originated from the tip of the patch pipette . Different concentrations of NAADP revealed a bell-shaped concentration-response curve with a maximum around 37.5 nm NAADP . Further evidence for a physiological role of NAADP in cardiac myocytes was obtained by determining endogenous NAADP concentration in extracts of whole hearts freshly prepared using an enzymatic assay with femtomole sensitivity (n = 3), a value well in accordance with data recently obtained in mouse heart . Furthermore, ryanodine also fully inhibited Ca2+ signals evoked by NAADP; whereas infusion of pipette solution (vehicle) resulted in a Fura-2 ratio of 0.912 , NAADP increased the Fura-2 ratio to 1.067 ± 0.033 . Co-infusion of NAADP and ryanodine resulted in a Fura-2 ratio of 0.876 ± 0.034 . Infusion of ryanodine alone resulted in a Fura-2 ratio of 0.900 ± 0.024 , respectively. Besides the endoplasmic reticulum, acidic Ca2+ stores have been postulated to be involved in NAADP signaling in different cell types (2+ gradients across the membrane of the acidic compartment (data not shown). Incubation of cardiac myocytes with bafilomycin A1 completely inhibited NAADP-mediated Ca2+ signaling, indicating that acidic stores are involved in the process of NAADP signaling in cardiac myocytes .As it has been established in different cell types that either NAADP activates RyR , 13 or C or RyRs , 8, 36, ll types , 36, 37.2+ signaling without any inhibitory effect on Ca2+ release induced by d-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose . These experiments in quiescent cardiac myocytes indicate a potential involvement of NAADP and acidic Ca2+ stores in RyR modulation during cardiac Ca2+ signaling.BZ194 (3-carboxy-1-octylcarbamoylmethylpyridinium bromide) is a specific antagonist of NAADP characterized in detail recently, and when applied directly on target, BZ194 inhibited NAADP-mediated CaP-ribose . In card2+ signaling to bafilomycin A1, ruthenium red, and ryanodine opens the possibility for acidic stores or RyR as direct NAADP targets. Thus, we analyzed a potential direct modulation of RyR. [3H]Ryanodine binding to cardiac heavy SR depended strongly on the free [Ca2+], although NAADP added at concentrations of >300 nm did not modulate [3H]ryanodine binding (a). Although a small but significant stimulatory effect of NAADP was detected at 125 and 300 nm NAADP, this was negligibly small when compared with the prototypical RyR activator caffeine (b), indicating minor activation of RyR2 by NAADP.The sensitivity of NAADP-evoked Ca binding a. Althoucaffeine b, indica2+ currents were almost unaffected by BZ194 even at a high concentration of 2 mm (c). SERCA activity was analyzed in permeabilized HEK293 cells upon ATP addition. The permeabilized cell preparation allowed BZ194 to directly act on any potential intracellular target (d). Addition of BZ194 (up to 400 μm) did not affect the rate of Ca2+ uptake, although SERCA inhibitor thapsigargin did (d and e). Thus, neither L-type Ca2+ currents nor SERCA activity was impaired by BZ194, further supporting the specificity of the NAADP-antagonist BZ194.Potential unspecific effects of BZ194 were assessed as follows. L-type Ca of 2 mm c. SERCA r target d. Additirgin did , d and e2+ signaling upon β-adrenergic stimulation, myocytes were electrically stimulated at 0.5 s−1. The vast majority (>80%) of control cells (addition of solvent only) responded with a single characteristic Ca2+ transient to electrical pacing . In the presence of 10 nm Iso, cells also showed regular single Ca2+ transients . 