AD),Parkinson’s disease (PD), amyotrophic lateral sclerosis(ALS), Huntington’s disease (HD), and spinocerebellarataxias (SCA) are very important both for fundamental science and forpractical medicine. Despite extensive research into the causes of these diseases, clinicalresearchers have had very limited progress and, as of now, there is still no cure for any ofthese diseases. One of the main obstacles in the way of creating treatments for these disordersis the fact that their etiology and pathophysiology still remain unclear. This paper reviewsresults that support the so–called “calcium hypothesis of neurodegenerativediseases.” The calcium hypothesis states that the atrophic and degenerative processes inthe neurons of AD, PD, ALS,HD, and SCA patients are accompanied by alterations incalcium homeostasis. Moreover, the calcium hypothesis states that this deregulation of calciumsignaling is one of the early–stage and key processes in the pathogenesis of thesediseases. Based on the results we reviewed, we conclude that the calcium channels and otherproteins involved in the neuronal calcium signaling system are potential drug targets forAD, PD, ALS, HD, andSCA therapy.Neurodegenerative disorders, such as Alzheimer’s disease ( Ca2+ signaling is required for short–term and long–termsynaptic plasticity. Because of its extreme importance, neurons use multiple mechanisms tocontrol intracellular levels of Ca2+, usually within local signaling microdomains.Calcium signaling in neurons connects membrane excitability with the biological function ofthe cell . Since С2+ channels are involved in neuronal Ca2+ signaling: thevoltage–dependant Ca2+ channels of the plasma membrane (VGCC ), NMDAreceptors, AMPA receptors, TRP channels, and depot–controlled channels. Release ofCa2+ from the intracellular ER depot is mediated byinositol–1,4,5–triphosphate receptors (InsP3R) and ryanodine receptors(RyanR). The SERCA pump in the ER, the Ca2+ pump of the plasmamembrane, and the Na+/Ca2+ exchanger of the plasma membrane are involvedin the accurate control of the Ca2+ level in the cytosol in a very narrow range. Themitochondria play a very important role in the formation of cytosolicСа2+ signals. The mitochondrial Ca2+ uniporter(MCU) is an ion channel involved in the rapid and massive entrance of calciuminto the mitochondria. A large number of Ca2+–binding proteins are involved inmaintaining a certain level of Ca2+ in the cytosol  and inside the ER  in neurons.A variety of Ca2+concentration, they have a whole range of Ca2+–dependent structures, includingproteins that are involved in the fusion of synaptic vesicles with the presynaptic membrane(such as synaptotagmins), Ca2+–dependant kinases and phosphatases ,Ca2+–dependent signaling enzymes (such as Ca2+–dependentadenylate cyclase and Ca2+–dependent NO–synthase), andCa2+–dependent transcription factors . Such avariety of Ca2+–dependent elements makes the fineCa2+–dependent regulation of a neural function on time–scales possiblein the microsecond range (as is the case of the Сa2+–dependent fusion ofa synaptic vesicle with the presynaptic membrane), in second and minute ranges (as in the caseof Ca2+–dependent phosphorylation and dephosphorylation), and up to day andyear ranges . TheseCa2+–dependent processes lead to short– and long–term changes inneuronal excitability (by changing the activity of ion channels and the expression pattern) andchanges in synaptic transduction (by modifying the synaptic machinery and forming or disjoiningsynaptic connections). Since neurons are extremely sensitive to changes in Ca2+signaling, even fine defects and deregulation of Ca2+ signaling can have destructiveconsequences [Since neurons are highly sensitive to changes in the intracellular CaAD),Parkinson’s disease (PD), amyotrophic lateral sclerosis(ALS), Huntington’s disease (HD), and spinocerbellarataxias (SCA), are a very important problem both for fundamental science andfor practical medicine. Despite extensive research into the causes of these diseases, clinicalresearchers have had very limited progress and as of now there is still no cure for any ofthese diseases. Therapeutic drugs used for treating these disorders have only a limited effect,causing only temporary relief of the symptoms or slowing the disease’s progression protein. This means that most experimental efforts are directed at lowering mutantHtt expression in the brain (such as by using interfering RNA or a antisenseknockdown).Neurodenerative disorders, such as Alzheimer’s disease  and the γ–secretase inhibitor tarenflurbil (Flurizan)were both unsuccessful. Clinical trials of amyloid–binding monoclonal antibodies(Bapineuzumab) had a very limited or even a negligible effect. The clinical trials ofapproaches for treating HD have problems with devising an adequate approachfor siRNA or antisense RNA delivery into the human brain. There is still no solution for thisproblem, and clinical trials cannot be performed. While the focused attention on amyloid andmutant Htt is understandable, it is worth mentioning that the collected dataindicate that these are targets which are very difficult to affect and that the creation ofsuccessful therapy based on these approaches will take a long time and take up considerableresources. In addition to developing methods for treatment, we propose a treatment that candelay the age at which the symptoms of the disease are manifested and/or lower the degree ofthe disease manifestation. Further, we will focus attention on the concept that the proteinsinvolved in the calcium signaling in neurons are promising targets for developing“disease–delaying” therapy for neurodegenerative pathologies. We surmise thatthe most promising approach for clinical treatment will be a combination of approachesdeveloped for each disease (such as amyloid–directed therapy for AD andhuntingtin–directed therapy for HD) and of Ca2+ blockers.Despite impressive scientific results, these approaches are hard to use in clinical treatment.For instance, in case of 2+–signaling system experiences changes during aging. These datahave been extensively published in the scientific press [2+ signaling has been proposed [2+ concentration viaactive Ca2+ release from the intracellular depot using InsP3R and RyanR,an increased release of Ca2+ through the L–type VGCC, an increase of the slowtrace hyperpolarization caused by the activation of Ca2+–dependent K+channels, a lowered involvement of NMDAR–mediated Ca2+entrance, and a lowered buffer capacity of the cytosol and activation of calcineurin andcalpains. Such changes in the neuronal Ca2+ dynamics lead to increased sensitivity,to the induction of long–term depression, and to the increased threshold frequencyrequired for long–term potentiation in aging neurons [Our neurons are the same age as us. Thus, it is not surprising that the risk ofneurodegenerative diseases increases with age .Comparaproposed . One of neurons . The imp neurons .2+ signalinghave still not been elucidated. One possible scenario is connected with the age–dependentdefects of mitochondrial function caused by the overall oxidative damage sustained by themitochondria. The mitochondria of aged neurons are depolarized and less effective in thecontrol of Ca2+ uptake [2+–signaling proteinswere also discovered [2+–signaling system on various levels.The mechanisms involved in age–dependent changes in neuronal CaHD) is a genetic disorder which is caused by asingle mutation: the expansion of the CAG (polyglutamine) repeat in the huntingtin(Htt) gene [MSN) in the striatum are cellsthat sustain the most damage during HD. Most researchers agree that the mutantprotein Httexp experiences a “gain of its toxicfunction” [2+ signaling is one of the toxic functions of theHttexp protein. Studies of HD patient’sbrains and also model experiments with mice show that the brain experiences sequential changesin the expression levels of Ca2+–signaling proteins [HD”[Httexp on Ca2+ signaling in MSN  gene  Table 1HD is a gunction” . The desproteins . We propins [HD”. There ag in MSN . Our labInsP3R1 . The asscreening . BindingorInsP3 . The keymodel HD , 12, andHD model . Recent nditions . These dreceptor . The incmembrane . Striatuculture , 16.Botffective .Memantipatients , and it  therapy . Riluzolccessful  Table 2HD is a g3R1 and to the NMDA–receptor,Httexp can also affect potential–dependent Ca2+channels (VGCC). Huntingtin directly binds the α2/δ accessory subunit of VGCC [HD–model fruit flies [HD–model mice showed an initial increase of the VGCC channel density,which was followed by a decrease in their density [2+ toxicity mechanisms duringHD are most often mediated by calpain activation and Ca2+accumulation in the mitochondria 3R1 and tHD treatment in the United States in 2008 was adopamine tetrabenzine antagonist (TBZ)  . TBZ is patients . Ourlabnditions . It was ignaling  Fig. 1)HD treatmpatients , which iHD, spinocerbellar ataxias (SCA) areautosomal dominant genetic disorders caused by the expansion of the polyglutamine sequence inataxin proteins (Atx) [2+ signaling can play arole in the pathogenesis of these diseases. Some of these data are presented further.Like in the case of ns (Atx) . There i2+–signaling proteins and Ca2+–binding proteins. A decreasein the Ca2+–binding proteins in Purkinje cells was also observed in patientswith early–stage SCA1 and in mouse models of this disease [2+–signaling proteins such as InsP3R1,Ca2+–channel TR PС3, and the ER pump SERCA2 during theearly stages of the disease [SCA1 leads to the degeneration of Purkinje cells of the cerebellum caused by the expansion ofCAG repeats in the cytosol/nuclear protein ataxin–1 . Purkinj disease . Crossin disease . The tra disease . Albeit 2+ signaling plays a very important role in the pathogenesis of this disease[3R1 similarly to the mutantform of Htt (article in print). We also demonstrated that inhibitors ofCa2+ signaling protected Purkinje cells from apoptosis during SCA2 under invitro conditions and had a pronounced positive effect in experiments on transgenicmice (article in print).During SCA2, the Purkinje cells of the cerebellum experience degeneration due to the expansionof CAG repeats in the cytosolic protein ataxin–2 . The gen disease. Our lab substantia nigra  and the pontine nucleiexperience degeneration as a result of the CAG repeat expansion in the ataxin–3 cytosolicprotein [3R1 similarly to how it binds the mutant form of theHtt protein [2+ stabilizer dantrolen facilitated the age–dependent deficit of motorcoordination in these mice and prevents the loss of neurons in the  substantia nigra and the pontine nuclei [During SCA3, neurons of the protein . Calpainprotein . Recentl protein . It was e nuclei .2+ channel) [2+channel in an expression system [2+ flow throughthe P/Q–type channels in dendrites [2+–signaling deregulation during SCA6 stillremains unresolved. Anomalous neuronal Ca2+ signaling is not limited to ataxias withexpanded polyglutamine repeats, but it can also play an important role in the ataxias of othertypes. The most recent genetic studies have shown that the cause of SCA15 is the loss of afragment of the InsP3R1 gene [SCA6 causes Purkinje cells of the cerebellum to degenerate as a result of the expansion of CAGrepeats in the C–terminus of the CaV2.1 (the pore–forming subunit of theP/Q–type Cachannel) . It wasn system . Howeverendrites . Thus, t3R1 gene .AD) is a neurodegenerative disorder which causesmemory loss. In most cases, AD appears sporadically and the first symptomsemerge in the elderly (after 60). A small fraction of cases (heritable AD(HAD)) are characterized by the early onset of symptoms and geneticinheritance.Alzheimer’s disease  should have some positive effect onthese disorders. Drugs which are targeted at the mitochondrial permeability transition pore(mPTP) should be extremely useful as a “last line of defence” forthe neuron delaying of the onset of neuronal dysfunction and cell death.The mitochondria in neurons of 2+ signaling and seems to be one of thefactors involved in pathogenesis during sporadic AD. Thus, it is expected thatblockers of Ca2+ signaling can have a positive effect on this disease. TheNMDA–receptor antagonist Memantin has demonstrated a certain degree of clinicalefficiency in AD treatment. The treatment of this disease requires thedevelopment and clinical trial of new Ca2+–signaling blockers by themselvesand as part of a complex therapy in combination with mitochondrial stabilizers and withmPTP inhibitors.The aging process affects neuronal CaAD pathogenesis.  