Heart failure (HF) affects over 5 million Americans and is characterized by impairment of cellular cardiac contractile function resulting in reduced ejection fraction in patients. Electrical stimulation such as cardiac resynchronization therapy (CRT) and cardiac contractility modulation (CCM) have shown some success in treating patients with HF. Computer simulations have the potential to help improve such therapy  as well as provide insight into the underlying mechanisms which could be beneficial. However, these myocyte models require a quantitatively accurate excitation-contraction coupling such that the electrical and contraction predictions are correct. While currently there are close to a hundred models describing the detailed electrophysiology of cardiac cells, the majority of cell models do not include the equations to reproduce contractile force or they have been added ad hoc. Here we present a systematic methodology to couple first generation contraction models into electrophysiological models via intracellular calcium and then compare the resulting model predictions to experimental data. This is done by using a post-extrasystolic pacing protocol, which captures essential dynamics of contractile forces. We found that modeling the dynamic intracellular calcium buffers is necessary in order to reproduce the experimental data. Furthermore, we demonstrate that in models the mechanism of the post-extrasystolic potentiation is highly dependent on the calcium released from the Sarcoplasmic Reticulum. Overall this study provides new insights into both specific and general determinants of cellular contractile force and provides a framework for incorporating contraction into electrophysiological models, both of which will be necessary to develop reliable simulations to optimize electrical therapies for HF. Heart failure (HF) affects over 5 million Americans and is characterized by impairment of cellular cardiac contractile function and reduced ejection fraction in patients. ElectriCRT is a current treatment for patients with congestive heart failure (CHF) for a wide QRS complex, where biventricular pacemakers are used to improve electrical synchrony and presumably ejection fraction,4. HowevAn alternate electrical therapy for HF is cardiac contractility modulation (CCM) which delivers electric fields to the heart during its refractory period. Although its mechanism of action was initially thought to result from increased calcium flux from Sarcoplasmic Reticulum (SR),8, recenIt is clear then, that optimization of CRT and CCM therapies require an understanding of the effects of electric fields on myocyte dynamics especially for inotropicity as regulated by intracellular calcium. Computer simulations have the potential to improve these therapies through clarification of the underlying mechanisms. For exaboth accurate. During the depolarization of an action potential, L-type Ca2+ channels are activated and the influx of Ca2+ current into the narrow dyadic space induces a large Ca2+ release from the Junctional Sarcoplasmic Reticulum (JSR) through the Ryanodine Receptor (RyR). Intracellular Ca2+ concentration is thus greatly increased immediately following the depolarization of the transmembrane action potential. Some of these Ca2+ ions bind to affinity sites on Troponin C therefore enable myosin heads, which contain cross-bridges, to attach to actin. Myosin heads ‘walk’ on actin generating force, transforming chemical energy to mechanical energy resulting in cell contraction. Then during the repolarization of the action potential, Ca2+ leaves the cytoplasmic compartment into either the Network Sarcoplasmic Reticulum (NSR) through the Ca-ATPase pump or out of the cell membrane though Na-Ca exchangers. The decreased intracellular Ca2+ concentration causes Ca2+ to detach from troponin C, inducing uncoupling of Myosin heads and actin, which ends the contraction process+[T*]). Equations represent the transitions among the four states in The muscle unit structure and cross-bridge dynamics are shown in T), troponin attached with cross-bridges (T*), troponin bound with calcium (TCa), and troponin bound with calcium attached with cross-bridges (TCa*); all with units of mM/ms. [TCa]eff is the effective [TCa] where cross-bridge attachment can occur and it’s dependent on the sarcomere length  action potential repolarization. The basic theory lies in the fact that Ca2+ release from the SR is determined by two factors: the recovery of Ryanodine Receptor’s (RyR’s) which is a function of interbeat interval; and the amount of Ca2+ in the SR. When ESI>PP, RyRs are more recovered from the last priming beat, resulting in a bigger Ca2+ release from SR for the ES beat. Consequently, the Ca2+ content in SR will be smaller compared to the priming period, and may lead to a smaller or larger Ca2+ release (compared to PP) for the next PES beat depending on the PESI.We applied the same PESP pacing protocol employed by Yue et al. , which wIn Yue’s experiments, they measured the maximum rate of pressure change in fourteen isolated perfused left canine ventricles under isovolumetric conditions. In the NL96 contraction model, force  is the variable to quantify the contractile strength; therefore to correlate with Yue’s experiment results, we assumed a linear relationship between force generated from single cell and ventricular pressure, and thuCa2+ troponin buffers can be found in We studied fourteen ventricular cell models, which include four species: guinea pig (n = 4)30]31][[31][30][[[31][30]Two of the fourteen models have contraction in their original versions: Iribe_etal_2006 and MatsCa2+ troponin (CaTRPN) a single-state-variable dynamic buffer :Ca2+]i is the intracellular Ca2+ concentration; Btroponinmax, is the total Troponin concentration; Kon and Koff are rate constants for the chemical reaction:Matsuoka_etal_2003 already has NL96 contraction in the original version. However, to investigate how much influence the mechanics has on this model, we remove the NL96 model and make Kon and Koff to the values of Y1 and Z1 and Btroponinmax, to the value in the original model. We call this new model Matuoska_etal_2003 without Contraction.We set CaTRPN is expressed by a single-state dynamic equation like Koff that depends on force, hence it is strongly coupled even for isometric contractions. Also the RWH99 model has six tropomyosin/cross-bridge states with rate constants that are functions of the Ca2+ troponin buffer. To simulate this model without the contraction part, we eliminated the six tropomyosin/cross-bridge states and set Koff for CaTRPN to a constant value by fixing the force in Koff expression to half of its maximum value. We then compare simulations of the original model and the version without contraction as well as one with NL96 contraction model (implemented as described in the next “Type Two” section).Iribe_etal_2006 includes the contraction model of Rice et al. (RWH99) in the original version . For RWHCa2+ troponin buffers in these models are the same as Ca2+ troponin buffer in the original model into two states: with (TCa*) or without (TCa) cross-bridges  for all the buffers: Shannon_etal_2004 and Grandges Eqs  and 4:dd T* Eqs . There aL96 paper except fCaTRPN but instantaneous forms for all the other buffers: Mahajan_etal_2008total was set to be 0.07mM, which is a standard value in most models. [Other]total = [General]total − [Troponin]total so that the concentration of the total intracellular Ca2+ buffer is the same as in the original model. If the model has other Ca2+ buffers (such as CMDN) but no CaTRPN , we keep the other buffers unchanged and add a CaTRPN buffer. For the new CaTRPN buffer we also set [Troponin]total = 0.07mM and Kd,CaTRPN = 0.6μM which are both common values in many models. After adding an instantaneous CaTRPN to the model, we follow the steps for Type Four to implement NL96.For this type of models, we first add instantaneous www.cellml.org in CELLML format. Then they were translated into.mat files (MATLAB files) using a PYCML program. Simu Simuwww. converge using fo converge the IyerOur pacing protocol is composed of three steps and similar to the pacing protocol from Yue et al  except tCa2+ concentrations vary considerably among models as shown in In this step, EP models without contractions were run with no stimulation current until quiescent steady states were reached . Initial values for state variables in each model were directly loaded from CellML code. The time for each model to reach the quiescent state is listed in Ca2+ troponin buffer were calculated by solving Eqs Ca2+]i. The total Ca2+ troponin buffer ([TCa]+[TCa*]) calculated using this method was verified to be similar to the quiescent value in the original EP model if the original model had a Ca2+ troponin buffer. We used this method to ensure that the initial conditions for the original models and the corresponding contraction models were as close as possible.During the priming pacing cycle, all models (with and without contractions) were paced with priming period T = 500ms starting from the quiescent states at the end of Step one, until a new “priming” steady state was achieved. The beat number required to reach this priming steady state for each model is listed in Ca2+]i, CaTRPN and generated force; we then changed ESI to a different value and repeated the process using the priming steady state as initial conditions. The shortest PESI is the refractory period of the ES beat so it is just long enough that the PES beat is separated from the ES beat. The longest PESI (ESI) value was chosen as 1200ms, 1500ms or 2000ms, depending on the time it took for the Postextrasystolic MRCpes (MRCes) to converge to a plateau level, as some models took longer than others to reach it.For all models, an extrasystolic (ES) and a postextrasystolic (PES) beat were delivered in sequence after the last priming beat as described in the Contraction Model and Experiment section. We held ESI at a constant value and varied PESI to record the transients of transmembrane potential, i; 2) maximum (systolic) [Ca2+]i; 3) the duration for which [Ca2+]i was above its half amplitude level, i.e. [Ca2+]i ≥ [Ca2+]diastolic + ([Ca2+]systolic − [Ca2+]diastolic) / 2; 4) the time at which [Ca2+]i reached its maximum systolic value (tpeak); and 5) the amplitude of the total Ca2+ charge delivered to the cytoplasmic compartment during one beat (Q) that was calculated by integrating the area underneath the [Ca2+]i − [Ca2+]diastolic transient. We quantified the differences of the parameters between original models and models with contractions by the relative difference, defined asCa2+]iPostextrasystolic Potentiation in i data in Ca2+]i transients for the last priming beat for six representative models with (dashed lines) and without (solid lines) contraction. They are chosen to include five of the different EP types: (a) Matsuoka_etal_2003 (Type One: with contraction(NL96)); (b) Iribe_etal_2006 (Type One: with contraction(RWH99)); (c) Shannon_etal_2004 (Type Two: full dynamic Ca2+ buffer); (d) Mahajan_etal_2008 (Type Three: dynamic Ca2+ troponin buffer); (e) Ohara_etal_2004 (Type Four: instantaneous Ca2+ troponin buffer); (f) TenTusscher_etal_2006 (Type Five: no Ca2+ troponin buffer). The corresponding plots for all the fourteen models are provided in Ca2+]i transient for all EP models without (with) contraction are provided in in We present the priming [Ca2+]i differs substantially among the original EP models (without contraction) as shown in Ca2+]i, Grandi_etal_2010 and Shannon_etal_2004 have values in the ×10−3mM range while Faber_etal_2000 is up to four times larger at 2.00×10−3mM; for diastolic [Ca2+]i, the maximum value is 2.00×10−3mM for Faber_etal_2000 and the minimum value is 3.82×10−4mM for Grandi_etal_2010, which is one fifth of the maximum value.