10 nm Iso increased peak amplitude by ∼70% and accelerated transient decay as compared with control . Increasing Iso to 25 nm started to induce SCT  showing a clear concentration-dependent effect with an EC50 of about 22 nm . Increases in amplitude and reciprocal time constant were already observed with EC50 of about 2 and 6 nm, respectively, suggesting the involvement of different signaling mechanisms or different signaling thresholds downstream of β-adrenoceptors.To analyze Caexchange protein activated by cAMP” (Epac) might be involved. Indeed, the PKA-selective cAMP-analog 6-Bnz-cAMP (d and e) in a fashion similar to high Iso concentrations (b and e), whereas the Epac-selective cAMP analog 8-pCPT (c and e). Furthermore, only 6-Bnz-cAMP induced a marked increase of amplitude and reciprocal time constant, again mimicking the effect of Iso (f and g).Because SCT occurred upon strong β-adrenergic stimulation, it was likely that cAMP generation and its downstream effectors PKA or “Bnz-cAMP  induced Bnz-cAMP , d and etrations , b and eg 8-pCPT  did not g 8-pCPT , c and et of Iso , f and g2+ transient amplitude, or reciprocal time constant . In stark contrast, preincubation with bafilomycin A1 almost completely blocked all SCT induced by high Iso, with the majority of cells showing only one Ca2+ transient per electrical stimulation (d versus c and e). However, bafilomycin A1 had no impact on Iso-stimulated Ca2+ amplitude (f) but induced a small, significant reduction of the reciprocal time constant from 11 to 8.6 s−1 (g).To investigate involvement of acidic stores in SCT, myocytes were preincubated with bafilomycin A1. In the absence of Iso, bafilomycin A1 had no significant effect on SCT numbers, Camplitude f but ind 8.6 s−1 g.2+]i was imaged at constant electrical stimulation in the presence and absence of Iso. A concentration-dependent decrease of Iso-induced SCT was obtained upon preincubation with BZ194, whereas Ca2+ transient amplitudes were almost unchanged (2+ transients in the absence of Iso (a–d and i–k), BZ194 ≥0.5 mm significantly reduced the number of SCT evoked by Iso (e–k). At 1 mm BZ194, only one transient per electrical stimulation was observed (h and i). At this concentration, BZ194 also induced a small but significant decrease of the reciprocal time constant from 10 to 8.5 s−1 (k).Having observed the strong reduction of SCT upon bafilomycin A1 treatment, we hypothesized that NAADP might be the endogenous stimulus for SCT. Because we directly demonstrated the inhibitory effect of BZ194 in quiescent cardiac myocytes infused with NAADP , cardiacnchanged . Althougd by Iso , e–k. Atobserved , h and i 8.5 s−1 k.a) nor in the presence of Iso  did BZ194 affect the shape or the duration of the action potentials. In particular, the resting membrane potential (c), the action potential amplitude (d), and the time course of early and late repolarization (e and f) were unaffected. This makes a direct effect of BZ194 on currents highly unlikely, e.g. the inward rectifying K+ current, the fast Na+ current, the L-type Ca2+ current , or the repolarizing K+ currents. Taken together, the results indicate that the decrease of SCTs by BZ194 is not a consequence of direct effects on these cardiac membrane currents.To address the possibility that BZ194 might directly affect cardiac plasma membrane currents, its effect on action potentials was assessed in isolated cardiac myocytes. Action potentials rely on a finely tuned interaction between all currents and thus represent a very sensitive readout for off-target effects. Neither in the absence a nor in  200 nm, b did BZ1otential c, the acmplitude d, and thrization , e and fsee also c, or the2+ concentration ([Ca2+]SR) upon electrical stimulation at 0.5s−1 (a). Under control conditions (cells preincubated with vehicle DMSO), the transient decreases of [Ca2+]SR showed a relatively slow recovery back to base-line values, thus resembling the slow decay of cytosolic Ca2+ transients under control conditions (a). 81% of cardiac myocytes displayed one transient decrease of [Ca2+]SR at each single electrical stimulation (b). Transient decreases in [Ca2+]SR in cardiac myocytes preincubated with BZ194 were of similar shape as compared with controls (a); moreover, a similar percentage of cells displaying one transient decrease of [Ca2+]SR at each single electrical stimulation was observed when compared with vehicle DMSO (b). Strong β-adrenergic stimulation influenced both the shape and number of transient decreases in [Ca2+]SR (a and b). The recovery back to base line of the transient decreases in [Ca2+]SR proceeded more rapidly as compared with controls (a), again resembling the changes in shape of cytosolic Ca2+ transients evoked by Iso . Importantly, spontaneous