The calcium hypothesis of The central idea forexplaining AD pathology is currently the amyloid hypothesis, which states thatthe main reason for neuron death and the decreased number of synapses during this disorder isthe increased production of Aβ42 amyloid peptide (or the increased Aβ42/40 ratio)[0 ratio).AD patients; (2) the heritable formof AD (HAD) is caused by nonsense–mutations in theβ–amyloid Aβ precursor protein (АРР); and (3)HAD is also caused by nonsense mutations in presenilins, which form thecatalytic subunit of γ–secretase, an enzyme that cleaves APP. Currently,amyloid–directed therapy is the central strategy in developing drugs forAD therapy. Recent clinical trials have shown that targets other than amyloidneed to be found in order to create an effective therapeutic solution for the treatment ofAD [2+ homeostasis in neurons plays a significantrole in AD pathogenesis. The data in favor of the calcium hypothesis ofAD development have been actively discussed in recent years [AD pathogenesis and Ca2+ is based on data whichstate that Aβ oligomers can form Ca2+–permeable channels in membranes[PtdS)[PtdS in neurons and thus facilitate the Aβ–mediatedformation of pores, the uptake of Ca2+, and cell death  amyloidplaques are accumulated in the brains of nt ofAD . A largent years . This hyembranes. The abis[PtdS), which oll death . In facttoxicity. The abiAPP mice . The autrphology . The morrphology . Based oreceptor , 47 2+ signald memory , 63 , the SERCA pump,calcineurin, and the mitochondrial Ca2+ regulation system.In conclusion, we must note that a large mass of experimental data demonstrate excessivelevels of CaonsetAD  (howeveronsetAD ). The pr targets. Thus, tgets[AD .There id EVT103 . An L–tyd EVT103 . Other p2+ hypothesis creates a basis for the development of a new class of drugs.The presented data constitute a new view on the therapy of neurodegenerative pathologies. Ourproposed CaMitochondrial stabilizers and antidepressants. Ketasyn, Creatine, coenzyme Q10(CoQ10), and MitoQ have all passed clinical trials for the therapy of AD andHD. Since mitochondria play a key role in the pathogenesis of these diseases[diseases, these cdiseases.Dimebon. Dimebon (Medivation Inc) showed promising results (based on cognitivetests performed on patients) in the second phase of AD–therapy clinicaltrials [HD therapy and demonstrated a weak effect on the brainactivity of patients ltrials . DimebonAD [AD werecompleted and deemed to be a complete failure .Currently, it is unclear whether Dimebon will be studied further as a treatment forAD and HD.Dimebon is a well known antihistaminic drug used throughout the world and in Russia which,according to reports, had a neuroprotective effect when used in picomolar concentrations via anew effect on the mitochondria . HoweverAD  wasprobAD . In MarcAntagonists of the NMDA–receptor. Memantine is noncompetitive antagonistof the NMDA–receptor which has been approved by the FDA for the treatment ofAD. Memantine is also in a clinical trial for the treatment ofHD. NR 2B–specific antagonists EVT101 and EVT103 (Evotec Inc) have beendeveloped for the treatment of AD and they are expected to be tested in secondstage clinical trials soon. The same drugs are also promising therapeutic compounds for thetreatment of HD.Riluzole. An antiglutamate agent which has been approved by the FDA for thetreatment of ALS, Riluzole has also competed third–stage clinical trialsfor the treatment of HD; however, it did not exhibit any reliable positiveeffect on the motor measurements performed on patients [patients .Antagonists of L–type VGCC. An “CNS–optimized”inhibitor of L–type VGCC MEM–1003  showed a moderatepositive effect in second stage clinical trials on AD patients.In conclusion, we must acknowledge that research in new directions of brain studies usingmodern molecular–biological and electrophysiological approaches will inevitably lead toan elucidation of the mechanisms behind highly effective informational flow and will also helpdiscover approaches for treating neurodegeneration.


Methods for storing and sharing biological models tend to focus on directly encoding a model and its equations. However, many models share the same equations, subject only to differences in parameter values or the number of instances of particular processes. We have therefore developed a mechanism within NeuroML to expreThe resulting system, known as LEMS (Low Entropy Model Specification), makes it possible to retro-fit most of the existing NeuroML version 1 definitions with domain independent definitions constructed from LEMS elements. This preserves the benefits of the existing semantically rich high-level concepts in NeuroML while providing the ability to add new concepts at the user level rather than requiring changes to the language. Simulator developers have a choice between directly supporting the library of core types, or supporting the underlying LEMS definitions. These options are not exclusive: a tool could provide hard-coded support for certain key components such as ion channels, and fall back to a generic (and probably less efficient) implementation for new component definitions.Component type definitions in LEMS consist of three main parts: a declaration of the static properties needed to define a particular type of model; specification of the dynamics of the component; and specification of the component's structure. The static definition includes parameters types, component nesting, and references between components. The dynamics contains the equations that govern the behavior of an instance of the model. The structure definition specifies how the static properties should be interpreted when a component is used. This may be as simple as creating a single instance of each of the state variables, but may also involve instantiating other components or connecting them together as in, for example, component types that express network structure or connectivity.As well as an extensive library of component types, and example models, NeuroML2/LEMS contains a reference interpreter that can run any LEMS model. Although initial development has been driven by the need to support the requirements of the NeuroML community, the resulting language only includes mathematical and structural elements and should be applicable to other model specification tasks.


Understanding the mechanisms of neuronal dysfunction and death represents a major frontier in contemporary medicine, involving the acute cell death in stroke, and the attrition of the major neurodegenerative diseases, including Parkinson's, Alzheimer's, Huntington's and Motoneuron diseases. A growing body of evidence implicates mitochondrial dysfunction as a key step in the pathogenesis of all these diseases, with the promise that mitochondrial processes represent valuable potential therapeutic targets. Each disease is characterised by the loss of a specific vulnerable population of cells—dopaminergic neurons in Parkinson's disease, spinal motoneurons in Motoneuron disease, for example. We discuss the possible roles of cell type-specific calcium signalling mechanisms in defining the pathological phenotype of each of these major diseases and review central mechanisms of calcium-dependent mitochondrial-mediated cell death. Local changes in [Ca2+]c are the very stuff of our thoughts and sensations, our memories and dreams. Neuronal energy supplies are meanwhile entirely predicated on mitochondrial oxidative phosphorylation—neurons are almost exclusively dependent on mitochondrial ATP generation and have almost no capacity to upregulate energy supply through glycolysis when oxidative phosphorylation is compromised c signal is used as the major medium of a continuous subtle dialogue between the cytosol and its mitochondrial population that regulates energy homeostasis. It is the [Ca2+]c signal that signals to mitochondria that more energy is required, and the [Ca2+]c signal that mediates the mechanisms that deliver increased energy. [Ca2+]c signals even tell mitochondria where to go in the labyrinthine maze of a complex neuron.The fine spatial and temporal organisation of intracellular calcium signals is fundamental to function in the CNS, perhaps more than in any other tissue. Signals are conveyed throughout the CNS by local changes in calcium concentration ) and Alzheimer's disease. These latter three diseases are unusual in this respect, as almost identical disease phenotypes are seen both as sporadic forms for which risk increases progressively with age, and as heritable familial forms, in which mutations of specific genes lead to almost identical disease phenotypes that usually occur in a younger age group of patients. The evidence for a mitochondrial pathophysiology is stronger in some of these diseases than in others, and a major challenge remains to define the processes that are primary mechanisms driving the pathophysiology, those which are contributors on the pathways to cell injury caused by other primary processes, and those that are epiphenomena, and ultimately to identify potentially useful therapeutic targets. These diseases are of huge importance as a major frontier in medical research, a major challenge to the pharmaceutical industry, and an enormous and terrifying threat to us all.2+ handling in neurons in the pathophysiology of these horrible and disabling diseases, with a focus on Ca2+-mediated cell death as a most extreme form of mitopathy that seems potentially tractable to therapeutic intervention, and that seems also to represent a ubiquitous form of cell injury that is recapitulated in many different disease contexts.In this short essay, I will explore in particular the role of mitochondrial CaOne of the abiding and critical unanswered questions in all the major neurodegenerative diseases is the basis for the selectivity and specificity of the cellular targets of degeneration: the death of motoneurons in ALS, of Purkinje neurons in the cerebellar ataxias, of dopaminergic neurons of the substantia nigra in Parkinson's disease (PD) and of retinal ganglion cells in optic atrophy and Leber's hereditary optic neuropathy. This is most difficult in those genetic diseases for which mutations of proteins have been identified, as, in all of these, the mutant proteins are ubiquitously expressed while only the target cells are affected. This signals a search for mechanisms or features of the target cells that makes them especially vulnerable to the effects of the mutation, but it has to be said that the questions remain in the most part unsatisfactorily answered if answered at all.2+ signalling—the overall theme of this special issue. This theme is perfectly exemplified in the CNS, where there is a striking diversity in the [Ca2+]c signalling pathways employed by different cell populations. Indeed, it