[Ca2+ troponin buffers (Type One: Ca2+]i transient after implementing contraction (adding NL96 model), but the models with instantaneous, or no Ca2+ troponin buffers (Type Four: panel (e); and Type Five: panel (f)), demonstrate large differences in the [Ca2+]i transient shape.The original models that include dynamic Ca2+]i, always accompanied by a decrease in the time of this peak (tpeak). As shown in Ca2+ troponin buffers have relative differences of less than 10% in systolic [Ca2+]i and five out them have relative differences less than 10% in tpeak. On the other hand, among the eight models with instantaneous or none Ca2+ troponin buffers, six models show differences larger than 50% and none has a difference less than 10% in systolic [Ca2+]i; in tpeak, one of the eight models  has a relative difference less than 10% and three of them have differences of more than 50%. In addition, except for one model , all of them have positive relative differences in systolic [Ca2+]i, indicating a systolic [Ca2+]i increase after the implementation of the NL96 contraction; and all eight models have negative Δtpeak, meaning the time of the peak is decreased by the implementation of contraction. The increase in maximum [Ca2+]i and the decrease in tpeak in models with instantaneous Ca2+ troponin buffers after the implementation of NL96  and after (dashed line) the NL96 implementation. It can be seen that after the implementation of NL96: (i) the instantaneous buffer factor β increases due to the elimination of the instantaneous Ca2+ troponin buffer from the denominator .The most significant change tended to be an increase in the maximum i (see Ca2+ troponin buffers show little change in t1/2 (less than 10%). Among the eight models with instantaneous or no Ca2+ troponin buffers, six have differences more than 50% and none has a difference less than 10%. Seven out of eight have negative Δt1/2, indicating the shortening in the time duration when [Ca2+]i stays above half the amplitude. This large difference in t1/2 is closely related to the change in systolic [Ca2+]i. A sharp increase in systolic [Ca2+]i greatly raises the half amplitude level ; this together with the large and narrow spike in [Ca2+]i generated by the inclusion of contraction, significantly decrease the time duration where [Ca2+]i stay above the half amplitude level.Results for the priming [2+]i see . All sixCa2+]i and the Ca2+ charge (Q) during one beat are the two parameters that do not change much after implementing NL96 contraction in all of the models. Thirteen out of fourteen models have diastolic [Ca2+]i changes of less than 10%, with only one model having a difference larger than 50% . Q for eleven out of fourteen models changes by less than 10% and only one model  has a change of more than 50%  models are shown in a to b; PES beats with a fixed PESI are labeled from a' to b' (see Step Three in Pacing Protocol in the a and a' (b and b') correspond to ES and PES paired beats for the shortest (longest) ESI.Postextrasystolic potentiation exists in intracellular Ca2+ troponin buffers show a significant postextrasystolic potentiation in the [Ca2+]i transients. As ESI increases, the amplitudes of the ES beats increase while the corresponding PES beats decrease. In addition, there’s no notable difference in [Ca2+]i transient before and after the implementation of NL96 contraction  / dF / dtmax (SS)) for six models are shown in MRCpes are given in Representative examples of MRCpes is well described  by a monotonically increasing curve with respect to PESI , four satisfy this condition, while among the eight models with instantaneous or none Ca2+ troponin buffers (Type Four and Five), only one satisfies C0 = 0  for ESI = 500ms and PESI ranges from 200ms to 1200ms in dF / dtmax stops decreasing when PESI is below 300ms and the minimum value is about 0.4. In order to fit this into C0 = 0.4. That is the reason for Mahajan_etal_2008 and other eight models, that C0 does not go to zero and also an example of how we choose the shortest ESI and PESI values.For all models  to PESI  as shown = 0 see . Here weMRCpes  decreased as ESI increased. From Ca2+ troponin buffers show unique plateau values for different ESIs while models with instantaneous or no Ca2+ troponin buffers converge to the same plateau values for different ESIs; this property will be further discussed in the PESPC subsection.The experimental results of Yue et al. showed tMRCpes  did not vary much as a function of ESI in the experiment of Yue et al.[MRCpes was observed as ESI decreased, presented as an increase of to,pes as ESI increased. The fourteen models behave differently regarding these two parameters. We will discuss these in detail in the to,pes and Tmrc,pes subsections.The time constant for ue et al.. In addiMRCes) as in Yue et al.[C0 ≠ 0 in the MRCpes, we have shifted the curves upward by C0 when plotting the PESPC (star *) on the same graph with the MRCes (open circle o) for comparison. We also use 95% confidence bounds as error bars for each data point on PESPC, which is the fitting confidence bound for CRpesmax, of each ESI value. Note that confidence bounds are relatively small compared to the scale of the graph.ue et al.. Note th2 values that are higher than 0.99. The double and the triple star (** and ***) means the 95% confidence bound overlaps Yue et al.[2 is less than 99% but more than 90% and the double-dagger (‡) means it’s less than 90%.A list of all parameters for the curve fitting of ue et al. data’s mCa2+ troponin buffers  have PESPCs well described by 2 values larger than 0.99. On the other hand, models with instantaneous or no Ca2+ troponin buffers (Type Four and Five) are not well fit by 2 values lower than 0.9 with one of them as low as 0.0048 . However, the poor quality of the fitting is not evident on The experiments of Yue et al.showed that PESPC decreased monoexponentially to a plateau level as ESI increases , meaningB) of CRThe plateau levels  have 95% confidence bounds overlap the mean±SD range of Yue et al.[Ca2+ troponin buffers (Type Four and Five) have 95% confidence bounds overlap the mean±2*SD range.ue et al., the meaue et al. and fourAParameter  has the least matching degree with Yue et al.[Ca2+ troponin buffers  have 95% confidence bounds overlap the mean±2*SD range from Yue paper and none of the models with instantaneous or none Ca2+ troponin buffers have confidence bounds overlapping this range  intercepts the line Ca2+ troponin buffers  and five out of eight models with instantaneous buffers (Type Four and Five) are in the mean±2*SD range. For this parameter, it seems that the dynamic buffers do not show much advantage over instantaneous buffers. However, in the Yue et al. paper, this interception is where MRCes intercepts with the line Ca2+ troponin buffers. But for most models with instantaneous buffers, this parameter is where MRCes intercepts with the line C0 ≠ 0. Therefore models with dynamic buffers still match this parameter better to the experimental data of Yue et al.[ue et al. is 284±3ue et al..Ttime constant for PESPC (pespc) is 176±18ms in Yue et al.[Ca2+ troponin buffers  and none of the models with instantaneous or none buffers (Type Four and Five) have 95% confidence bounds that overlap the experimental mean±2*SD range. Ten other models have time constants much larger(50% more) than Yue et al.[Ca2+ troponin buffers; five out of eight models have 95% confidence bounds larger than 50% of the center values, indicating a poor fit.The mean value (± SD) of the ue et al.. Only twue et al.. This pato,pes for different ESI values. Error bars are 95% confidence bounds. Figures for all models are provided in the to,pes increased as ESI increased. They explained this trend by suggesting that the refractory period is an increasing function of ESI[to,pes increasing exponentially with ESI; only the Iribe_etal_2006 model shows a non-monotonic behavior. Three out of eight models with instantaneous buffers (Type Four and Five) show the same trend as Yue et al.  while three models show a monotonically decreasing trend  and two  do not have monotonic behaviors. The Ranges of to,pes of the models match Yue et al. paper well as thirteen out of fourteen models fall within the experimental range.In the Yue et al. paper, to,peson of ESI. Five ouMRCpes  vs ESI for 6 models, where the dash line indicates the mean Tmrc,es from the Yue et al. paper; the grey area denotes the range of the mean±SD range. Open circles (o) are Tmrc,pes from curve fitting for different ESI values. Error bars are 95% confidence bounds. Figures for all the models are in Tmrc,pes. First, Tmrc,pes varied little with ESI; the mean (± SD) Tmrc,es was 181±41ms and the mean normalized Tmrc,pes  was 1.01±0.12. In our simulations, all of the models have their normalized time constant values overlap the mean±2*SD range from Yue paper while ten of the models have their Tmrc,es  overlap the mean±2*SD range from Yue paper. Therefore our contraction models fit this property with Yue paper quite well. The second major result from Yue paper is that the time constants of PESPC and MRCpes were close; the mean difference between the two time constants (± SD) was 13±23ms. Models with dynamic Ca2+ troponin buffers  are more consistent with the experiments than instantaneous buffers (Type Four and Five) in this respect. Three out of six models with dynamic buffers have 95% confidence bounds that overlap Yue’s mean±2*SD range while only one out of eight models with instantaneous buffers overlaps that. Six of them have large 95% confidence bounds inherited from the large Tpespc bounds, so we marked them by a symbol ※, indicating the 95% confidence bounds overlap Yue’s mean±SD but the confidence bound is so large (more than 3*SD) that we do not consider this as a real overlapping.Ca2+ dynamic and force dynamics. And since the Ca2+ released from SR (Jrel) is the main source for intracellular Ca2+, we propose that Jrel dynamics, which depends on RyR, There is a clear resemblance between the intracellular Ca2+ released from SR (Jrel) and the contractile strength on