diastolic transient decreases in [Ca2+]SR (SDC) were observed upon strong β-adrenergic stimulation (a). The percentage of myocytes displaying SDC significantly increased from 19% (DMSO) to 57% (b). Importantly, preincubation of myocytes with BZ194 and subsequent strong β-adrenergic stimulation resulted in a significant decrease in the percentage of cells displaying SDC to 24% (b). At the end of each experiment, 10 mm caffeine was added to deplete the SR (a). Taken together, the data indicate that strong β-adrenergic stimulation resulted in spontaneous diastolic transient decreases in [Ca2+]SR (SDC), most likely due to repetitive opening of sensitized RyR2.Ventricular myocytes loaded with mag-Fura-2 showed single transient decreases of the sarcoplasmic reticular-luminal Cat 0.5s−1 a. Under nditions a. 81% ofmulation b. Transicontrols a; moreovcle DMSO b. Strong[Ca2+]SR , a and bcontrols a, again mulation a. The pe) to 57% b. ImportC to 24% b. At thee the SR a. Taken 2+ signaling by β-adrenergic stimulation translate into protection against arrhythmias in the intact organism, we investigated the consequences of Iso on cardiac electrophysiology with or without administration of BZ194 (180 mg/kg body weight) in awake mice (a). Administration of BZ194 induced a prominent but transient tachycardia that reached its maximum ∼60 min after injection and then slowly faded. BZ194 had no effect on the morphology of the ECG (b) and did not cause arrhythmic events in any animal. Four hours later, all animals received two intraperitoneal injections of Iso (2 mg/kg body weight) within 30 min, and ECGs were recorded for 120 min after the last injection (a). Iso rapidly increased heart rate and induced a variety of arrhythmias, including premature ventricular beats (PVB) (c), salves of PVBs (d), bigeminy (e), and ventricular tachycardia . Ventricular fibrillation was not observed. Each animal developed arrhythmias after Iso; ∼90% of the observed events were PVBs (g and h). DMSO, used as a solvent for BZ194, did not affect the occurrence of any of the arrhythmic events (g and h) evoked by Iso. In contrast, when BZ194 was injected 240 min prior to the first dose of Iso, the frequency of PVBs and bigeminy was reduced by 91 ± 2% (g) and that of salves of PVBs and VTs was reduced by 88 ± 11% (h and i). The number of events observed after combined Iso and BZ194 treatment was not significantly different from controls (i). After completion of the in vivo β-adrenergic provocative testing, all mice were monitored over the following week. All animals, except for those treated with Iso in combination with BZ194, recovered well with no signs of physical or behavioral impairment. In contrast, mice treated with Iso and BZ194 showed a reduced motor activity and an impaired general condition, and three of the six animals died 1–4 days after β-adrenergic provocative testing.To determine whether the observed inhibitory properties of BZ194 on alterations in myocardial Caake mice a. Admini the ECG b and didnjection a. Iso rats (PVB) c, salves of PVBs d, bigemibigeminy e, and verdia VT, f. Ventriere PVBs , g and hc events , g and h 91 ± 2% g and tha88 ± 11% , h and icontrols i. After 2+-releasing messenger known to date, induced Ca2+ signaling in quiescent cardiac myocytes, a process sensitive to antagonists of RyRs and NAADP signaling and to inhibitors of H+-ATPase of acidic stores. Furthermore, SCT observed in the presence of high concentrations of Iso in electrically driven myocytes were sensitive to both inhibition of Ca2+ loading of acidic stores and NAADP antagonism. Spontaneous Ca2+ release events were directly observed by live cell imaging of both [Ca2+]i and [Ca2+]SR. Our data suggest that NAADP-induced Ca2+ release from (micro)domains, e.g. acidic stores, close to SERCA and/or RyRs increases the sensitivity of the RyR. The sensitized Ca2+ release system in turn leads to SCT even in the absence of the regular trigger, Ca2+ entry via L-type Ca2+ channels. The observation that the NAADP antagonist BZ194 largely ameliorated Iso-induced arrhythmias in awake mice provides strong evidence that NAADP-mediated modulation of couplon activity