seems very plausible that the differences in expression of specific elements in the [Ca2+]c-signalling toolkit may be, at least in part, responsible for the cell type specificity of injury seen in most of the neurodegenerative diseases.There is growing evidence that the answer, at least in some cases, may be embedded in the cell-specific aspects of mitochondrial Ca2+ influx through voltage- and ligand-gated ion channels, with smaller modulations by intracellular signalling pathways, while [Ca2+]c signalling in glial cells is dominated by Ca2+ release from ER using IP3 as a second messenger system. This very crude classification leads to further sub-specialisations, depending on the classes of ion channels expressed by the neurons, neuronal architecture, cell size, Ca2+ buffering power and patterns of excitability. Thus, the specific Ca2+ signalling physiology of a cerebellar Purkinje cell is radically different from that of the neighbouring cerebellar granule cell, although both are ‘neurons’, reflecting differences in size and complexity and the requirement for local amplifying signalling mechanisms. Spinal motoneurons differ in many respects from many other cell types in their large volume, their enormously long axons and in their expression of Ca2+ permeant ‘AMPA’ subclass of glutamate receptors c interacts with the outer mitochondrial protein Miro, inhibiting trafficking through the kinesin motor KIF5, so that mitochondria stop where they are, and remain localized at areas where [Ca2+]c is high—presumably the places where energy demand will be highest and where local Ca2+ buffering is also likely to be important c as the ion enters through the Ca2+-permeant NMDA channels, generating NO which in combination with the [Ca2+]c signal is toxic to the cells. Importantly, cells could be rescued by nNOS inhibition or by decoupling the nNOS from the NMDA receptor.A decisive element in the neuronal signalling machinery is the expression of the glutamate receptor families. Of these, two are highly Catoxicity . Not onls lethal . His gros lethal . The Ca2This process is probably not an experimental oddity of neurons in tissue culture but is thought to occur in the brain under pathological conditions. The clearest role is in ischaemic disease in the CNS—in stroke for example—where glutamate can rapidly accumulate in the extracellular space to toxic concentrations of hundreds of micromolar. It is believed that this mechanism is likely responsible for the progression of a stroke over the first few days after an ischaemic episode, extending the areas of brain injury beyond the core ischaemic zone into a penumbra. What completed the circle very nicely was the demonstration by Tymianski and colleagues that decoupling nNOS from the NMDA receptors using small peptides rendered membrane permeant with the HIV TAT sequence rescued CNS tissue in an in vivo stroke model .2+ loads and mitochondrial Ca2+-dependent injury, whilst activation of synaptic receptors tends to cause smaller Ca2+ elevations that play a prosurvival role c-dependent toxicity is the ‘delayed deregulation of [Ca2+]c homeostasis’ —a secondary rise in [Ca2+]c that can be delayed by anything from 1 or 2 to 10 min after the initial exposure to glutamate  c loads by NO donors c rises so high that the cytosolic component overwhelms the mitochondrial specific signal, which is probably saturated anyway. It has been shown that brief exposure to uncoupler around the time of glutamate application can be protective [2+ uptake. Even though the change in [Ca2+]c was if anything enhanced, cell viability was improved, arguing that mitochondrial Ca2+ uptake plays a critical role in driving cell death. This is a tricky experiment as uncouplers can only too easily kill the cells, and so the timing is critical.Key questions then are what are the mechanisms of DCD and of loss of mitochondrial membrane potential? Once mitochondrial potential is lost in a neuron, that cell is destined to die as neurons have almost no capacity to increase glycolytic ATP once oxidative phosphorylation is lost , and so otective . The log2+ uniporter using Ru360 prevented the loss of mitochondrial membrane potential and decoupled DCD and mitochondrial depolarization, as DCD proceeded even though mitochondrial potential was preserved [2+ overload as a determinant of mitochondrial dysfunction . It wou2+]m and dependent on nitrosative stress immediately flags involvement of the mitochondrial permeability transition pore (mPTP) for any mitochondrial biologist. Therefore it is timely to introduce the permeability transition pore into this text. This is an extraordinary phenomenon, which seems to underlie Ca2+-dependent mitochondrial cell death in most tissues c, probably related to ER disruption (VAPB is an ER protein). The local elevation of the [Ca2+]c signal regulates Miro1-mediated axonal mitochondrial transport (see above) leading to impaired mitochondrial trafficking and distribution. It is not clear why this defect should selectively affect motoneurons, nor why it should lead ultimately to motoneuron degeneration.There is also substantial evidence pointing to a disturbance in mitochondrial axonal transport in fALS. Thus, the VAPB mutation also seems to target CaA mutant . The VAP2+ signalling pathways in different cell types may play a major role in shaping the patterns of cell loss in each of the major neurodegenerative diseases. Understanding these pathways brings the promise of defining new potential therapeutic targets for these dreadful diseases.Mitochondrial dysfunction is clearly implicated in the pathogenesis of the major neurodegenerative diseases and plays a critical role as a determinant of irreversible cell injury following stroke. Increasing evidence suggests that specialisations of Ca


In a proof-of-principle study, we imaged isolated Langendorff-perfused mouse hearts with reversible regional myocardial infarctions.We present an easily implementable method for measuring Fura-2 fluorescence from isolated mouse hearts using a commercially available switching light source and CCD camera. After calibration, it provides a good estimate of intracellular [CaIsolated mouse hearts were perfused in the Landendorff-mode and loaded with Fura-2. Hearts were then paced rapidly and subjected to 15 minutes of regional ischemia by ligation of the left anterior descending coronary artery, following which the ligation was removed to allow reperfusion for 15 minutes. Fura-2 fluorescence was recorded at regular intervals using a high-speed CCD camera. The two wavelengths of excitation light were interleaved at a rate of 1 KHz with a computer controlled switching light source to illuminate the heart.2+ transients from different hearts. Ligating the coronary artery rapidly generated a well defined region with a dramatic rise in diastolic Ca2+ without a significant change in transient amplitude; Ca2+ handling normalized during reperfusion. Conduction velocity was reduced by around 50% during ischemia, and did not recover significantly when monitored for 15 minutes following reperfusion.Fura-2 produced consistent Ca2+ levels in cardiac tissue. The persistent change in the conduction velocities indicates that changes to tissue connectivity rather than altered intracellular Ca2+ handling may be underlying the electrical instabilities commonly seen in patients following a myocardial infarction.Our method of imaging Fura-2 from isolated whole hearts is capable of detecting pathological changes in intracellular Ca Coronary heart disease affects approximately 1 in 8 adults and causes 1 in 6 deaths in the United States ,3. Patie+-K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase (SERCA) to directly change intracellular Ca2+ homeostasis and the homeostasis of other ions including Na+ and K+ state . Low inta+ and K+,7. Slowiular Ca2+.2+ homeostasis has various effects as Ca2+ is arguably the most important ion in cardiac tissue  from Langendorff-perfused mouse hearts with high spatial and temporal resolution using the ratiometric dye Fura-2. A switching light source is used to alternately excite the dye at two wavelengths and a CCD camera is synchronized to record the resulting fluorescence. Timing between the excitation source and CCD camera, critical for calculating ratios, is handled by a computer-controlled hardware timer. Our method allows monitoring of fluorescence from 1600 discreet spatial points while switching illumination wavelengths at an effective rate of 1000 Hz, significantly improving on current ratiometric imaging systems without using custom hardware. This spatiotemporal resolution is sufficient to determine the speed and patterns of conduction on the heart’s surface. If two cameras are available, the system described here could be easily modified to allow concurrent ratiometric Ca2+ measurements along with pH or transmembrane potential.In this study we present a simple quantitative method for imaging intracellular  were generated by determining the minima of the average of 4 transients taken from each point on the surface of the heart. These minimas were stored in an array to form a map of diastolic [Ca2+] over the heart.Maps of diastolic  dyes, taking the ratio of Fura-2 fluorescence at each pixel produces a homogeneous emission across the surface of the heart. This corrects the misleading heterogeneity in fluorescence seen while using single-wavelength dyes and permits the study of pathological spatial heterogeneities in mouse hearts. Furthermore, whereas leakage and photobleaching of single-wavelength dyes can cause temporal changes to emission intensity, the ratio of Fura-2 emission stays constant regardless of intracellular concentration.The acetoxymethylester (AM) form of Fura-2 can be loaded into Langendorff-perfused mouse hearts using a 25 minute loading protocol. Whereas correcting for the heterogeneous uptake of dye in the heart is difficult when using single-wavelength , but the damage caused cannot be reversed by simply lowering [Ca2+]. Prolonged slowing of CV would render the myocardium pro-arrhythmic and potentially contributes the increased incidence of arrhythmias seen in patients following heart attacks. However, as we have demonstrated here, correcting for the changing kd – necessary in ischemic preparations – is difficult in intact tissue. We believe that our method would be best utilized in settings where tissue parameters to not change, and the Fura-2 ratios can be directly compared.In this study we have imaged the spatial extent and effects of ischemia-reperfusion injury in hearts with LAD ligations. Ligation of the LAD, similar to a heart attack, produces a region of ischemic myocardium. Ischemia causes a rapid significant rise in diastolic Ca2+ cycling. Secondary effects may involve both changes to tissue properties and Ca2+ cycling. Based on the trends seen in our experiments  for Fura-2; SERCA: Sarcoplasmic Reticulum Ca2+-ATPase; SR: Sarcoplasmic Reticulum; Vm: Transmembrane voltage.The authors declare that they have no competing interests.RV: Participated in designing the study. Performed surgeries and experiments. Developed data analysis techniques and analyzed data. Made figures and drafted the manuscript. MH: Implemented the timing scheme used to synchronize camera and light source. RH: Developed hardware to measure light source-camera sync and participated in editing the manuscript. BCK: Contributed to the protocol to calibrate the ratiometric dye Fura-2. FB: Conceived of and designed the study. Developed data analysis techniques. Participated in drafting the manuscript and final edits. All authors read and approved the final manuscript.


RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca2+]i rise in iPSC-ECs from normal individuals but a sustained [Ca2+]i elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red , and a specific TRPV2 channel inhibitor, tranilast (100 µM), abolished the sustained phase of hypotonicity-induced [Ca2+]i rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca2+]i rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca2+]i elevation in HGPS-iPSC-ECs under hypotonicity, consequently resulting in apoptotic cell death. This mechanism may contribute to the pathogenesis of vascular diseases in HGPS patients.Hutchinson-Gillford Progeria Syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP) channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90) iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca Mechano-activation of these TRP channels results in Ca2+ influx. Excessive Ca2+ influx or Ca2+ overload are known to contribute to apoptotic and necrotic vascular cell death Transient receptor potential (TRP) channels are nonselective cation channels that comprise of six subfamilies: TRPC , TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), TRPA (ankyrin) and TRPV (vanilloid) Our recent studies showed that human induced pluripotent stem cell (iPSC) could be derived from normal subjects as well as from patients with HGPS 2+]i change. 2+]i in the iPSC-ECs derived from normal individuals (IMR90-iPSC-ECs) and HGPS patients (HGPS-iPSC-ECs), respectively. Upon perfusion with hypotonic solution (210 mOsm), [Ca2+]i increased rapidly and peaked within 54±7 sec in IMR90-iPSC-ECs and 67±4 sec in HGPS-iPSC-ECs. Subsequently, elevated [Ca2+]i gradually reverted back to its resting level in IMR90-iPSC-ECs with F1/F0 value of 1.0±0.1 (n  =  6). In contrast, [Ca2+]i elevation in HGPS-iPSC-ECs sustained with the F1/F0 value maintained at ∼3.0 over 10 min (n  =  7)  only induced transient [Ca2+]i rise in both cell types. The magnitude of transient [Ca2+]i response to ATP was slightly higher in HGPS-iPSC-ECs than that in IMR90-iPSC-ECs  . In the ll types , suggestiPSC-ECs . [Ca2+]iiPSC-ECs . Howeverp<0.05) than in IMR90-iPSC-ECs (n  =  5)  in both cell types . In partn  =  5) . The spen  =  5) , similarn  =  5) .2+]i influx, a nonselective TRPV inhibitor ruthenium red (RuR) and a TRPV2-specific inhibitor tranilast were used. RuR (20 µM) completely abolished the sustained phase of hypotonicity-induced [Ca2+]i rise in HGPS-iPSC-ECs  and 56%  in IMR90-iPSC-ECs and HGPS-iPSC-ECs, respectively  The sustained phase of hypotonicity-induced [Ca2+]i rise was completely abrogated by tranilast or RuR in iPSC-ECs from HGPS patients. Furthermore, the transient phase of hypotonicity-induced [Ca2+]i rise was partially inhibited by RuR or tranilast. 4) Hypotonicity treatment for 10 min caused substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in iPSC-ECs from normal individuals. Tranilast could inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data strongly suggested that TRPV2 was a major mechanosensitive channel that contributed to hypotonicity-induced [Ca2+]i rise in iPSC-ECs. More importantly, this channel was exclusively responsible for sustained Ca2+ influx under hypotonicity and contributed to apoptotic cell death in HGPS-iPSC-ECs.Major findings of the present study are as follows: 1) iPSC-ECs from HGPS patients expressed much higher levels of TRPV2 mRNAs and proteins compared to iPSC-ECs from normal individuals. 2) Hypotonicity induced a transient i elevation in HGPS-iPSC-ECs. The results showed that both RuR and tranilast could abolish the sustained phase of hypotonicity-induced [Ca2+]i rise, suggesting that TRPV2 was the sole component in mediating sustained Ca2+ entry in response to hypotonicity in these cells. Both RuR and tranilast could also substantially reduce the transient phase of hypotonicity-induced [Ca2+]i rise by 50–60% in both cell types, indicating that TRPV2 also contributed significantly to the transient phase of [Ca2+]i rise. The residual component (∼40%) in the transient phase of [Ca2+]i rise after RuR or tranilast treatment might be mediated by other mechanosensitive Ca2+-permeable channels, such as TRPC1, TRPV4, TRPM7 and TRPP2. Our data agreed with several previous reports, which showed TRPV2 being a hypotonocity-activated channel in other cell types, including murine aortic smooth muscle It has been well documented that most TRP channels are Ca2+ entry, leading to Ca2+ overload and subsequent vascular cell death. Indeed, our data clearly showed that TRPV2 mediated the hypotonicity-induced activation of caspase 8, an index for apoptosis, in iPSC-ECs from HGPS patients. These data were in agreement with the generally accepted notion that Ca2+ overload could result in cell death 2+ overloading was previously reported to contribute to muscular dystrophy and/or cardiomyopathy in humans and animal models Our findings may have significant pathological significance. HGPS cells displayed an increase in mechanosensitivity 2+]i elevation under mechanical stress in iPSC-ECs from HGPS patients. This could be an underlying reason for mechanical stress-induced vascular cell death in HGPS patients.In conclusion, we found that an upregulation in TRPV2 expression is responsible for the sustained i measurement was performed in NPSS, isotonic solution, or hypotonic solution. NPSS contained in mM: 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 10 HEPES, pH 7.4. Isotonic solution contained in mM: 65 Na-aspartate, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, 140 mannitol, pH 7.4 with NaOH, ∼300 mOsm. Hypotonic solution contained in mM: 65 Na-aspartate, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4 with NaOH, ∼210 mOsm. Fluo-4 fluorescence was recorded and analyzed by FV1000 laser scanning confocal imaging system. [Ca2+]i response was expressed as a ratio of real-time fluorescence  relative to the intensity at the beginning of the experiment (F0), namely F1/F0. The peak magnitude of the transient phase of hypotonicity-induced [Ca2+]i rise was measured from the onset of hypotonic stimulation to the peak of [Ca2+]i rise. The magnitude of sustain phase of [Ca2+]i rise was measured at 400 sec after hypotonicity treatment. Fura-2 dye was used to measure basal [Ca2+]i level. The Ca2+-bound and -unbound Fura-2 fluorescence signals were measured by dual excitation wavelengths at 340 and 380 nm using InCyt Basic Fluorescence Imaging System . Resultant fluorescence signals were collected at 510 nm and was used to calculate [Ca2+]i as described by Grynkiewicz et al. 2+ concentration standard with precisely known Ca2+ concentration . Rmin and Rmax for Fura-2 were obtained by permeabilizing the cells with 10 µM ionomycin in the presence of 5 mM EGTA and 5 mM CaCl2, respectively The cells were loaded with 5 µM Fluo-4/AM or 10 µM Fura-2/AM in the presence of 0.02% pluronic F-12 for 1 hr, and maintained in normal physiological saline solution (NPSS). [CaThe cells were seeded on 96-well plate  coated with Matrigel™  with mTeSR™ medium . Cells were grown for 48 hrs and then were incubated with hypotonic or isotonic solution in the presence or absence of tranilast (100 µM) for 10 minutes. Cells were then washed and incubated with culture medium for 1, 6, or 24 hr to determine the optimal time that was needed for apoptosis to develop, following by luminescent-based caspase 8 assay . We found that 24 hr was the optical time for caspase 8 activity to increase. Thus, all experiments were performed with 24 hr incubation.t-test and differences were considered significant at *P<0.05.Data were expressed as mean ± SE. Statistics was performed using Prisms 4.0 software . Statistical significances were determined by the unpaired student


Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC). PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK), a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs) in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in concert with antioxidants, MMP inhibitors and cytokine antagonists to reduce VSM hyperactivity in certain forms of HTN that do not respond to Ca2+ channel blockers.Blood pressure (BP) is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM) and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN). In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca Hypertension (HTN) is a major cardiovascular and renal disease affecting a large proportion of the population in the Western World. Several factors contribute to increased blood pressure (BP) including neuronal, hormonal, renal and vascular mechanisms. Understanding the physiological mechanisms that control BP would help define the pathological changes in HTN, and help design specific approaches to manage the increases in BP and HTN.Blood vessels play a major role in the control of vascular tone. The blood vessel wall has three layers; the tunica intima made of a single layer of endothelial cells (ECs), the tunica media made of several layers of vascular smooth muscle cells (VSMCs), and the adventitia made of fibroblasts, connective tissue and extracellular matrix (ECM). ECs release vasodilator and vasoconstrictor mediators that control the vessel diameter. The ability of VSMCs to contract and relax plays an important role in the regulation of the vessel diameter and blood flow to various tissues and organs. The ECM proteins provide structural integrity to the vessel wall and are regulated by proteolytic enzymes such as matrix metalloproteinases (MMPs).This review will focus on how vasoconstrictor agonists affect the mechanisms of VSM contraction particularly protein kinase C (PKC), and the changes in these mechanisms in vascular disease such as HTN. The role of PKC in the regulation of EC function and ECM will be briefly discussed. In addition to changes in vasoactive factors, changes in reactive oxygen species (ROS) ,2,3, MMP2+ is a major determinant of VSM contraction. VSM activation by physiological or pharmacological agonist triggers an increase in intracellular free Ca2+ concentration ([Ca2+]i) due to Ca2+ release from the sarcoplasmic reticulum and Ca2+ influx from the extracellular space through plasma membrane Ca2+ channels. Four Ca2+ ions bind to calmodulin (CAM) to form a Ca2+-CAM complex, which activates myosin light chain (MLC) kinase, and in turn causes the phosphorylation of the 20 kDa MLC, stimulates actin-myosin interaction and promotes VSM contraction  and the Na+-Ca2+ exchanger, as well as Ca2+ reuptake via the sarcoplasmic reticulum Ca2+ pump (SERCA). The decrease in [Ca2+]i allows the dissociation of the Ca2+-CAM complex, and the remaining phosphorylated MLC is dephosphorylated by MLC phosphatase, leading to detachment of actin-myosin crossbridges and VSM relaxation i (nanomolar), the opening of VGCCs facilitates Ca2+ influx, and leads to MLC phosphorylation and VSM contraction. In contrast with membrane depolarization, physiological agonists such as norepinephrine, prostaglandin F2α and thromboxane A2 activate other intracellular signaling pathways in addition to Ca2+ channels. In VSM, the interaction of an agonist with its specific receptor causes activation of phospholipase C (PLC), and promotes the hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) i and VSM contraction. Agonists also activate receptor-operated (ROCs) and store-operated Ca2+ channels (SOCs), causing maintained Ca2+ influx, increased [Ca2+]i, MLC phosphorylation and VSM contraction  i ,22,23,24 C (PKC) ,18.2+-activated phospholipid-dependent protein kinase i i . PKC maynization ,57.2+-dependent actin-myosin interaction and VSM contraction  for its translocation to the plasma membrane, thus allowing a spatially confined graded response to external stimuli and plasmalemmal localization of PKC I, and a role of Ca2+-independent ε-PKC has been suggested i could exert trophic effects in both the vasculature and the heart, leading to narrowing of the arterial lumen and cardiac hypertrophy in long-standing HTN [2+-independent ε-PKC may enhance the myofilament force sensitivity to [Ca2+]i in VSM and promote the vasoconstriction associated with HTN PDBu) binding and PKC concentration in both the cytosolic and membrane fractions i and PKC activity. Polymorphisms in human tyrosine hydroxylase gene have been associated with increased sympathetic activity, norepinephrine release and HTN i and enhanced VSM contraction. Other vasoactive factors such as soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) may be released during reduction of uteroplacental perfusion pressure [Experimental studies have suggested that reduction in uteroplacental perfusion pressure and the ensuing placental ischemia or hypoxia during late pregnancy may increase the release of cytokines into the maternal circulation, which in turn cause generalized vascular changes and HTN ,169,170.pressure ,177 and in vivo effects of PKC inhibitors have not been fully examined. Dahl-salt-sensitive rats on high NaCl (8%) diet exhibit an increase in BP, excretion of the endogenous inhibitor of α1 Na/K-ATPase marinobufagenin, left ventricular weight, and myocardial Na/K-ATPase and βII-PKC and δ-PKC. Treatment of Dahl-salt rats with cicletanine causes reduction in BP and left ventricular weight, decreased sensitivity of Na/K-ATPase to marinobufagenin, no increase in βII-PKC, and reduced phorbol diacetate-induced Na/K-ATPase phosphorylation. The cicletanine-induced decrease in BP may be due to targeting of PKC-induced phosphorylation of cardiac α1 Na/K-ATPase [The effects of PKC inhibitors on VSM contraction has been examined in isolated blood vessels, but the K-ATPase . In isolK-ATPase .2+ channel blockers on vasoconstriction. Targeting Ca2+-independent PKCs could be beneficial in Ca2+ antagonist-resistant forms of HTN. The effects of PKC inhibitors in reducing vasoconstriction and BP could also be potentiated by Rho-kinase and MAPK inhibitors. RhoA/Rho-kinase causes inhibition of MLC phosphatase and thereby enhances Ca2+-MLC kinase dependent VSM contraction, and may play a role in the development and progression of HTN [2+ antagonist-insensitive forms of HTN.It is important to note that HTN is a multifactorial disease, and PKC inhibitors alone may not be sufficient to manage HTN. However, PKC inhibitors may decrease the VSM growth and hyperactivity associated with HTN particularly when used with other therapeutic modalities. PKC inhibitors could potentiate the inhibitory effects of Can of HTN ,179. Thein vivo and in animal models of HTN with other co-morbidities such as hypercholesterolemia and diabetes. Although the first generation of PKC inhibitors may not be very selective, newly-developed PKC inhibitors are more specific, and further experimental studies and clinical trials are needed before these compounds can be used safely in human. Certain PKC inhibitors such as ruboxistaurin (LY333531), a selective β-PKC inhibitor, have shown promise in clinical trials for diabetic retinopathy, macular edema and microvascular complications [Upregulation of PKC could play a role not only in vascular disease such as HTN and atherogenesis, but also in metabolic disorders, insulin resistance and cancer in what has been termed as the “PKC syndrome” . Therefoications ,182,183.