pacing intervals. In this study we have shown two sets of curves demonstrating the potentiation strength: the Postextrasystolic Mechanical Restitution Curve (MRCpes) and Postextrasystolic Potentiation Curve (PESPC). The MRCpes demonstrates how postextrasystolic contractile strength changes with respect to various PESI for a fixed ESI. From A) in PESPC captures this difference. Since we are interested in what causes the difference in models, we studied the correlation between the Ca2+ released from SR (Jrel) and the contractile strength of the postextrasystoles with various ESI and fixed PESI. We designed an analogous parameter AJ for the Jrel amplitude. Similar to our dF / dtmax fit, for a fixed ESI, we first fit the normalized Jrel_max (to the prime beats) of the postextrasystolic beats to a mono-exponentially increasing curve with respect to PESI . For most models, Jrel_max was well described by a mono-exponential increasing curve, similar to the contractile strength. Then we fit the plateau values of these curves to a mono-exponentially decreasing curve with respect to ESI ; we define the amplitude of this curve as AJ. Models with larger AJ suggest that their fully-restituted Jrel_max vary with respect to ESI. Therefore the correlation between A and AJ can be used as a gauge for the correlation between the dependence of Jrel and the contractile strength on pacing intervals. We performed a linear regression for AJ and A for thirteen (out of the fourteen) electromechanical models as shown in 2 = 0.848) between the two parameters. In addition, data from models with instantaneous buffers clustered around the origin while data from models with dynamic buffers were distributed along the regression curve. JThis means for models with instantaneous buffers, the insufficient variation in the fully-restituted postextrasystolic contractile strength responding to various ESI is a direct result of the reldynamics. Note that the model excluded from the linear regression was TenTusscher_etal_2006  require models that accurately reproduce the excitation-contraction coupling that occurs in the cardiac myocyte. While action potential duration restitution curves tend to be used to help validate EP models, there has been no systematic method to validate cellular contraction models. We suggest that the PESP protocol can be used in the validation process of mathematical models of cellular electromechanics and we present a methodology to include mechanics into cellular EP models of various types and compare results among the models with and without contraction.Ca2+ troponin buffers are much better candidates to implement contraction than EP models with instantaneous or none Ca2+ troponin buffers.The results from this study suggest four reasons for which electrophysiology models with dynamic Ca2+ troponin buffers. Type Two and Type three models only take one step in the implementation flowchart while Type Four and Type Five require one or two more steps to add in instantaneous Ca2+ troponin buffers and change the instantaneous ones into a dynamic form ; Postextrasystolic Potentiation Curve (PESPC); Minimum-value Axis Intercept Curve  and Time Constant Curve ) and their various parameters.Fourth, contraction properties of models with dynamic s better  . This means for models with instantaneous buffers, the insufficient variation in contractile strength responding to various pacing cycle length is a direct result of the Jrel dynamics. The interpretation for this result is twofold. First, Ca2+ release from SR is the main source for intracellular Ca2+ therefore Jrel can be the direct reason of contraction behaviors. Second, time constants play a crucial role in cell contraction. The time constants of Ca2+ binding with troponin C in Ca2+, CaTRPN and tension development. The RyR release kinetics is highly timing sensitive and strongly coupled to the CaTRPN binding process. Therefore models with dynamic buffers, which can describe the timing of interaction among different cell compartments more accurately, reproduce correct contraction behaviors. This has biological significance since most electrophysiological models incorporate instantaneous buffering due to their simplicity and lower computational cost.To unveil the mechanism why model with dynamic buffers reproduce PESP better than models with instantaneous buffers, we studied the correlation between contraction and calcium release from SR. We found a strong positive correlation  into dynamic buffers using the same strategy described in the Method section. We found that doing these changes not only affected the calcium as before but in addition the voltage AP, both by large amount (not shown).S1 File(DOC)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S6 File(DOCX)Click here for additional data file.S7 File(DOCX)Click here for additional data file.S8 File(DOCX)Click here for additional data file.S9 File(DOCX)Click here for additional data file.


Activation of metabotropic glutamate receptor 5 (mGluR5) by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. In contrast, the consequences of mGluR5 inhibition are less well understood. Here, we used BV-2 cells, retaining key characteristics of primary mouse microglia, to examine whether mGluR5 inhibition by 2-methyl-6-(phenylethynyl)-pyridine (MPEP) enhances cellular stress and production of inflammatory mediators.2+ ([Ca2+]i) were measured using luminescence and fluorescence assays.BV-2 cells were treated with MPEP, followed by determination of cellular stress using fluorescent dyes and high-content imaging. The expression of inflammatory mediators, endoplasmic reticulum (ER)-stress markers and phosphorylated AMPKα was analyzed by quantitative PCR, ELISA and Western blotting. Additionally, phospholipase C (PLC) activity, cellular ATP content and changes in intracellular free Ca2+]i, following Ca2+ release from the ER, mainly via inositol triphosphate-induced receptors (IP3R). The MPEP-induced ER-stress could be blocked by pretreatment with the chemical chaperone 4-phenylbutyrate and the Ca2+ chelator BAPTA-AM. Pretreatment with the AMPK agonist AICAR partially abolished, whilst the inhibitor compound C potentiated, the MPEP-dependent ER-stress. Importantly, the PLC inhibitor U-73122 and the Gi-protein inhibitor pertussis toxin (PTX) blocked the MPEP-induced increase in [Ca2+]i. Moreover, pretreatment of microglia with AICAR, BAPTA-AM, U-73122 and PTX prevented the MPEP-induced generation of oxidative stress and inflammatory mediators, further supporting a role for Gi-protein-mediated activation of PLC.Treatment of BV-2 microglia with 100 μM MPEP increased intracellular reactive oxygen species (ROS), mitochondrial superoxide, mitochondrial mass as well as inducible nitric oxide synthase (iNOS) and IL-6 expression. Furthermore, MPEP reduced cellular ATP and induced AMPKα phosphorylation and the expression of the ER-stress markers CHOP, GRP78 and GRP96. The MPEP-dependent effects were preceded by a rapid concentration-dependent elevation of [Ca2+-dependent pathway in microglia. The induction of cellular stress and inflammatory mediators involves PTX-sensitive Gi-proteins and subsequent activation of PLC, IP3R and Ca2+ release from the ER.The results emphasize the potential pathophysiological role of mGluR5 antagonism in mediating oxidative stress, ER-stress and inflammation through a CaThe online version of this article (doi:10.1186/s12974-014-0190-7) contains supplementary material, which is available to authorized users. Neuroinflammation involves the activation and recruitment of immune cells, including microglia, macrophages and lymphocytes, as well as an expression of factors designed to respond to injury and aid in repair. Microglia are resident immune cells in the central nervous system and play an integral role in the neuroinflammatory response. However, excessive microglial activation is a hallmark of neurodegenerative diseases .2+ mobilization and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) downstream signaling pathways i) i) ,26, whic2+]i have been implicated in the regulation of several activities of microglia, including proliferation, migration, cytokine release and ROS generation [2+]i, either by Ca2+ release from the ER or by entry through the plasma membrane i increase has not, to our knowledge, been reported in microglia.GPCRs play an important role in controlling microglial cell activity. The activation of mGluR5 in microglia has been suggested to involve the Gαq-protein signal transduction pathway through PLC, PKC and Ca2+ . Upon st2+ . Howeverin vitro [3R-dependent increase in [Ca2+]i following pharmacological mGluR5 inhibition. Importantly, the mGluR5-dependent effects could be blocked by PTX, indicating the involvement of a Gi protein-dependent signal transduction pathway.In order to elucidate the effects of the pharmacological inhibition of mGluR5 on cellular stress and inflammatory mediators in microglia, we tested the responsiveness of BV-2 mouse microglia to the selective mGluR5 antagonist MPEP. There is a close resemblance between BV2 cells and primary mouse microglia in terms of their inflammatory signaling pathways; therefore, BV-2 microglial cells can be used as a model for the activation of microglia in vitro ,41. In tDMEM, RPMI-1640 medium, penicillin, streptomycin, 0.05% (w/v) trypsin/EDTA, non-essential amino acids (NEAA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Hank’s balanced salt solution (HBSS), EnzChek Direct PLC assay kit, Hoechst-33342, dihydroethidium (DHE), Mitotracker Red CMXRos, MitoSOX Red, Fluo-4 AM and probenecid were obtained from Invitrogen . FBS was obtained from Connectorate , Cell Titer Glo® assay kit from Promega  and thapsigargin, AICAR, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), 6-methyl-2-(phenylethynyl)-pyridine (MPEP), BAPTA-AM, pertussis toxin (PTX), dantrolene sodium and sodium phenylbutyrate (PBA) from Sigma-Aldrich . Compound C and xestospongin C were obtained from Calbiochem . Specific antibodies against GRP78, AMPK, and its phosphorylated form were obtained from Cell Signaling Technology . Specific antibodies against β-actin and goat anti-rabbit IgG-horseradish peroxidase were obtained from Santa Cruz Biotechnology .2 flasks in RPMI-1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 μg/mL streptomycin and 100 U/mL penicillin, 10 mM HEPES and 0.1 mM NEAA at 37°C, 5% CO2. Prior to initiating the experiments, cells were resuspended in DMEM supplemented with 10% FBS, 1 g/L glucose, 2 mM glutamine, 100 μg/mL streptomycin and 100 U/mL penicillin, 10 mM HEPES and 0.1 mM NEAA, and seeded again to reach 70% confluence. Cells were then distributed to 96-well (1 × 103 cells/well), 24-well (2 × 104 cells/well), 12-well (5 × 105 cells/well) or 6-well (1.0 × 106 cells/well) plates. Cells in the exponential growth phase (60 to 70% confluence) were used for the experiments.Murine BV-2 microglia, immortalized by infection with v-raf/c-myc recombinant retrovirus , were ki-(ΔCt sample-ΔCt control), as described previously i in a concentration-dependent manner. Importantly, pretreatment of cells with 5 μM of the PLC inhibitor U-73122 completely abolished the effect of 100 μM MPEP. Furthermore, the MPEP-dependent induction of CHOP, GRP78 and GRP96 mRNA expression, measured after 24 hours, was also prevented by cotreatment of the cells with U-73122 , a specific inhibitor of Gi/o-type G-proteins. As shown in Figure 2+]i, suggesting the involvement of Gi/o-proteins in [Ca2+]i responses to MPEP. Pretreatment of BV-2 cells with PTX prevented the MPEP-dependent activation of PLC, while PTX alone did not affect basal activity of PLC .Several studies have demonstrated that G-protein coupled receptors may exert their effects through activation of PLC, which hydrolyzes phospholipids in cell membranes to IP2+ release from the ER, thereby producing cellular stress and enhancing the expression of inflammatory cytokines.In order to further study the impact of the intracellular signaling pathways that were shown to be influenced by mGluR5 above, we pretreated BV-2 cells with the various modulators and measured the effects of MPEP exposure on the regulation of cellular stress and inflammatory mediators. The levels of intracellular ROS, mitochondrial mass, mitochondrial superoxide production, iNOS mRNA as well as IL-6 mRNA and protein were determined. Pretreatment of cells with AICAR reversed the effects of MPEP on cellular stress parameters and cytokine expression, whereas compound C potentiated them Figure . BAPTA-Ain vitro and in vivo. It has been shown that the mGluR5 agonist CHPG diminished NF-κB activation, NADPH oxidase activity and ROS production in lipopolysaccharide (LPS)-stimulated microglia and improved functional recovery after traumatic brain injury i, with peak concentrations reached 15 minutes after addition of MPEP and return to baseline after 40 minutes. The fact that pre-exposure of cells to thapsigargin completely abolished the MPEP-induced increase in [Ca2+]i, and vice versa pre-incubation with MPEP reduced thapsigargin-induced [Ca2+]i increase, indicated that Ca2+ was released from the ER. Furthermore, the IP3R blocker xestospongin C effectively prevented the MPEP-induced Ca2+ release from the ER, whereas the RyR antagonist dantrolene was less effective, suggesting a major role for IP3R in the mGluR5-dependent signaling pathway.To begin to understand why mGluR5 blockade results in enhanced cellular stress in microglia, we investigated the mGluR5-mediated signaling pathway in BV-2 cells upon treatment with MPEP. Our results revealed a rapid increase in [Ca2+]i represent a major signaling pathway in microglia. Exposure of microglia to LPS leads to chronically elevated [Ca2+]i and it was shown that the Ca2+ chelator BAPTA-AM could prevent the LPS-induced release of NO and the cytokines TNF-α, IL-6 and IL-12 [3R by xestospongin C has been shown to abolish the production of TNF-α and IL-6 in activated microglia [Changes in [Cand IL-12 . Importaicroglia .2+-mediated signal transduction was observed under pathological conditions. Exposure to amyloid-β increased [Ca2+]i in cultured microglia, and basal [Ca2+]i was higher in microglia isolated from the brain of Alzheimer’s patients compared with microglia from non-demented individuals [2+]i may act directly on the mitochondrial membrane, leading to enhanced ROS production i by Ca2+ reuptake into the ER and/or mitochondrial dysfunction and impaired ATP production. The MPEP-dependent ATP depletion was ameliorated by pretreatment of cells with the AMPK inducer AICAR, indicating an involvement of AMPK in the modulation of the mGluR5-mediated effects. The depletion of ATP is likely to occur prior to the induction of AMPKα phosphorylation, in line with the role of AMPK in restoring cellular ATP levels i.The excessive Cand GRP96 . In this2+]i (data not shown), suggesting that the Ca2+ release from the ER precedes ATP depletion and AMPK activation. The protective effects of pretreatment of cells with AICAR may be explained by an increased cytoplasmic ATP concentration, which may facilitate energy dependent transport of Ca2+ back into the ER following mGluR5 blockade.Impaired AMPK signaling has been associated with ER-stress in different cell types ,38,70. W2+]i and subsequent induction of ER-stress markers and mitochondrial dysfunction involved the activation of PLC and could be prevented by pretreatment of BV-2 cells with the PLC inhibitor U-73122. Importantly, an increased PLC activity could be measured following incubation of microglia with MPEP, suggesting that mGluR5 inhibition caused cellular stress via the excessive activation of the PLC-IP3 pathway. It was reported that the mGluR5 agonist CHPG reduced the LPS-dependent inflammatory response and activated PLC, thereby increasing [Ca2+]i in microglia i was attenuated by the Gi-protein inhibitor PTX, the PLC inhibitor U-73122 and the IP3R antagonist xestospongin C, thus suggesting crosstalk between mGluR5 receptors and Gi-protein subunits to modulate PLC and induce IP3R-mediated Ca2+ release from the ER. Several studies in a variety of cells and tissues demonstrated an increase in [Ca2+]i following stimulation of Gi-coupled receptors i upon inhibition of mGluR5 was followed by a depletion of cellular ATP and activation of AMPK, resulting in the induction of pro-inflammatory mediators. The results suggest that xenobiotics inhibiting mGluR5 disrupt Ca2+ signaling and redox regulation in microglia, thereby promoting neuroinflammation.The present study demonstrates for the first time that antagonism of mGluR5 in BV-2 microglia results in increased cellular stress with mitochondrial dysfunction and ER-stress caused by an activation of PTX-sensitive Gi-proteins and subsequent PLC activation and IP


Petersburg Nuclear Physics Institute (PNPI). The spectral efficiency of the silicon UCN detector was measured by means of a gravitational spectrometer at Institut Max von Laue – Paul Langevin (ILL). The sandwich-type detector from two silicon plates with a 6LiF converter placed between them was also studied. Using this type of technology the UCN detector with analysis of polarization was developed and tested. The analyzing power of this detector assembly reaches up 75 % for the main part of UCN spectrum. This UCN detector with analysis of UCN polarization can be used in the new electric dipole moment (EDM) spectrometer.A silicon ultracold neutron (UCN) detector with an area of 45 cm It allows one to keep the amplitude of the signal at a sufficiently high level with respect to detector noise. The surface of the detector was coated with 6LiF (80 % 6Li enrichment) with a thickness of 0.6 mg/cm2 to 0.8 mg/cm2. The energy dependence of the registration efficiency was measured using the gravitational spectrometer.For the production of the Si UCN detector with large area we used a Si wafer of 78 mm diameter. The area of the detector (48 cm3He UCN detector with an Al window (100 µm thickness) and for the Si detector with a single 6LiF (80 % 6Li enrichment) converter on its surface. Efficiencies of both detectors were compared in the upper part of the spectrum when detectors were in the down position.3He detector although the boundary velocity of 6LiF (80 % 6Li enrichment) is higher than the boundary velocity of the 3He detector Al window. It is connected with that the absorption coefficient of materials with a high capture cross section is sufficiently bigger for the low energy region and that the density of coated 6LiF is 30 % lower than the density of a LiF crystal [The efficiency of the Si detector is better than that of the  crystal .6LiF converter by signal summation from the first and the second detectors and application of coincidence and anticoincidence techniques.This detector was developed at PNPI and investigated in an experiment at ILL. It allowed one to obtain the fraction of α-particles lost in the 6LiF converter. The thickness of the converter was between 0.6 mg/cm2 and 0.8 mg/cm2.The fraction of signals without coincidence is about 28 % and corresponds to events for which the triton was registered and the α-particle was absorbed in the 6LiF converter looses 14 % of events due to α-particle absorption in the 6LiF converter. Thus, the registration efficiency of this detector is about 86 %. This conclusion does not contradict with the results of the registration efficiency measurements carried out with the gravitational spectrometer.The results of the measurements allow us to conclude that the detector with one Si wafer and 3He detector with an Al window.The Si sandwich UCN detector is fully efficient for tritons, but decreases the flux of falling UCN with energies of 200 neV to 300 neV by about 20 % to 25 %. Thus, the Si sandwich detector has 75 % to 80 % efficiency for UCN with energies of 200 neV to 300 neV. It should be used with a vertical guide to accelerate UCN in the gravitational field, like the The scheme of this detector is presented in Nup is the count rate of the upper detector, Nd is the count rate of the down detector, P is the UCN polarization, and A is the analyzing power. In the ideal case this ratio is equal to 1 when the flux is completely polarized (the superconducting solenoid-polarizer is switched on) and it is equal to 0 when the flux is unpolarized (the solenoid is switched off). In reality there are several circumstances which disturb these correlations: the registration efficiencies of the detectors are different because of different areas; there is an effect of reflection of UCN from materials which does not depend on the polarization of the neutrons and decreases the efficiency of the polarization analysis; and the analyzing power of the detector depends on the properties of the analyzing ferromagnetic foil. Different materials which could be used as a substrate for thin ferromagnetic films were studied: Al foils  and Si wafers.The analyzing power of the detector can be defined by the polarizing ratio:H = 4 T) and for unpolarized flux . When we change the UCN polarization from 1 to 0, the polarizing ratio is changed by 0.8. Overall this result is not bad. The imperfection in splitting of spin components is connected with the effect of UCN reflection from the detector and the substrate of the analyzer. Measurements of the detector count rate ratios with an Al substrate but without ferromagnetic film showed that the albedo effect is determinative. Using the ratio obtained in these measurements as a correction allows one to reconstruct almost completely the full polarization value. The empty squares and circles are polarization ratios for polarized and unpolarized flux after correcting for the albedo effect.In conclusion, the newly developed UCN detector with polarization analysis is very promising, e.g., for an application in the nEDM experiment. Obtained parameters of this detector are sufficient to solve the task, but further improvements are expedient.