plays a pivotal role for triggering arrhythmias in the intact animal. Importantly, because systolic Ca2+ transients were not affected regarding transient amplitude and only slightly regarding reciprocal time constant, NAADP signaling may become an attractive target for antiarrhythmic therapy.Here, we describe a novel mechanism involved in catecholamine-induced arrhythmias in mouse heart. NAADP, the most potent CaNAADP has been proposed to act via RyRs , 12, 13,2+ releasing activity of NAADP was sensitive to a functional H+ gradient across the membranes of acidic stores. Maneuvers resulting in a breakdown of the H+ gradient also destroyed the secondary gradient of Ca2+ ions leading to unresponsiveness of cells toward NAADP  upon Iso stimulation; importantly, BZ194 prevented these spontaneous diastolic Ca2+ release events , it appears likely that the antiarrhythmic effect of NAADP antagonism resulted from suppression of diastolic Ca2+ release from the SR. As detailed above, the magnitude of Ca2+ diastolic leak from the SR is critically dependent on the sensitivity of RyR2.Corresponding to the inhibitory effect of NAADP antagonism on the development of SCT Unexpectedly, we observed signs of physical and behavioral impairment in mice that had received both Iso and BZ194, and about 50% of these animals died within a few days after treatment. The mechanisms leading to these severe side effects are currently unclear and may be explained by mechanism-related as well as off-target actions of the administered drugs. In preliminary experiments, we had tested the tolerability of an intraperitoneal injection of 180 mg/kg BZ194 in four mice, which all survived. This suggests that drug interactions between Iso and BZ194 might have played a major role in the pathogenesis of the observed high lethality. Clearly, the data indicate that BZ194 itself is unsuitable for treatment in human patients but do not argue against the proposed mechanism of action. In any case, further detailed pharmacological studies on the safety of BZ194 are mandatory.in vitro and a high degree of antagonism of ventricular arrhythmogenic events in vivo by the NAADP antagonist BZ194 suggesting that the NAADP signaling pathway in cardiac myocytes may become suitable as a target for pharmaceutical intervention in cardiac arrhythmias.In summary, a novel pathological role of NAADP in the generation of cellular arrhythmias induced upon strong β-adrenergic stimulation was discovered. We demonstrate full antagonism of SCT in cardiac myocytes


NICR is mediated by type 1 ryanodine receptor (RyR1), a Ca2+ release channel expressed in endoplasmic-reticular membrane. Furthermore, NICR is indicated to be dependent on S-nitrosylation of RyR1, and involved in synaptic plasticity in the cerebellum. In this review, molecular mechanisms and functional significance of NICR, as well as non-enzymatic PTM of proteins by gaseous signals, are described.Nitric oxide (NO) is a typical gaseous messenger involved in a wide range of biological processes. In our classical knowledge, effects of NO are largely achieved by activation of soluble guanylyl cyclase to form cyclic guanosine-3′, 5′-monophosphate. However, emerging evidences have suggested another signaling mechanism mediated by NO: “S-nitrosylation” of target proteins. S-nitrosylation is a covalent addition of an NO group to a cysteine thiol/sulfhydryl (RSH), and categorized into non-enzymatic post-translational modification (PTM) of proteins, contrasted to enzymatic PTM of proteins, such as phosphorylation mediated by various protein kinases. Very recently, we found novel intracellular calcium (Ca Primary structure of proteins obtained from genome analysis is not sufficient to explain their various biological functions: while it is estimated that the human genome, for example, comprises ∼27,000 genes, the total number of proteins in the human proteome is estimated at over one million. In addition to changes at the transcriptional and mRNA levels, it is now increasingly recognized that “post-translational modification (PTM) of proteins” provide important roles in a wide range of signaling pathways, including