Predominantly neutrophilic inflammatory infiltrates are seen in a subset of chronic urticaria patients, with lesions that tend to be less itchy and poorly responsive to antihistamine therapy.,2 There Patient 1: A 47-year old female presented with urticaria and associated night sweats, fevers and polyarticular arthritis. Acute phase reactants were elevated with worsening of symptoms. Initial treatment with topical and systemic corticosteroids, antihistamines, and immunosuppressants was unsuccessful. 100% clinical resolution was achieved with anakinra, an Il-1 receptor antagonist. Patient 2: A 26-year old female presented with urticaria and associated joint pain and swelling. Initial treatment included antihistamines, colchicine, and dapsone. Only colchicine provided moderate benefit, but was stopped due to significant GI-discomfort. Anakinra was initiated; the patient now has 100% control on daily therapy.The cases described in this report represent a multi-systemic inflammatory entity: neutrophilic urticaria with systemic inflammation (NUSI). In NUSI, there is abrupt onset of neutrophilic urticaria occurring in tandem with inflammatory arthritis and other systemic symptoms, and complete control of symptoms is achieved with IL-1 blockade. NUSI likely lies on a spectrum with clinically similar autoinflammatory diseases, with IL-1 dysregulation and subsequent neutrophil-driven inflammation leading to cutaneous symptoms progressing from varied severity of neutrophilic urticaria to include Sweet’s syndrome to possibly pyoderma gangrenosum. The diagnosis of NUSI is an important one to consider in patients who present with antihistamine-resistant urticaria in combination with systemic inflammatory symptoms, with IL-1 blockade a viable option for therapy.H. Belani: None declared, K. Leslie Consultant for: Novartis


P<0.0001), ERK phosphorylation (P<0.0001) and FOXM1 (P = 0.001) were frequently increased and associated with high-grade tumors, as well as a high tendency in association with advanced stage ovarian cancer by immunohistochemical analysis. Intriguingly, the expressions of GRB7 (P<0.0001), ERK phosphorylation (P<0.001) and FOXM1 (P<0.001) showed a significant stepwise increase pattern along Grade 1 to Grade 3 ovarian cancers. Biochemical studies using western blot analysis demonstrated that enforced expression or knockdown of GRB7 showed GRB7 could elevate the levels of ERK phosphorylation and FOXM1, whereas enforced expression of FOXM1 could not alter levels of GRB7 and ERK phosphorylation. But inhibition of ERK signaling by U0126 or PD98059 could reduce the level of FOXM1 in GRB7-overexpressing ovarian cancer cells, suggesting that GRB7, ERK and FOXM1 are regulated orderly. Moreover, inhibition of ERK activity by U0126 or PD98059, or decreased FOXM1 expression by Thiostrepton significantly inhibited cell migration/invasion, tumor growth in vitro and in vivo. Collectively, our findings confer that targeting GRB7/ERK/FOXM1 signaling cascade may be a promising molecular therapeutic choice in combating ovarian cancer.Ovarian cancer is a highly lethal disease with poor prognosis and especially in high-grade tumor. Emerging evidence has reported that aberrant upregulation and activation of GRB7, ERK as well as FOXM1 are closely associated with aggresivenesss of human cancers. However, the interplay between these factors in the pathogenesis of human cancers still remains unclear. In this study, we found that GRB7 ( Ovarian cancer is the most lethal disease among all gynecological malignancies Growth factor receptor-bound protein 7 (GRB7) is a signaling adaptor which functions to couple signals from cell surface receptors to specific signaling pathways in vitro and in vivo tumor growth. Our results emphasize the importance of the GRB7/ERK/FOXM1 pathway in tumorigenic properties and argue this pathway for a promising therapeutic target in high-grade ovarian cancer.In this study, we show that the expressions of GRB7, ERK and FOXM1 were significantly correlated with the progression of ovarian cancer. We also describe the regulatory mechanism of GRB7/ERK/FOXM1 signaling cascade and the inhibition of this signaling using either the chemical inhibitor U0126 or FOXM1 inhibitor Thiostrepton could remarkably abrogate the ovarian cancer cell migration/invasion, and 2 in minimum essential medium or Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum. MAPK/ERK kinase 1/2 (MEK1/2) inhibitors PD98059 and U0126, and FOXM1 inhibitor Thiostrepton were obtained from Calbiochem .Two ovarian cancer cell lines: A2780cp  The pEGFP/GRB7 expressing plasmids were used as previously Immunohistochemical (IHC) staining for GRB7, ERK phosphorylation and FOXM1 was performed on an ovarian cancer tissue array (OVC1021)  using primary polyclonal anti-GRB7 , anti-phospho-ERK , and anti-FOXM1 . The percentage of immuno-positive cells in tumors and normal epithelia was assessed by the proportions of immuno-positive cells ranged from 10 to 100%, and the intensity of staining scored as 0 (negative), 1 (faint), 2 (moderate), 3 (strong) and 4 (marked). The immunoreactivity for each case was scored as a percentage of the proportions of immuno-positive cells multiplied by the intensity of staining. The fold change of each staining was obtained by dividing the expression level of each cancer sample by the mean immunoreactive staining value of normal ovaries and borderline mixed cystadenoma. The quantification of immunohistochemical staining was scored blindly at least by two independent observers.β-actin . Blots were then incubated with goat anti-rabbit or anti-mouse secondary antibody conjugate with horseradish peroxidase  and visualized by enhanced chemiluminescence (ECL) (Amersham).For Western blot analysis, cells were lysed with Cell Lysis Buffer  containing Protease Inhibitor Cocktail  and PMSF (phenylmethylsulphonyl fluoride) . The samples were resolved by SDS-PAGE and electroblotted onto Immobilon-P Transfer Membrane . Blots were blocked with 5% skim milk, followed by incubation with anti-GRB7, FOXM1 (Santa Cruz), GFP (Abcam), phospho-ERK, ERK , and Cell viability was measured by Cell Proliferation Kit II (XTT) for 5 days according to the manufacturer’s instructions (Roche). The data were collected from at least three independent experiments.5 cells were resuspended in serum free culture medium  and seeded on the upper chamber, whereas the full medium was placed in the lower chamber as chemo-attractant. The plate was incubated at 37°C and 5% CO2 for 12 hrs for cell migration assay, and 48 hrs for cell invasion assay allowing cells to pass through the pores in the membrane. The cell migrated and invaded rates were calculated after cell staining and counting by using at least three different fields for each transwell filter. The experiments were repeated thrice.To quantify the cell migratory and invasive capacities of ovarian cancer cells, the Transwell cell migration and cell invasion assay kits  were used in this study according to the manufacturer’s instructions. In brief, 1.5×10in vivo effects of U0126 and Thiostrepton on tumor development, 5×106 A2780cp cells were inoculated s.c. into BALB/c nu/nu female mice of 3–4 weeks of age and in groups of five. The tumor formation in nude mice was monitored for every 3 days. 25 to 50 µmol/kg of U0126 or 200 to 300 µmol/kg Thiostrepton  was administered i.p. once for every 3 days with total of 4 injections into five nude mice when every tumor size became ∼3 mm in diameter. As a control group, DMSO alone was administrated i.p. for the same time of treatment. The tumor sizes were measured using slide calipers and were calculated by the following formula: volume = (width) 2*length*π/6. The tumor growth curves were plotted from the mean volume±SEM of tumors from 5 mice. The side effects such as body weight changes were monitored closely. All the animal experiments were approved by the University of Hong Kong Committee on the Use of Live Animals in Teaching and Research (CULATR No.2560-11).To examine the t-test was used to analyze the cell viability, migration/invasion and in vivo tumor growth results. A p-value was considered significant when less than 0.05.The clinical parameters were analyzed by SPSS 13.0 software . Fisher’s exact test (for parametric data) and Mann-Whitney test (for non-parametric data) were used to compare the values between subgroups. The Student’s P<0.0001, Fisher’s exact test) and FOXM1 (>3 folds)  , ERK phosphorylation , and FOXM1  were significantly correlated with high-grade tumor and had a high tendency in association with advanced stage ovarian cancer  , ERK phosphorylation , and FOXM1  expression pattern was observed from Grade 1 to Grade 3 tumors (We have previously reported that GRB7 ct test) . In addict test) . It was 3 tumors  and a le3 tumors , suggestGiven that the increased GRB7, ERK phosphorylation and FOXM1 significantly correlate with high-grade ovarian tumor, they may be regulated coordinately in order to mediate oncogenic functions in ovarian cancer progression. We were thus interested to investigate the regulatory mechanism amongst these factors in ovarian cancer cells. For this purpose, both A2780cp and OVCA433 cells were firstly treated with U0126 MEK1/2 inhibitor. Western blotting showed that ERK phosphorylation and FOXM1 were remarkably reduced, but no change in GRB7 expression was observed . BesidesIt has been documented that high-grade ovarian tumor exhibits high capacity in cell migration/invasion P = 0.02, Student t-test) and OVCA433  exhibited significant reduction in cell proliferation rate as compared with their controls  and OVCA433  also showed a profound reduction in cell proliferation rate as compared with their controls   and 50 µM/kg  respectively  (in vitro and in vivo.We next examined the  t-test) . We nextectively . Besides t-test) . These fin vitro and in vivo. These findings clearly evidence that the activated GRB7/ERK/FOXM1 signaling cascade plays an important role in the pathogenesis of high-grade ovarian cancer.In this study, we present evidence that GRB7, ERK phosphorylation and FOXM1 are increased in ovarian cancer. Interestingly, the concomitant increase of GRB7, ERK phosphorylation and FOXM1 is associated with high-grade ovarian cancers. Importantly, we show that these factors are coordinately regulated in GRB7/ERK/FOXM1 signaling axis in ovarian cancer cells. Functionally, inhibiting ERK activity by U0126 or PD98059 and FOXM1 expression by Thiostrepton remarkably inhibit ovarian cancer cell migration/invasion and tumor growth Constitutive activation of ERK signaling pathway has been involved in human cancer development in vitro and in vivo tumorigenic studies clearly show that the ovarian cancer cell growth and cell migration/invasion are remarkably reduced upon treatments of PD98059 or U01260, and Thiostrepton via targeting MEK/ERK and FOXM1 activities respectively. More importantly, the tumor suppressive effects derived from either U0126/PD98059 or Thiostrepton are equivalent. These findings seem to provide an alternative approach when developing GRB7-targeted therapeutics in ovarian cancer.GRB7 is a known adaptor which relays signals from cell surface receptors to specific downstream signaling cascades via the protein-protein interaction of its Src-homology 2 (SH2) domain to a variety of tyrosine kinases In summary, this study shows that aberrant activation of GRB7/ERK/FOXM1 signaling cascade is significantly correlated with the development of ovarian cancer. Targeting this signaling axis by MEK/ERK or FOXM1 inhibitors could obtain promising effect on the signal transduction-based therapy for this disease.Figure S1Immunohistochemical analysis showed increased expressions of GRB7, ERK phosphorylation and FOXM1 were associated with advanced stage ovarian cancers.(TIF)Click here for additional data file.Figure S2Enforced expression of GRB7 increases tumor growth in mouse xenograft model. The GRB7 stably expressing A2780cp cells (Acp-GRB7) and empty vector control A2780cp cells (Acp-V) were subcutaneously injected into the right flank of nude mice (5 mice per group). The tumor size was monitored for every 3-day. The representative pictures and bar charts show the average tumor weight of each group taken on day 18. .(TIF)Click here for additional data file.