To dissect the regulatory network of cardiac Ca2+ handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca2+ extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca2+ uptake and accelerates the transfer of Ca2+ from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca2+ sparks and thereby inhibits Ca2+ overload-induced erratic Ca2+ waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca2+ uptake in the regulation of cardiac rhythmicity.Tightly regulated CaDOI:http://dx.doi.org/10.7554/eLife.04801.001 The heart is a large muscle that pumps blood around the body by maintaining a regular rhythm of contraction and relaxation. If the heart loses this regular rhythm it works less efficiently, which can lead to life-threatening conditions.Regular heart rhythms are maintained by changes in the concentration of calcium ions in the cytoplasm of the heart muscle cells. These changes are synchronised so that the heart cells contract in a controlled manner. In each cell, a contraction begins when calcium ions from outside the cell enter the cytoplasm by passing through a channel protein in the membrane that surrounds the cell. This triggers the release of even more calcium ions into the cytoplasm from stores within the cell. For the cells to relax, the calcium ions must then be pumped out of the cytoplasm to lower the calcium ion concentration back to the original level.tremblor—that has irregular heart rhythms because its heart muscle cells are unable to efficiently remove calcium ions from the cytoplasm. Embryos of the tremblor mutant were treated with a wide variety of chemical compounds with the aim of finding some that could correct the heart defect.Shimizu et al. studied a zebrafish mutant—called tremblor mutants. Efsevin binds to a pump protein called VDAC2, which is found in compartments called mitochondria within the cell. Although mitochondria are best known for their role in supplying energy for the cell, they also act as internal stores for calcium. By binding to VDAC2, efsevin increases the rate at which calcium ions are pumped from the cytoplasm into the mitochondria. This restores rhythmic calcium ion cycling in the cytoplasm and enables the heart muscle cells to develop regular rhythms of contraction and relaxation. Increasing the levels of VDAC2 or another similar calcium ion pump protein in the heart cells can also restore a regular heart rhythm.A compound called efsevin restores regular heart rhythms in Efsevin can also correct irregular heart rhythms in human and mouse heart muscle cells, therefore the new role for mitochondria in controlling heart rhythms found by Shimizu et al. appears to be shared in other animals. The experiments have also identified the VDAC family of proteins as potential new targets for drug therapies to treat people with irregular heart rhythms.DOI:http://dx.doi.org/10.7554/eLife.04801.002 Ca2+ influx after activation of the L-type Ca2+ channel in the plasma membrane induces the release of Ca2+ from the SR via ryanodine receptor (RyR) channels, which leads to an increase of the intracellular Ca2+ concentration and cardiac contraction. During diastolic relaxation, Ca2+ is transferred back into the SR by the SR Ca2+ pump or extruded from the cell through NCX1. Defects in cardiac Ca2+ handling and Ca2+ overload, for example during cardiac ischemia/reperfusion or in long QT syndrome, are well known causes of contractile dysfunction and many types of arrhythmias including early and delayed afterdepolarizations and Torsade des pointes  and the mitochondrial Ca2+ uniporter (MCU) serve as primary routes for Ca2+ entry through the outer and inner mitochondrial membranes, respectively  locus encodes a cardiac-specific isoform of the Na+/Ca2+ exchanger 1, NCX1h   . The treillation . In thistre mutant embryos suffer from Ca2+ extrusion defects and manifest chaotic cardiac contractions resembling fibrillation  establish a regular contraction pattern with rhythmic Ca2+ transients  (ncx1h deficient embryos (LB). A 32kD protein species was detected from zebrafish lysate due to its binding ability to efsevinLB and OK-C125LB, an active efsevin derivative conjugated to beads, but not to beads capped with ethanolamine alone or beads conjugated with an inactive efsevin analog (OK-C19LB)  . This mo embryos  and was K-C19LB) . FurtherK-C125LB . Mass spK-C125LB .10.7554/2+ handling. To examine this possibility, we injected in vitro synthesized VDAC2 RNA into tre embryos and found that the majority of these embryos had coordinated cardiac contractions similar to those subjected to efsevin treatment (myl7:VDAC2 transgenic fish in which VDAC2 expression can be induced in the heart by tebufenozide (TBF)  . KnockinO hearts . Convers efsevin . Further efsevin . Similar embryos . These f2+ flux  MEFs where VDAC2 is the only VDAC isoform being expressed  were maintained and bred as described previously  and cloned into pCS2+ or pCS2+3XFLAG. Full length cDNA fragments of zebrafish MCU (Accession number: JX424822) and MICU1 (JX42823) were amplified from 2 dpf embryos and cloned into pCS2+. For mRNA synthesis, plasmids were linearized and mRNA was synthesized using the SP6 mMESSAGE mMachine kit according to the manufacturers manual .tretc318 heterozygotes. Cardiac performance was analyzed by visual inspection on 1 dpf. The tre mutant embryos were identified either by observing the fibrillation phenotype at 2–3 dpf or by genotyping as previously described (VDAC2 mRNA and morpholino antisense oligos (5′-GGGAACGGCCATTTTATCTGTTAAA-3′)  were injected into one-cell stage embryos collected from crosses of escribed .tre embryos. 12 embryos collected from crosses of tretc318 heterozygotes were raised in the presence of individual compounds at a concentration of 10 µM from 4 hpf (tretc318 embryos manifest a chaotic movement resembling cardiac fibrillation with intermittent contractions in rare occasion (tre mutant embryos and NCX1h morphants (>500 embryos).Chemicals from a synthetic library  and fromom 4 hpf . Cardiacoccasion . Compounmyl7:GFP hearts were taken at 30 frames per second. Line-scan analysis was performed along a line through the atria or the ventricles of these hearts  treatment. Human EBs were differentiated for 15 days and treated with 5 mM CaCl2 for 10 min before DMSO or efsevin (5 μM) treatment. Images of beating EBs were acquired at a rate of 30 frames/s and analyzed by motion-detection software. For calcium recording, the EBs were loaded with 10 μM fluo-4 AM in culture media for 30 min at 37°C. Line-scan analysis was performed and fluorescent signals were acquired by a Zeiss LSM510 confocal microscope.The mouse E14Tg2a ESC and human H9 ESC line were cultured and differentiated as previously described . At day tre, and efsevin-treated tre embryos were placed on uncoated, microelectrode arrays (MEAs) containing 120 integrated TiN electrodes . Local field potentials (LFPs) at each electrode were collected for three trials per embryo type over a period of three minutes at a sampling rate of 1 kHz using the MEA2100-HS120 system . Raw data was low-pass filtered at a cutoff frequency of 10 Hz using a third-order Butterworth filter. Data analysis was carried out using the MC_DataTool (Multichannel Systems) and Matlab (MathWorks).2-day-old wild type, 2. For the recording of Ca2+ sparks and transients, the external solution contained 2 mM CaCl2. For Ca2+ transients, cells were field stimulated at 0.5 Hz with a 5 ms pulse at a voltage of 20% above contraction threshold. For all measurements, efsevin was added 2 hr prior to the actual experiment. Images were recorded on a Zeiss LSM 5 Pascal confocal microscope. Data analysis was carried out using the Zeiss LSM Image Browser and ImageJ with the SparkMaster plugin (Murine ventricular cardiomyocytes were isolated as previously described . Cells wr plugin . Cells w4HCO3 (30 min at 56°C) and 100 mM iodoacetamide (45 min in dark), respectively. Gel plugs were washed with 50 mM NH4HCO3, dehydrated with ACN, and dried down in a Speedvac. Gel pieces were then swollen in digestion buffer containing 50 mM NH4HCO3, and 20.0 ng/μl of chymotrypsin . Peptides were extracted with 0.1% TFA in 50% ACN solution, dried down and resuspended in LC buffer A .For pull down assays mono-N-Boc protected 2,2'-(ethylenedioxy)bis(ethylamine) was attached to the carboxylic ester of efsevin and its derivatives through the amide bond. After removal of the Boc group using TFA, the primary amine was coupled to the carboxylic acid of Affi-Gel 10 Gel . 