intercellular signaling pathways such as endocrine systems as well as intracellular pathways. PTMs are covalent processing events that change the properties of a protein by proteolytic cleavage or by addition of a modifying group to one or more amino acids. PTMs of proteins are indicated to be involved in various biological events through changes in protein activity, their cellular locations and dynamic interactions with other proteins , 2.More than 300 different types of PTMs are currently known, and new ones are regularly discovered –7. Consein situ by S-nitrosylation (SNO-proteins) participate in a wide range of biological process including those involved in cellular trafficking  is now well established as a major source of NO bioactivity –12, and fficking , muscle fficking , apoptosfficking , 16, andfficking . In addi2S)  –23.Probably most prevalent is the “S-nitrosylation” by NO. NO is produced enzymatically in cells expressing NO synthase (NOS) . Additio2+ release from sarcoplasmic reticulum (SR) isolated from skeletal and cardiac muscles (S-nitrosocysteine (cysNO), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitrosylated glutathione (GSNO), induced Ca2+ release from the isolated SR vesicles. Correspondingly, application of SNAP increased open probability of SR channels in lipid bilayer -3-ethyl-1-triazene (NOC12) (an NO donor) do not activate or S-nitrosylate RyR2 while GSNO induce activation and S-nitrosylation of RyR2  and endothelial NOS (eNOS), respectively , 28, and muscles . The com bilayer . The effu et al.  showed t bilayer . In thissylation . However of RyR2 . Thus, a channel .2+ release channel was demonstrated. In these studies, NO-sensitivity of the channels had been studied extensively in ambient O2 tension (pO2 ∼150 mmHg), whereas tissue pO2 is ∼10–20 mmHg and even lower in exercising muscle , was activated and S-nitrosylated by submicromolar concentration of NO (2 was at tissue level (∼10 mmHg) but not at ambient level (∼150 mmHg). However, when the concentration of NO was increased to micromolar ranges, the channels were activated by NO even in the ambient O2 levels  under pathological conditions  inputs: climbing fiber, originate from inferior olive, and parallel fiber (PF), axon of cerebellar granule cells . The PF-N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC7), an NO donor, induced Ca2+ elevation in cerebellar PC in acute slice preparation from young-adult (1- to 2-month-old) mice  [inhibitors of sarco/endoplasmic-reticulum Ca2+ ATPase (SERCA)], and dantrolene (a specific inhibitor for RyR1), but insensitive to chelating the extracellular Ca2+ and heparin (a specific inhibitor for IP3Rs) ”  mice . Subsequout mice . Moreove by cGMP . Instead (NICR)” .2+ levels in PCs were transiently but clearly elevated. This BS-induced Ca2+ increase was indicated to be Ca2+ release mediated by RyR1, because the Ca2+ increase was abolished by bath application of thapsigargin, CPA, and dantrolene. Furthermore, BS-induced Ca2+ elevation was inhibited by NG-nitro-l-arginine methyl ester (l-NAME), a broad antagonist of NOSs, and abolished also in nNOS-knockout mice , although the concentration of uric acid (100 μM) and FeTPPS (10 μM) were thought to be high enough. In accordance with the insensitivity of NICR to these reagents, neither uric acid nor FeTPPs impaired the induction of PF-LTP induced by BS. These results do not support the involvement of peroxynitrite in NICR and PF-LTP induction, and NICR is indicated to by induced directly by endogenous NO , or pipette application of heparin inhibited neither NICR nor PF-LTP. Taken together, NICR is revealed to be induced by physiological activity of PF and essential for the induction of PF-LTP, and these observations strongly indicate that Ca2+ release mediated by S-nitrosylation of RyR1 is induced in living cells and has physiological function(s)    is much lower than the ambient level (∼150 mmHg), and the submicromolar levels of NO is indicated to induce S-nitrosylation and activation of RyR1 at the tissue pO2 levels  are observed in various regions of the brain , 60. FurThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.