Although proanthocyanidin extract from blueberries reportedly affects Ca2+ buffering capacity, there are no reports on the effects of proanthocyanidin on glutamate-induced [Ca2+]i or cell death. In the present study, the effects of grape seed proanthocyanidin extract (GSPE) on glutamate-induced excitotoxicity was investigated through calcium signals and nitric oxide (NO) in cultured rat hippocampal neurons.Proanthocyanidin is a polyphenolic bioflavonoid with known antioxidant activity. Some flavonoids have a modulatory effect on [Ca2+]i increase normally induced by treatment with glutamate (100 μM) for 1 min, in a concentration-dependent manner. Pretreatment with GSPE (6 μg/ml) for 5 min significantly decreased the [Ca2+]i increase normally induced by two ionotropic glutamate receptor agonists, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). GSPE further decreased AMPA-induced response in the presence of 1 μM nimodipine. However, GSPE did not affect the 50 mM K+-induced increase in [Ca2+]i. GSPE significantly decreased the metabotropic glutamate receptor agonist (RS)-3,5-Dihydroxyphenylglycine-induced increase in [Ca2+]i, but it did not affect caffeine-induced response. GSPE (0.3-6 μg/ml) significantly inhibited synaptically induced [Ca2+]i spikes by 0.1 mM [Mg2+]o. In addition, pretreatment with GSPE (6 μg/ml) for 5 min inhibited 0.1 mM [Mg2+]o- and glutamate-induced formation of NO. Treatment with GSPE (6 μg/ml) significantly inhibited 0.1 mM [Mg2+]o- and oxygen glucose deprivation-induced neuronal cell death.Pretreatment with GSPE (0.3-10 μg/ml) for 5 min inhibited the [Ca2+]o- and oxygen glucose deprivation-induced neurotoxicity through inhibition of calcium signals and NO formation in cultured rat hippocampal neurons.All these data suggest that GSPE inhibits 0.1 mM [Mg Proanthocyanidins are polymers of flavonoid molecules that are widely available in fruits, vegetables, nuts, seeds, flowers, and bark, and especially in grape seeds . These c2+ concentration ([Ca2+]i) in neurons by activating ionotropic and metabotropic glutamate receptors. In pathological conditions, including epilepsy and ischemia, a massive glutamate release leads to glutamate neurotoxicity [2+ concentration ([Ca2+]i) and subsequent neuronal cell death [2+]i following NMDA receptor activation stimulates nitric oxide synthase (NOS), an enzyme that induces formation of nitric oxide (NO) in neurons i. (-)-Epigallocatechine gallate (EGCG) increase [Ca2+]i in U87 cells [2+]i increase in PC12 cells i or cell death in cultured rat hippocampal neurons.Some flavonoids have modulatory effects on o) and oxygen glucose deprivation.The present study determined whether grape seed proanthocyanidin extract (GSPE) affected glutamate-induced Ca2+]i is one of the major causes of glutamate excitotoxicity [2+]i increase in cultured rat hippocampal neurons. Treatment with glutamate (100 μM) for 1 min caused [Ca2+]i increase. Reproducible response could be elicited by applying glutamate (100 μM) for 1 min at 30-min intervals    Figure . Pretrea) Figure . Pretrea) Figure . Therefon humans .2+]i increase, the present study used two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA. Application of AMPA increased [Ca2+]i by activating AMPA/kainate channels and then voltage-gated Ca2+ channels in neurons. Reproducible increase in [Ca2+]i was induced by treatment with (S)-AMPA (10 μM) for 1 min at 10-min intervals . Pretreatment with GSPE (6 μg/ml) for 5 min significantly inhibited the AMPA-induced [Ca2+]i response   Figure .2+-permeable AMPA receptors are expressed in hippocampal neurons early in development [2+-permeable AMPA-receptor-mediated Ca2+ influx. Pretreatment with nimodipine (1 μM) for 5 min inhibited the AMPA-induced [Ca2+]i response . Pretreatment with GSPE (6 μg/ml) for 5 min further inhibited AMPA-induced response in the presence of nimodipine (1 μM)   Figure .2+]i increase was induced by treatment with NMDA (100 μM) for 1 min at 20-min intervals . Pretreatment with GSPE (6 μg/ml) for 5 min also significantly inhibited NMDA-induced [Ca2+]i response   Figure .+ (partly Ca2+) into neurons and depolarized the neurons. This depolarization induced secondary activation of voltage-gated Ca2+ channels i increase by 50 mM K+ HEPES-HBSS . Treatment with GSPE (6 μg/ml) for 5 min did not affect high K+-induced [Ca2+]i response   Figure .3) and diacylglycerol. Binding of IP3 to the IP3 receptors initiates release of Ca2+ from intracellular stores [2+]i increase. Reproducible [Ca2+]i increase was induced by treatment with DHPG (100 μM) for 1 min at 30-min intervals . Pretreatment with GSPE (6 μg/ml) for 5 min significantly inhibited DHPG-induced [Ca2+]i response   for 2 min at 10-min intervals . Pretreatment with GSPE (6 μg/ml) for 5 min did not significantly affect caffeine-induced [Ca2+]i response  . At 10 min after exposure to GSPE, the frequency of [Ca2+]i spikes was 12.8 ± 8.7% of the initial frequency  or low [Mg2+]o for 1 h significantly increased DAF-2T fluorescence. While the 0.1 mM [Mg2+]o-induced increase in NO formation was markedly inhibited by pretreatment with GSPE (6 μg/ml) for 5 min, and the glutamate-induced NO formation was slowly inhibited at a later phase    increased cell survival to 81.3 ± 7.3% of the control.Reduction of [Mgll death -30. The t Figure . In the ) Figure . A simil) Figure . However) Figure . The eff) Figure . The celin vitro rat hippocampal culture model to determine the inhibitory mechanisms of GSPE in low [Mg2+]o or oxygen glucose deprivation-induced neuronal cell death. GSPE reduced the glutamate-induced [Ca2+]i increase by inhibiting the AMPA, NMDA, and DHPG-induced [Ca2+]i increase in hippocampal neurons. Synaptically mediated low [Mg2+]o-induced [Ca2+]i spikes were also inhibited by GSPE. GSPE inhibited low [Mg2+]o or oxygen glucose deprivation-induced neuronal cell death by inhibition of both [Ca2+]i increase and Ca2+-dependent NO formation.The present study used an + (partly Ca2+) through non-NMDA receptors, which secondarily activate voltage-gated Ca2+ channels and induce Ca2+ influx i increase, but it did not affect the depolarization-induced [Ca2+]i increase from 50 mM K+ HEPES-HBSS, suggesting that GSPE inhibits AMPA-induced [Ca2+]i increase by inhibiting Ca2+ influx directly through Ca2+-permeable AMPA receptors. In fact, Ca2+-permeable AMPA receptors are strongly expressed in hippocampal neurons, especially early in development i increase i increase. Although the working mechanism of GSPE is not obvious, GSPE may inhibit DHPG-induced Ca2+ release from IP3-sensitive stores or DHPG-induced activation of PLC. Therefore, further research is needed to determine whether proanthocyanidin inhibits release of Ca2+ from IP3-sensitive stores or metabotropic glutamate receptor-induced activation of PLC.The group I metabotropic glutamate receptor agonist, DHPG, induces a release of Cating PLC ,32. In t2+]i increase by inhibiting AMPA, NMDA, and metabotropic glutamate receptor-induced [Ca2+]i increase. Reduction of [Mg2+]o in cultured central nervous system neurons to 0.1 mM elicited [Ca2+]i spikes that depend on glutaminergic synaptic transmission [2+]o-induced [Ca2+]i spikes. All these data suggest a possibility that proanthocyanidin can inhibit glutaminergic synaptic transmission in hippocampal neurons. In the present study, GSPE did not affect the depolarization-induced [Ca2+]i increase induced by high K+, which is involved in neurotransmitter release in the synaptic terminal. Thus, it is not clear whether proanthocyanidin inhibited synaptic transmission by decreasing glutamate release in presynaptic sites.In the present study, GSPE inhibited glutamate-induced o-induced NO formation, and it slightly inhibited glutamate-induced formation. GSPE reportedly has potent inhibitory action on NO production presumably through of the inhibition of Ca2+-dependent nitric oxide synthase o-induced [Ca2+]i increase.In the present study, GSPE completely inhibited low o- and oxygen glucose deprivation-induced neuronal cell death as well as both [Ca2+]i increase and Ca2+-dependent NO formation. Ischemic insults have reportedly induced [Ca2+]i increase and formation of NO in neurons i signalling and NO formation as well as antioxidant effects and antiapoptotic effects.Previous investigations have reported that proanthocyanidin protects multiple target organs from drug- and chemical-induced toxicity. GSPE protects cells against acetaminophen-induced hepato- and nephrotoxicity, amiodarone-induced lung toxicity, doxorubicin-induced cardiotoxicity, and dimethylnitrosamine-induced spleenotoxicity . GSPE in neurons ,12,40,41 neurons . These rThe daily intake of proanthocyanidins may vary from tens to several hundred mg/day depending on diet . Proanth2+]o- and oxygen glucose deprivation-induced neurotoxicity in cultured rat hippocampal neurons. The neuroprotective effects of proanthocyanidin might have been mediated by inhibition of glutamate-induced calcium signalling and NO formation. These results demonstrated that proanthocyanidin, and especially oligomeric polyphenolic compounds, may have future utility as neuroprotective agents or as supplements against glutamate excitotoxicity-related neurologic disorders such as epilepsy, traumatic brain injury, and ischemia.The results of the present study showed that IH636 grape seed proanthocyanidin extract protected neuronal cells against the low i, hippocampal cells were incubated in 4 μM fura-2 AM in HEPES-buffered Hank's salt solution  containing 0.5% bovine serum albumin for 45 min at 37°C. The cover glass was then mounted in a flow-through chamber that was superfused at a rate of 1.5 ml/min. Digital calcium imaging was performed as described by Rhie et al. [2+]i was calculated from the ratio of the background-subtracted digital images. Cells were delimited by producing a mask that contained pixel values above a certain threshold applied to the 380 nm image. Background images were collected at the beginning of each experiment after removing cells from another area to the coverslip. Autofluorescence from cells not loaded with the dye was less than 5% and thus not corrected. Ratio values were converted to free [Ca2+]i by the equation [Ca2+]i = Kdβ(R-Rmin)/(Rmax-R), in which R was the 340/380 nm fluorescence emission ratio and d K= 224 nM was the dissociation constant for fura-2. Rmin, Rmax, and β was determined in ionomycin-permeabilized cells in calcium-free and saturated solutions .To measure [Cae et al. . The cha2+]i spikes were measured using fura-2-based-microfluorimetry [2+]i spikes were induced by HHSS containing 0.1 mM MgCl2 and 10 μM glycine. [Ca2+]i was calibrated by the same method that was used for the digital [Ca2+]i imaging. Rmin, Rmax, and β were 0.86, 14.89, and 7.42, respectively.i: intracellular free Ca2+ concentration; DAF-2DA: 4,5-diaminofluorescein diacetate; DHPG: (RS)-3,5-Dihydroxyphenylglycine; DMEM: Dulbecco's modified Eagle's medium; EGCG: (-)-epigallocatechine gallate; FBS: fetal bovine serum; GSPE: grape seed proanthocyanidin extract; HHSS: HEPES-buffered Hank's salt solution; IP3: inositol 1,4,5-trisphosphate; [Mg2+]o, extracellular Mg2+ concentration; NMDA: N-methyl-D-aspartate; NO: nitric oxide; NOS: nitric oxide synthase; OGD: oxygen glucose deprivation; PLC: phospholipase CAM: acetoxymethyl ester; AMPA: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; [CaSHY designed the study. S-HA, HJK, IJ, YJ H, M-JK, D-JR and SJH performed the experiments and analyses. S-HA, HJK, Y-HJ, and SHY wrote the paper. All authors read and approved the final manuscript.