2-day-old zebrafish embryos were deyolked by centrifugation before being lysed with Rubinfeld's lysis buffer . The lysDanio rerio IPI database v3.45 using the SEQUEST algorithm in the BioWorks software program version 3.3.1 SP1. All spectra used for identification had deltaCN>0.1 and met the following Xcorr criteria: >2 (+1), >3 (+2), >4 (+3), and >5 (+4). Searches required full cleavage with the enzyme, <4 missed cleavages and were performed with the differential modifications of carbamidomethylation on cysteine and methionine oxidation.Extracted peptides were analyzed by nano-flow LC/MS/MS on a Thermo Orbitrap with dedicated Eksigent nanopump using a reversed phase column . The flow rate was 200 nl/min for separation: mobile phase A contained 0.1% formic acid, 2% ACN in water, and mobile phase B contained 0.1% formic acid, 20% water in ACN. The gradient used for analyses was linear from 5% B to 50% B over 60 min, then to 95% B over 15 min, and finally keeping constant 95% B for 10 min. Spectra were acquired in data-dependent mode with dynamic exclusion where the instrument selects the top six most abundant ions in the parent spectra for fragmentation. Data were searched against the In situ hybridization was performed as previously described . DIG-labHeLa cells were transfected with a C-terminally flag-tagged zebrafish VDAC1 or VDAC2 in plasmid pCS2+ using Lipofectamine 2000 (Invitrogen). After staining with MitoTracker Orange (Invitrogen) cells were fixed in 3.7% formaldehyde and permeabilized with acetone. Immunostaining was performed using primary antibody ANTI-FLAG M2  at 1:100 and secondary antibody Anti-Mouse IgG1-FITC  at 1:200. Cells were mounted and counterstained using Vectashield Hard Set with DAPI .2+ indicator preferentially localized in mitochondria, for 1 hr at 15°C followed by a 30 min de-esterification period at 37°C. Subsequently, cells were permeabilized with 100 µM digitonin for 1 min at room temperature. Fluorescence changes in Rhod2  immediately after the addition of Ca2+  were monitored in internal buffer  using a FLUOSTAR plate reader .HeLa cells were transfected with zebrafish VDAC2 using Lipofectamine 2000 . 36 hrs after transfection, cells were loaded with 5 µM Rhod2-AM (Invitrogen), a Ca2+]c in suspensions of permeabilized cells or imaging of [Ca2+]m simultaneously with [Ca2+]c in intact single cells. Permeabilization of the plasma membrane was performed by digitonin (40 μM/ml). Changes in [Ca2+] in the cytoplasmic buffer upon IP3 (7.5 μM) addition in the presence or absence of ruthenium red (3 μM) was measured by fura2 in a fluorometer  and subsequently transferred to the microscope stage. Stimulation with 1 μM ATP was carried out in a norminally Ca2+ free buffer. Changes in [Ca2+]c and [Ca2+]m were imaged using fura2 (ratio of ex:340 nm–380 nm) and mitochondria-targeted inverse pericam (ex: 495 nm), respectively . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and 2 reviewers.Thank you for sending your work entitled “Activation of VDAC2 regulates mitochondrial Ca2+ uptake and cardiac rhythmicity” for consideration at The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Jodi Nunnari (Reviewing editor); Rosario Rizzuto (peer reviewer 1). Reviewer 2 remains anonymous.The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. All agree that the work is of high quality and represents a significant advance in our understanding of the role of VDAC in calcium handling and cardiac physiology. A majority of the comments pasted below can be addressed by a careful revision of the manuscript. However, as pointed out by reviewer 2, the experiments do not adequately test the authors' conclusion that Ca2+ is directly transferred between the ER and mitochondria. Additional experimentation, for example examining the effect of addition of a Ca2+ chelator.Reviewer #1:The authors point to mitochondrial Ca2+ buffering as a protective mechanism against erratic electrical activity, and indicates a prominent role of VDAC2 as a “facilitator” of Ca2+ transfer through MCU channels. In this respect, the authors could consider investigating whether additional, possibly isoform-specific mechanisms add to the role of VDAC2 in Ca2+ permeation across the outer mitochondrial membrane.1) Does VDAC 2 interact with MCU and/or with some of its regulators ?2) Is the expression of MCU and its regulators (“MCU complex”) altered in the tre mutants?3) Does pharmacological or molecular stimulation of VDAC2 affect the expression of the MCU complex?Reviewer #2:The manuscript reports exciting results but needs to be improved in the following ways:1) The results in 2) The authors state “... efsevin restores rhythmic cardiac contraction in tre by potentiating VDAC2 activity.” The authors do not measure VDAC2 activity and thus this conclusion is unnecessary speculation. VDAC2 is not an enzyme and thus its action is not so easily defined in this context. The first part of the last sentence on that page is the appropriate summarizing statement.3) The authors state: “VDAC is an abundant channel located on the outer mitochondrial membrane serving as a primary passageway for metabolites and ions including Ca2+  Rapizzi.“ The au4) In addition, the authors state “We examined whether Ca2+ released from intracellular stores could be directly transported into mitochondria through VDAC2 and whether this process could be modulated by efsevin...” The experiments reported do not distinguish between Ca++ release into the medium and quickly taken up my mitochondria or Ca++ directly traveling between the ER and the mitochondria. That could have been tested in 3C by having a strong chelator in the medium. Thus, with the current data the authors cannot claim evidence for direct transfer. They can claim a more effective uptake of Ca++ into mitochondria after efsevin treatment.Based on the results reported in 5B, the authors conclude that Ca++ has been transported into mitochondria in the permeabilized cells, ignoring the ER. The choice of Rhod2 was presumably based on its affinity for Ca++ and its likely saturation with Ca++ in the ER, so that observed changes are likely due to the mitochondria. The authors make no effort to point this out to the reader. It should be addressed.5) In the conclusions the authors state: “In this model, VDAC-dependent Ca2+ uptake controls the duration and the diffusion of cytosolic Ca2+ near the Ca2+ release sites...” How, physically, can VDAC control the “duration of cytosolic Ca++” and the “diffusion of cytosolic Ca++”? VDAC cannot control diffusion nor can it control the duration of diffusion. What I hope the authors want to say is that VDAC2 facilitates Ca++ uptake via MCU thus reducing the local Ca++ concentration more rapidly than otherwise. We appreciate the reviewers’ insightful and constructive suggestions to help us improve our manuscript. We have taken the reviewers’ advice and revised the manuscript accordingly. In addition to clarifying the text, we revised Reviewer #1 questioned whether the expression of MCU complex would be affected by NCX1 mutation or by the molecular stimulation of VDAC2.tremblor hearts with and without efsevin treatment. These findings are presented in We address this question by in situ hybridization. We find that the expression levels of MCU and MICU1 are comparable between wild type and Reviewer#1 also questioned whether VDAC2 interacts with MCU and its regulators.tremblor requires the activity of both VDAC2 and MCU provides genetic evidence supporting a functional interaction between VDAC2 and MCU  MICU1 is expressed in the developing heart  overal VDAC2 , and 3) al VDAC2 . These fReviewer #2 made a few suggestions toThe reviewer thought that the sample Mass Spec image presented inwas too small for inspection, and questioned the identities of peptides detected in our Mass Spec analysis.We thank the reviewer for pointing out this problem. In the original The reviewer also questioned whether we identified any other proteins interacting with efsevin.tremblor and knocking down VDAC2 abolishes the responsiveness of tremblor to efsevin. Together, these findings support VDAC2 as a target of efsevin.In the course of our study, we inspected six sets of data. VDAC2 was the only protein that was not present in the controls but was consistently identified from the efsevin affinity column. Vitellogenin 1 (vtg1) and NADPH quinine 1 (nqo1) were frequently found in both the control and efsevin affinity columns and thus were considered as contaminants. More importantly, our genetic analysis showed that overexpression of VDAC2 recapitulates the rescue effect of efsevin on Reviewer #2 raised a few questions regarding the mitochondrial Ca2+uptake analyses.The reviewer questioned the experimental detail about the mitochondrial Ca2+uptake experiment in HeLa cells.2+ into the samples to make the final Ca2+ concentration as approximately 10 μM. We have now clarified this information in the Methods and Figure Legend.We apologize for the confusing description. In this experiment, we added CaThe reviewer thought it would be helpful to point out how Rhod2 functions as a mitochondrial Ca2+indicator.We appreciate the reviewer’s concern and include this information in the Methods section.The reviewer thought that “examine whether Ca2+released from intracellular stores could be ‘locally’ transported into mitochondria through VDAC2” would be a better description of our analysis.2+ transfer through VDAC2 .We thank the reviewer for the suggestions. We have revised the manuscript accordingly.