In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca2+) homeostasis has been linked to presenilins (PS) , the catWe used neuroblastoma SH-SY5Y cell lines stably overexpressing human APP695 cDNA harboring or not the Swedish double mutation (K670M→N671L) (APPswe and APP695 respectively) and Tg2576 mice carrying human APPswe cDNA under the control of the hamster Prion promoter. We analyzed subcellular Ca2+ signals by using aequorin targeted Ca2+ probes, Fura2, AM dye and patch clamp experiments. We analyzed the expression of the Ryanodine receptor (RyR) by western blot and quantitative RT-PCR. We studied the impact of RyR blockade by dantrolene on APP metabolism and Amyloid beta peptide production using biochemistry, ELISA and immunohistochemistry approaches and investigated the underlying mechanisms through the study of APP phosphorylation, and beta- and gamma secretase expression and activities. We finally studied pre- and post-synaptic proteins expression and learning and memory paradigms in Tg2576 mice upon vehicle or dantrolene treatment.We show that overexpression of APP695, or APPswe triggers increased Ryanodine receptors (RyR) expression and enhances RyR-mediated ER Ca2+ release in SH-SY5Y neuroblastoma cells and in APPswe-expressing (Tg2576) mice. Interestingly, dantrolene-induced lowering of RyR-mediated Ca2+ release leads to the reduction of both intracellular and extracellular amyloid beta load in neuroblastoma cells as well as in primary cultured neurons derived from Tg2576 mice. This Aβ reduction can be accounted for by decreased Thr-668-dependent APP phosphorylation and β- and gammaological lesions and slows down learning and memory deficits in Tg2576 mice [Overall, our data document a key role of RyR in Aβ production and learning and memory performances, and delineate RyR-mediated control of Ca2+ homeostasis as a physiological paradigm that could be targeted for innovative therapeutic approaches.


Accordingly, the objective of this study was to develop a mathematical model of the human atrial myocyte that, in addition to the sarcolemmal (SL) ion currents, accounts for the heterogeneity of intracellular Ca2+ dynamics emerging from a structurally detailed sarcoplasmic reticulum (SR). Based on the simulation results, our model convincingly reproduces the principal characteristics of Ca2+ dynamics: 1) the biphasic increment during the upstroke of the Ca2+ transient resulting from the delay between the peripheral and central SR Ca2+ release, and 2) the relative contribution of SL Ca2+ current and SR Ca2+ release to the Ca2+ transient. In line with experimental findings, the model also replicates the strong impact of intracellular Ca2+ dynamics on the shape of the action potential. The simulation results suggest that the peripheral SR Ca2+ release sites define the interface between Ca2+ and AP, whereas the central release sites are important for the fire-diffuse-fire propagation of Ca2+ diffusion. Furthermore, our analysis predicts that the modulation of the action potential duration due to increasing heart rate is largely mediated by changes in the intracellular Na+ concentration. Finally, the results indicate that the SR Ca2+ release is a strong modulator of AP duration and, consequently, myocyte refractoriness/excitability. We conclude that the developed model is robust and reproduces many fundamental aspects of the tight coupling between SL ion currents and intracellular Ca2+ signaling. Thus, the model provides a useful framework for future studies of excitation-contraction coupling in human atrial myocytes.Electrophysiological studies of the human heart face the fundamental challenge that experimental data can be acquired only from patients with underlying heart disease. Regarding human atria, there exist sizable gaps in the understanding of the functional role of cellular Ca In the human heart, the contraction of atrial and ventricular muscle cells is based largely on common mechanisms. There is, however, a fundamental difference in the cellular calcium dynamics that underlie the contractile function. Here, we have developed a computational model of the human atrial cell that convincingly reproduces the experimentally observed characteristics of the electrical activity and the cyclic fluctuations of the intracellular calcium concentration. With the model, we evaluate the relative roles of the most important cellular calcium transport mechanisms and their impact on the electrical behavior of the cell. Our simulations predict that the amount of calcium released from the cellular stores during each electrical cycle crucially regulates the excitability of the human atrial cell. Furthermore, the results indicate that the cellular sodium accumulation related to faster heart rates is one of the main mechanisms driving the adaptation of cardiac electrical activity. Finally, we conclude that the presented model also provides a useful framework for future studies of human atrial cells. The majority of Ca2+ is re-circulated back to the SR by the SR Ca2+-ATPase (SERCA), leaving a smaller fraction of Ca2+ to be extruded from the cell by the Na+/Ca2+ exchanger (NCX) and plasmalemmal Ca2+-ATPase (PMCA).In cardiac myocytes, the process triggered by the action potential (AP) and resulting in the contraction of the myocyte is commonly referred to as excitation-contraction coupling (ECC) 2+]i in both ventricular and atrial myocytes, there are substantial spatiotemporal differences in the properties of the atrial and ventricular Ca2+ transients 2+ release that is virtually uniform throughout the cell 2+ wave arises in the periphery  and then propagates to the center of the cell, activating secondary release from the corbular  SR compartments Whilst the same CICR mechanism initiates the transient elevation of i, sarcolemmal (SL) ion currents, and the SR Ca2+ release, the aim of this study was to elucidate to what extent intracellular Ca2+ regulates the AP waveform, cellular excitability, and rate-dependent electrophysiological mechanisms. The results indicate that the peripheral  SR Ca2+ release sites define the interface between Ca2+ and AP, whereas the central  release sites are important for the fire-diffuse-fire propagation of Ca2+ diffusion. Moreover, our analysis suggests that the rising intracellular Na+ concentration is an important modulator of the action potential duration at increasing heart rates. Finally, the results predict that SR Ca2+ release is a strong modulator of AP duration and thus affects the human atrial myocyte refractoriness.Facing the complexity of a highly integrated and interdependent system, mathematical modeling has become an established complement to the experimental approach in elucidation of the mechanisms that underlie cardiac electrophysiology The presented mathematical model is a set of ordinary differential equations that were implemented to the Matlab (The MathWorks) environment of technical programming. A brief description of the novel and modified model components are given below. The complete mathematical formulation of the model and parameters are shown in 2+ dynamics, the computational load of the model is rather low: a 10-second simulation at 1 Hz pacing takes about 10 seconds to run on a normal desktop PC .Simulation results were obtained by numerically integrating the model equations with a stiff ordinary differential equation solver method (ode15s). Despite the detailed description of intracellular Ca2+, Na+, and K+ and SR Ca2+ concentrations  was fitted based on experimental SR Ca2+ content 2+ diffusion in the cytosol and in the SR was set to 50% of the total volume of the compartments 2+ diffusion between junctional and bulk cytosol was set to 50% of the total area between these compartments.The Ca2+ in the SR is 8–9 µm2/s 2+ buffering in the SR 2+ in the SR is 44 µm2/s, which yields an effective coefficient of 8–12 µm2/s in the relevant range of [Ca2+]SR (0.3–0.6 mM).The diffusion coefficients are shown in 2+ buffering have not been characterized from atrial myocytes in the same detail as in ventricular myocytes 2+ buffering 2+ transients with the experimental SR Ca2+ content 2+ buffers are the sarcolemma, which is assumed to be as in ventricular myocytes based on 2+ transient kinetics and SR Ca2+ content. The rest of the buffering, consisting most likely of troponin, calmodulin and myosin, is modeled as an single arbitrary mobile buffer, which has the mobility and Kd of calmodulin The components of cytosolic Ca2+ buffering to and uptake by the SERCA and the passive Ca2+ leak from the SR to the cytosol were modeled as previously The Ca2+ release flux through the RyR channel, we developed a novel phenomenological model  and outward (repolarizing) ion channel currents  at different stages of repolarization . In line with experimental findings 2+]i increased by 52% and 88%, respectively.In our model, the average cytosolic Ca 0.58 µM  at 1 Hz 2+ release homogenizes the cytosolic Ca2+ signal, the Ca2+ transients at different parts of the cytosol differ from the average signal . A possible source for the difference between the integrated NCX value and the actual SR Ca2+ content is the fraction of Ca2+ that is extruded from the cell by the PMCA, which was not considered in the experimental analysis In human atrial myocytes, the diastolic SR Ca2+ release from the SR generates 79±6% of the Ca2+ transient amplitude in human atrial myocytes 2+ release is generated in the junctional compartment  is decreased by 10%  and amplitude of the Ca2+ transient  and then a gradual  decrease in the APD of atrial myocytes; this has been shown in numerous studies. Experimental findings indicate that this adaptation coincides with functional changes in the LTCC following calcium overload + compartment (model variant: “vCaNass”) to the developed model (vCa), and we also included the recently updated description of K+ currents according to 2+]i dynamics: diastolic and systolic [Ca2+]i increase and decrease with faster pacing, respectively, and accumulation of intracellular Na+. The simulated values of APD30 that were calculated for each BCL fall within the range of reported experimental values  fits well to the in vitro values of Boutjdir et al. et al. To account for the other rate-dependent mechanisms that affect the APD, we implemented two additional variants of the myocyte that are described in detail in the Supporting Information . Brieflyl values . The stehe model . Althoug+]i, we wanted to study the rate dependence of AP with a second pacing protocol, in which simulation is always started from the quiescent steady-state and continued for five minutes for each BCL, separately , the effect of continued fast pacing on the APD is dramatically reduced . The enhanced pumping function of NKA lead to a dramatic increase in the current (INKA)  and found that fast pacing resulted in a similar increase in INKA (data not shown).Simulation results suggest that the mechanism that links AP shortening  to Na+ a (INKA) . Accordi+ accumulation due to fast pacing has on the Ca2+ dynamics via modulation of the NCX function, we simulated a previously used pacing protocol 2+ dynamics of the model  during fast pacing, the flux via reverse mode of NCX is increased considerably . However, since relative contribution of NCX is much smaller, we performed an additional simulation analysis to quantitate its role in rate-dependent modulation of Ca2+ dynamics. In line with a previous study 2+]i by 1.8 ms  when the pacing frequency is 1 Hz. This effect is more pronounced during fast pacing: the delay increases to 2.4 and 3.5 ms with 2 and 3 Hz, respectively. Thus, the results suggest that the rate-dependent modulation of NCX could mediate a shortening of the delay between the electrical excitation and the peak of Ca2+ transient.To further study the role of NCX in the rate dependence of Ca2+]i dynamics. The presented results indicate that the model accurately replicates crucial experimental findings: instead of a homogenous Ca2+ release, ECC is driven by a Ca2+ wave that arises first in the periphery and then propagates to the cell center 2+]i, sarcolemmal ion currents, and SR Ca2+ release. The results highlight the importance of the junctional SR Ca2+ release sites in defining the interface between Ca2+ and AP, whereas the non-junctional release sites are significant for the fire-diffuse-fire propagation of Ca2+ diffusion. Furthermore, our analysis indicates that intracellular Ca2+ dynamics are strongly linked to the AP morphology via modulation of NCX current, thus also affecting the refractoriness and excitability. The presented simulations also suggest that one of the main mechanisms of AP rate dependence is the accumulation of [Na+]i that modulates the function of NKA during continued fast pacing.We have developed a mathematical model of the human atrial myocyte with a spatially detailed and atria-specific description of intracellular i homeostasis may initiate electrical remodeling during atrial fibrillation, which is characterized by a marked shortening of the action potential plateau phase 2+ signaling, has thus great potential in elucidating the function of the remodeled cells with altered Ca2+ homeostasis and SL ion currents.Cumulative evidence suggests that changes in the [Ca2+]i has been well established in experimental setups of both ventricular 2+ transient produce significant changes to the APD compared to the control situation. Changes in [Ca2+]i in the vicinity of the SL affects both INCX and ICaL. However, while both increased amplitude and decelerated decay of the Ca2+ transient enhance the inward INCX, the inactivation of ICaL is affected only by the former. These findings indicate that in human atrial myocytes the NCX is more important than the LTCC in linking the amount of Ca2+ released from the SR to changes in the APD. This scheme also concurs with a previously reported role of NCX as the main mediator of the inotropic effect of AP prolongation in canine atrial myocytes 2+ transient) but also situations such as hypothyroidism (slowed decay of the Ca2+ transient) to APD modulations.The tight coupling of SL ion currents, which underlie the AP shape, and the i increases with continued fast pacing.The primary physiological context, in which the duration of the AP is modulated, relates to changes in heart rate +]i in response to continued fast pacing, the APD is decreased less compared to the normal situation, in which both of them sense an increased [Na+]i. A similar phenomenon is apparent in our atrial model: the mere increase in pacing frequency (or reduction in BCL) does not produce a maximal shortening of the AP. Rather, the increase of [Na+]i and the consequent change in the NKA function, caused by the continued high frequency pacing, promote a substantial further decrease of APD. Furthermore, the results indicate that the other ionic determinants of APD, emerging from the rate dependence of Isus2+ dynamics, are effective already within a shorter time-scale (∼10 s) of adaptation compared to that of Na+ (∼minutes). Thus, our findings underline the importance of considering the effect of Na+ accumulation in both in vitro and in silico studies of AP rate dependence when results obtained with different protocols are interpreted or compared.As previously demonstrated in ventricular myocytes 2+ transient shortens the AP (via inactivation ICaL). Interestingly, our simulation results indicate that, in this respect, the human atrial myocytes behave similar to animal with short AP. That is, increased Ca2+ transient amplitude (due to e.g. enhanced SR Ca2+ release) promotes an inward INCX, thus lengthening the AP.In myocytes with long AP, the increase of Ca2+ release has been studied extensively in ventricular myocytes during recent years; the findings, however, are controversial 2+ entry via the reverse mode of NCX is significant in physiological conditions The role of the NCX as a trigger of SR Ca2+ influx is increasingly significant at higher pacing rates, even though the bulk of the flux goes via the LTCC. However, when the temporal role of these two mechanisms is compared, it is apparent that during the first few milliseconds of the CICR process the NCX contributes rather equally to the Ca2+ influx. Accordingly, the acute inhibition of the reverse mode of NCX delays the peak of [Ca2+]i by a few milliseconds, depending on the pacing rate. These findings are in line with the reported delay of the Ca2+ transient if the Ca2+ entry via the NCX is inhibited in ventricular myocytes 2+]i with increasing heart rate, even though LTCC is the primary trigger of CICR, as suggested previously in vivo significance of the delay modulation by the NCX presents an interesting question for future simulation studies in tissue and/or whole-atria models.Our simulation results suggest that in atrial myocytes the contribution of NCX to the Cain vitro data. Instead, it is more essential that the simulation results agree qualitatively and semiquantitatively with the majority of the measured results, which has been established for the AP morphology in our model. There is a further challenge for validation of the Ca2+ dynamics due to incomplete experimental data. To compensate for this, we have also compared the model behavior to ventricular data and measurements obtained with atrial animal models.Variability of experimental results imposes a fundamental challenge for modeling studies that utilize data measured from isolated human atrial tissues or cells. It would thus be an irrelevant and futile effort to try to fit the electrophysiological characteristics of the model perfectly to one single set of 2+ release, sharing the general limitations ‘common pool’ models. Thus, the justification for most of the chosen parameter values cannot be derived directly from the biophysical properties of RyR channels ad hoc parameter values of the RyR modules are based on indirect fitting: the dynamics of intracellular Ca2+ were adjusted to be in line with macroscopic experimental observations 2+ release characterize the emergent properties of atrial Ca2+ dynamics: biphasity of the increasing Ca2+ concentration during the Ca2+ transient, and the relative contribution of SL and SR Ca2+ fluxes to the Ca2+ transient. A more complex RyR module might be needed in future studies if the simulations are run beyond the conditions that were investigated in this study.The developed RyR module of the myocyte model is an approximate description of macroscopic SR Ca2+/calmodulin-dependent protein kinase (CaMK)II that has been studied extensively in ventricular myocytes Although our model can reproduce rather accurately the rate-dependent changes in ion dynamics, it should be noted that the description of the underlying mechanisms is by no means exhaustive. For example, one potential regulatory pathway is the CaWe have chosen to use Hodgkin-Huxley formalism for the ion currents. We acknowledge that Markovian models would allow for a more detailed description of the complex kinetics of processes  that the channels exhibit. However, it can be very difficult to meet the information requirements for defining the transitional rate constants 2+ release and intracellular Na+ that are subject to strong modulation under physiological conditions. Furthermore, with a physiologically accurate description of intracellular Ca2+ dynamics the model is a potential tool, for example, in the elucidation of mechanisms that link changes in Ca2+ homeostasis to pathophysiological conditions. Thus, it offers attractive possibilities to study the electrical remodeling during atrial fibrillation, and to find potential targets that affect the refractoriness of the AP. Finally, as the computational cost of the model is low, it is also a feasible component for multi-scale models of tissue and/or heart.We conclude that the novel myocyte model provides significant insight into the excitation and ion dynamics of human atria. Our results underline the tight coupling of AP morphology to SR CaFigure S12+ current (ICaL) characteristics. (A) The time-constant for gate f1 was fitted to experimental data 2+ dependent inactivation gate is shown as a function of [Ca2+] in the junctional subspace. As the lower panel shows, this formulation reproduces qualitatively the result that blocking of SR Ca2+ release decreases the rate of LTCC inactivation significantly in human atrial myocytes CaL that was recorded in voltage clamp simulation; holding potential = −80mV.The L-type Ca(0.14 MB TIF)Click here for additional data file.Figure S2f. Experimental conditions were replicated from f were determined by application of hyperpolarizing voltage steps from −40 to −140 mV in 10 mV steps, the holding potential was −40 mV. The If model was fitted to the left atrium wall data from Steady-state activation and time-constant of I(0.07 MB TIF)Click here for additional data file.Figure S3Na and ICaL. (D) The Ito (black line) and Isus (grey line) generate large repolarizing currents in the beginning of the AP. (E) The late repolarization is carried mostly by IK1 (F). As demonstrated by the time courses of If (black solid line), IKs (black dashed line), and IKr (grey line), they contribute very little to the AP, compared to Ito and Isus. (G) During the late phase of the AP, the INCX (grey line) generates a significant depolarizing current, while the amplitude of IPMCA (black line) is much smaller.The principal outputs of the myocyte model at 1 Hz pacing. (A) The model reproduces an AP that is characteristic for human atrial myocytes: a large initial peak with a narrow early plateau is followed by a late, low amplitude, plateau phase; a so-called triangular shape. (B) and (C) The major depolarizing currents in the initial phase of the AP are the I(0.17 MB TIF)Click here for additional data file.Table S1Modified parameter values of the previously published model components.(0.01 MB PDF)Click here for additional data file.Table S2Parameters of the novel RyR model.(0.01 MB PDF)Click here for additional data file.Table S3Comparison of AP characteristics of the developed model (vCa) and the two extended variants (vCaNass and vCaNassIk) to experiments.(0.01 MB PDF)Click here for additional data file.Table S4Initial values for the differential variables at 1 Hz pacing steady-state.(0.01 MB PDF)Click here for additional data file.Text S1Model implementation.(0.16 MB PDF)Click here for additional data file.