We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ΔCm and Q suggests that Ca2+-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ∼10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca Failure of the beta-cells to release enough insulin is an important factor in the development of type-2 diabetes. Recent genetic data have demonstrated that the expression of genes involved in the final steps of insulin secretion is reduced in patients with type-2 diabetes 2+ influx through voltage-gated channels m) using the voltage-clamp mode of the patch-clamp technique As mentioned, the release of insulin from the secretory granules is a result of calcium-triggered exocytosis, which follows Ca2+ channels 2+2+ currents, Ca2+ influx shows a biphasic pattern resembling the biphasic exocytotic response In murine beta-cells, the rate of exocytosis is typically higher in response to shorter than to longer depolarizations, resulting in a biphasic capacitance pattern that has been suggested to correspond to biphasic insulin secretion, and to be caused by depletion of an immediately releasable pool (IRP) of granules located near Ca2+ channels in single human beta-cells in situ show a decline in the exocytotic response In isolated human beta-cells the rate of the exocytotic response does not decrease but rather increases with prolonged stimulation 2+ influx rather than of time 2+ influx, Q, and the resulting increase in capacitance, ΔCm, suggests pool depletion Q and ΔCm in beta-cells QRecently, a detailed theoretical study showed that to investigate whether pool depletion occurs, depolarization-evoked exocytosis should be studied as a function of CaMixed-effects statistical modeling m as a function of Q to investigate whether depletion of a pool of granules underlies the decline of the capacitance pattern in response to depolarizations of different lengths in INS-1 832/13 cells. We find no evidence of pool depletion contributing to the exocytotic profile and suggest that the reduced rate of exocytosis is due to inactivation of the Ca2+ current. Inclusion of high concentrations of the Ca2+ buffer ethylene glycol tetraacetic acid (EGTA) does not interfere with the IRP but lowers the sensitivity of later exocytosis to Ca2+ entry, indicating that later fusion occurs away from Ca2+ channels.Here we investigated the exocytotic response in INS-1 832/13 to obtain a deeper understanding of the dynamics of exocytosis. The INS-1 832/13 cells are rat insulinoma cells expressing human insulin with good glucose responsiveness 2 and cultured in RPMI 1640 media  with 11.1 mM glucose and supplemented with 10% FBS (wt/vol), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM β-mercaptoethanol. At ∼80% confluence the cells were split 1∶8 using Trypsin-EDTA and seeded into 35 mm petri dishes, where they were left overnight prior to patch-clamp experiments.Rat insulinoma INS 832/13 cells http://www.heka.com) and the software Pulse . Exocytosis was recorded as changes in membrane capacitance using the standard whole-cell configuration of the patch-clamp technique.Whole cell currents and exocytosis were recorded using an EPC-9 patch-clamp amplifier , 5.6 KCl, 2.6 CaCl2,1.2 MgCl2, 5 glucose, and 5 HEPES (pH 7.4 using NaOH). The standard pipette solution (IC1) consisted of the following (in mM): 125 Cs-glutamate, 10 NaCl, 10 CsCl, 1 MgCl2, 0.05 EGTA, 3 Mg-ATP, 0.1 cAMP and 10 HEPES (pH 7.15 with CsOH). In other experiments Ca2+ buffering was increased by including 10 mM EGTA and 2.5 (IC2) or 7 mM CaCl2 (IC3), respectively. The free Ca2+ concentration was estimated using the software Maxchelator : 118 NaCl, 20 tetra-ethyl-ammonium chloride  of the amplifier. Exocytosis was elicited by depolarizations from −70 to 0 mV at varying pulse durations . In another series exocytosis was evoked by a double pulse protocol, meaning that two 50-ms depolarizations from −70 to 0 mV separated by a 100-ms interval were applied. The double pulse experiments were part of a larger protocol where variations of the double-pulse and pulse-length protocols were used. In this larger series all depolarizations of varying pulse duration  were preceded by a 50-ms depolarization from −70 mV to 0 mV. The interval between the depolarization of varying length and the pre-pulse was 100 ms. In addition, a protocol using the combined pre-pulse pulse length protocol was used and each pair of depolarizations was followed by a third 500-ms depolarization from −70 to 0 mV applied either 200 ms (protocol I) or 10 s (protocol II) after the end of the second pulse. Finally, the responses to a train of ten 500 ms depolarizations delivered at 1 Hz were studied (train protocol). Pulses of different lengths were given in varying order, and no dependency on the order of the pulses was found. All experiments were conducted at 30–32°C.2+-currents were inspected visually, and data with large leak currents or high amounts of noise were discarded for the analysis. An in-house MATLAB (Mathworks Inc.) script was used to extract total calcium influx, Q, and increase in capacitance, ΔCm, for each experiment  to be compared to the control (“CTRL“) group for the final analyses.We found no statistical differences between the groups with high EGTA concentrations and ∼60 nM or ∼460 nM free CaThe final model for analysis of the standard pulse-length protocol with depolarizations of 5, 10, 20, 40, 80, 160, 320 and 640 ms was of the formm0 is the intercept representing an increase in capacitance in the limit of zero Ca2+ entry, βGROUP  is the slope of the relation between Q and ΔCm representing the efficacy of Ca2+ entry in the control and EGTA groups. Moreover, εij is a normally distributed error term, GROUPi is a covariate indicating whether cell i was in the “CTRL“ or “EGTA” group, b0i and b1i are factors allowing for cell-to-cell variation, and the parameters ΔCm0, βCTRL, βEGTA, σ, σb0 and σb1 are to be estimated.for observation j of cell i, where ΔCThe mixed-effects model describing the data from the two 50 ms pulses of the double-pulse protocol was m0,k) is allowed to differ between the two pulses.for pulse k = 1,2 of cell i. Parameters are as in Eq. 1, except that the intercept  was preceded by a 50 ms prepulse, the data from the pulses of varying lengths following the prepulse were well-described by a model with no intercept of the formfor observation j of cell i, Parameters are as in Eq. 1.The final model for the data from the third pulses, following 50 ms prepulses and pulses of varying length , and either 200 ms (protocol I) or 10 s (protocol II) rest, did not include any effect of the protocol, 2+ current sensitivity β was the same for all cells, since these protocols were only applied under control conditions.for observation j of cell i, Parameters are as in Eq. 1, except that the Cam)ij  =  ΔCm,i1 +… + ΔCm,ii, to cumulative calcium entry, (ΣQ)ij  =  Qi1 +… + Qij, for pulse j of cell i. The final model was similar to Eq. 4,Data from the train protocol was analyzed by relating cumulative increase in membrane capacitance, (ΣΔCQij2 to the models describing pulse-length data without (Eq. 1) or with (Eq. 3) a prepulse, to test whether there was a significant concave (downward curvature) deviation from linearity in the data, which would be indicative of pool depletion To investigate whether pool depletion occurred, we added a quadratic term γThe statistical software R m) reflecting exocytosis showed a biphasic relation to the pulse length such that the average rate of exocytosis was higher during short than during longer pulses  that enters the cell during each depolarization does not have a simple relationship to pulse length (m to Q rather than to pulse length 2+ influx measured as charge (Q). Plotting ΔCm to Q in this case gave a linear relationship m to Q relation  was included in the patch-pipette. Under these conditions the general patterns of ΔCm and Q were unchanged, but the amount of exocytosis was reduced while Ca2+ entry increased. EGTA is a relatively slow Ca2+ buffer, and 10 mM EGTA chelates Ca2+ ions at a typical distance of ∼100 nm away from Ca2+ channels m as a function of Q, from ∼0.8 fF/pC in control cells to ∼0.3 fF/pC in the presence of intracellular EGTA. Interesting, there was no difference between the responses in groups with 10 mM EGTA and ∼60 nM or ∼460 nM free Ca2+, excluding that the effect of EGTA was because of lower Ca2+-stimulated granule recruitment due to a reduction of basal [Ca2+]. We therefore united the two EGTA pools in To obtain spatial information of the exocytotic machinery, the CaThe data representation in 2+ current sensitivity in the control and EGTA group, respectively. We use the terminology “Ca2+ current sensitivity” to mean “the sensitivity of exocytosis to Ca2+ entry via Ca2+ channels”, which is different from the biochemical Ca2+ sensitivity of exocytosis The data was well described by a linear mixed-effects model with fixed effects describing the Ca2+] (p = 0.89). Moreover, the data could be fitted with a model with common intercept (ΔCm0) for the control and EGTA groups. This intercept reflects exocytosis in the limit of zero Ca2+ entry. The random effects describe cell-deviation from the group estimates for the intercept and Ca2+ current sensitivity . Thus, EGTA interfered with granule fusion caused by larger amounts of Ca2+ entry, suggesting that later exocytosis occurred away from Ca2+ channels 2+ buffering by EGTA did not interfere with the small pool, which is likely located very near Ca2+ channels, and corresponds to IRP. There was substantial cell-to-cell variation with estimated standard deviations of 6.7 fF and 0.32 fF/pC for the random effects of the intercept and the Ca2+ current sensitivity, respectively. There was no evidence for a deviation from linearity in the data (p = 0.39).The common intercept for the control and EGTA groups was estimated to be 6.5±1.6 fF, significantly larger than zero , corresponding to ∼10 granules based on a mean single-granule capacitance of 0.6 fF in INS-1 832/13 cells An alternative to the pulse-length protocol for the study of pool depletion is the double-pulse protocol 2+ current inactivation, we analyzed the capacitance increases ΔCm as a function of Ca2+ entry Q, derived under control conditions and in the presence of EGTA. A simple linear regression model with slope depending on both the group (CTRL/EGTA) and the pulse number revealed no statistically significant dependence on the pulse number (estimated slopes for the CTRL group: 1.18±0.28 vs. 0.42±0.31 fF/pC (p = 0.075); EGTA: 0.33±0.19 vs. 0.25±0.21 fF/pC (p = 0.79)). The data from the two groups were therefore further analyzed by a linear mixed-effects model with a slope depending on whether EGTA was present or not, but independent of the pulse number. In contrast, the intercept depended on the pulse number, but was not influenced by EGTA, and therefore common for the control and EGTA groups (2+ current sensitivity from 0.92±0.28 fF/pC in the control group to 0.26±0.19 fF/pC in the EGTA group (p<0.001). Together, these results confirm the presence of an IRP of ∼7 fF, which is unaffected by EGTA and depleted by the first depolarization. The exocytotic response to higher amounts of Ca2+ entry is reduced by EGTA and similar for the first and the second pulse, indicating that the first pulse does not deplete the pool responsible for later exocytosis.To circumvent this issue without neglecting the problem of CaA groups . For theThe double-pulse data set analyzed above is a subset of a larger dataset where 50 ms prepulses followed by 100 ms resting periods at −70 mV and depolarizations of varying lengths , were applied to each cell .2+ current sensitivity of the second pulses of varying length compared to the case of the pulse-length protocol with no prepulse , while it was significantly lower in the EGTA group . Cell-to-cell variation was rather large as quantified by the standard deviation of 0.62 fF/pC for the random effect on Ca2+ current sensitivity. In summary, in the control group the Ca2+ current sensitivity was not reduced following a 50 ms prepulse . Similarly, the application of a prepulse did not affect the Ca2+ current sensitivity in the EGTA group . These results speak against pool depletion causing the capacitance pattern seen in Linear mixed-effects modeling of ΔCnd pulse  showed tm-Q relation did not show any concave deviation from linearity as expected in the absence of pool depletion, but rather a convex relation as indicated by a positive estimate (0.0013±0.0004 fF/(pC∧2), p = 0.0021, n = 31 cells) of the coefficient γ of the quadratic term (see Methods). Such a convex relation between ΔCm and Q can arise from exocytosis occurring away from Ca2+ channels in a more general submembrane domain 2+ elevations and exocytosis mainly away from Ca2+ channels.Interestingly, the ΔC2+ channel inactivation is the cause of the declined exocytotic response from another perspective, we generated another set of data. In this experiment the protocol started with two depolarizations, prepulse plus pulse-length protocol, as in the previous experiment. The difference was that these two depolarizations were followed by a third 500-ms depolarization to 0 mV after a rest interval at −70 mV lasting either 200 ms (protocol I) or 10 seconds (protocol II). Our idea was based on the fact that the Ca2+ current recovers much faster 2+ current sensitivity between protocols I and II for the third depolarization to be equal, and Ca2+ current sensitivity similar to the pulse-length protocols investigated above. In contrast, if the current recovers in 10 seconds but the pool does not, then the Ca2+ current sensitivity should be lower for the third pulse in protocol II than for the pulse-length protocols. Moreover, the current would recover to some extent while the pool would not during a 200 ms rest period. Thus, the Ca2+ current sensitivity for the third pulse should be even lower in protocol I than in protocol II if the pool of granules is depleted.To address the question whether pool depletion or Ca2+ influx during the first 50 ms of the third pulse , and related it to the Ca2+ influx during the 50 ms prepulse (Q1). Mean recovery was then defined as the average of the ratios Q3,50 ms/Q1. We found that following a 50 ms prepulse and a 50 or 100 ms second pulse, the Ca2+ current did not inactivate much, and hence recovered substantially . In summary, the exocytotic response recovers in parallel to Ca2+ currents, and the Ca2+ current sensitivity is unaffected by the two preceding pulses. These findings speak against pool depletion.Since our argumentation is based on substantial CaHaving established that short depolarizations deplete a small IRP of ∼10 fF, but do not exhaust subsequent exocytosis resulting from a larger pool, we analyzed if more intense stimulation, in the form of the widely used train protocol consisting of 500 ms pulses delivered at 1 Hz, could deplete the larger pool, likely the RRP.2+ entry Previous results 2+ current sensitivity was estimated to be 1.81±0.48 fF/pC. However, when we analyzed the responses to the first three pulses only, it was clear that the data showed a non-zero intercept , because the first pulse released relatively more granules than the second and third pulses. This finding is in agreement with the IRP from the data analyzed in the previous sections. The Ca2+ current sensitivity was estimated to be 1.46±0.62 fF/pC for the first three pulses. Cell-to-cell variation was big with estimated standard deviations of the random effects amounting to 17.9 fF for the intercept, and 1.55 for the Ca2+ current sensitivity. In the full data set including all ten pulses the non-zero intercept was masqueraded by an upward curvature towards the end of the train, since a linear fit tends to lower the intercept and increase the slope in order to approximate the last data points. Our results are similar to the patterns observed in rat beta-cells stimulated by a train of 40 ms depolarizations delivered at 10 Hz When analyzing all ten pulses of the train, we found no sign of pool depletion, and an intercept not different from zero . The Ca2+ entry to insulin release using an accepted model cell system, with the main finding that the RRP in INS-1 832/13 cells is not as easily depleted as previously thought, and that Ca2+ current inactivation can masquerade as pool depletion. INS-1 832/13 cells are a commonly used cell line in confirming human findings. We are most aware that cell lines are not primary cells and that there are large differences between murine and human beta-cells http://www.nc3rs.org.uk) we should strive to work as much as possible in cell lines In this work we have carefully studied the basic mechanisms underlying the coupling of Cam is related to pulse duration  is situated near Ca2+ channels, and is similar to IRP measured in murine cells, although ten times smaller in size. Our finding that EGTA does not interfere with release from this small pool supports this conclusion. Similarly, Yang and Gillis We found evidence of a tiny pool of granules of ∼7 fF, or ∼10 granules, that could be released my minimal amounts of entering Ca2+ current sensitivity of exocytosis by more than 50%. This suggests that in INS-1 832/13 cells most exocytosis occurs away from Ca2+ channels from a granule pool that is not depleted by depolarizations lasting even up to a second. Either this pool is very large or is very rapidly refilled. The exocytotic responses is of similar magnitude in INS-1 832/13 cells as in primary cells, but electron micrographs from insulinoma cells demonstrate that these cells have fewer granules and of smaller size 2+-induced exocytosis, is ∼100 fF in INS-1 cells. Accordingly, one half of this pool is released by a 640 ms depolarization  and a large cell-to-cell variation (the standard deviation of the random effect was 17.9 fF), indicating that the train protocol is not appropriate for determining the size of the IRP.Interestingly, and supporting our conclusion, repeated 500-ms pulses did not lead to the exhaustion of the increases in membrane capacitance in INS-1 832/13 cells  [12]. Ho2+ entry was taken into account. This convex ΔCm vs. Q relation could result for example from residual Ca2+ from the first pulses increasing the Ca2+ current sensitivity , or residual Ca2+ could increase the rate of docking and refilling, which are Ca2+ dependent processes. Moreover, in INS-1 cells a train of 500-ms depolarizations did not provoke pool depletion at temperatures similar to the ones used here, but at lower temperature (24°C) capacitance increases were markedly reduced after the first two pulses Late exocytosis was not reduced by the train of pulses, but rather tended to increase when Ca2+ channels the Ca2+ concentration reaches a few μM 2+ channels, the fusing granules must be “highly Ca2+ sensitive” in order to be released. Yang and Gillis 2+ sensitive pool (HCSP) in INS-1 cells, and it seems likely that the HCSP is also present in INS-1 832/13 cells and is responsible for most of the exocytosis seen in our experiments. Moreover, the data suggest that this pool in INS-1 832/13 cells not only have high Ca2+ sensitivity but is also difficult to deplete.Away from CaIt has been suggested m and Q, as observed in the INS-1 832/13 cells, is also seen in pulse-length data from mouse beta-cells ++A linear relation between ΔC2+ dynamics must be taken into account for a better elucidation of the biphasic nature of insulin secretion.In summary, we demonstrate that the release mechanism in INS-1 832/13 is different from primary cells, in particular that the RRP in INS-1 832/13 is not easily depleted. Our findings underline that the interplay between RRP depletion and phasic CaData S1m vs. Pulse length protocol (5–640 ms) ΔCQ data ( and ).(DAT)Click here for additional data file.Data S2m vs. 50 ms prepulse followed by pulse length protocol (50–800 ms) ΔCQ data (and).(DAT)Click here for additional data file.Data S350 ms prepulse followed by pulse length protocol (50–800 ms) and a third 500 ms pulse after 200 ms or 10 s (and).(DAT)Click here for additional data file.Data S4m vs. Train ΔCQ data .(DAT)Click here for additional data file.


There is growing evidence of clinical data recently for successful outcomes of non-operative management (NOM) for blunt hepatic and spleen injuries (BHSI). However, the effectiveness of NOM for high-grade BHSI remains undefined. The aim of the present study was to review our experience with NOM in high-grade BHSI and compare results with the existing related data worldwide.In this retrospectively protocol-driven study, 150 patients with grade 3–5 BHSI were enrolled during a 3-year period. Patients were divided into immediate laparotomy (immediate OP) and initial non-operative  groups according to hemodynamic status judged by duty trauma surgeon. Patients who received initial NOM were divided into successful NOM (s-NOM) and failed NOM (f-NOM) subgroups according to conservative treatment failure. We analyzed the clinical characteristics and the outcomes of patients.Twenty-eight (18.7%) patients underwent immediate operations, and the remaining 122 (81.3%) were initially treated with NOM. Compared with the initial NOM group, the immediate OP group had significantly lower hemoglobin levels, a higher incidence of tube thoracostomy, contrast extravasation and large hemoperitoneum on computed tomography, a higher injury severity score, increased need for transfusions, and longer length of stay (LOS) in the intensive care unit (ICU) and hospitalization. Further analysis of the initial NOM group indicated that NOM had failed in 6 (4.9%) cases. Compared with the s-NOM subgroup, f-NOM patients had significantly lower hemoglobin levels, more hospitalized transfusions, and longer ICU LOS.NOM of high-grade BHSI in selected patients is a feasible strategy. Notwithstanding, patients with initial low hemoglobin level and a high number of blood transfusions in the ICU are associated with a high risk for NOM failure. Blunt abdominal trauma (BAT) resulting from a traffic accident, fall, assault, or occupational accident is not unusual in the emergency room. The prevalence of intra-abdominal injury after BAT has been reported to be high at 12-15%. Since tPatients admitted to Kaohsiung Chang Gung Memorial Hospital with BHSI between January 2010 to December 2012 were retrospectively reviewed. All patients were initially assessed and resuscitated at the emergency room (ER) according to the advanced trauma life support (ATLS) guidelines. The selection of patients for a nonsurgical management protocol was baseAlthough this was a retrospective study, data on age, gender, mechanism of blunt trauma, initial vital signs , hemoglobin level, Glasgow coma scale (GCS), alcohol intoxication, incidences of endotracheal intubation and tube thoracostomy, CT findings, Injury Severity Score (ISS), blood transfusion at ER and during admission, length of stay (LOS) at intensive care unit (ICU), duration of hospitalization, and outcomes, including morbidities and mortalities, were prospectively collected. ER transfusions included units of blood transfused during resuscitation at ER or before transfer from a local clinic, whereas admission transfusions referred to all units administered during hospitalization, except resuscitation at ER. The severity of BHSI was graded according to the classification of the American Association for the Surgery of Trauma (1994 revision). Patients with concomitant liver and spleen injuries were assigned to either liver or splenic injury group according to the organ with higher injury grading. The presence of intra-abdomen fluid was determined using CT. The amount of hemoperitoneum was quantified as follows: minimal, perihepatic blood in subphrenic or subhepatic space or perisplenic fossae (<500 mL); moderate, minimal plus blood along paracolic gutter (500-1000 mL); and large, moderate plus blood accumulating in pelvic cavity (>1000 mL). Patients who died at ER, those without available abdominal CT, and those with CT findings consistent with grade I or II injuries were excluded from the present study. High-grade injury referred grade III-VI in blunt hepatic injurIES (BHI) and grade III-V in blunt spleinc injuries (BSI).The patients were initially categorized into two groups: those initially treated non-operatively were included in the initial NOM group and those receiving early laparotomy at ER because of hemodynamic instability or suspected peritonitis were included in the immediate OP group. Patients in the initial NOM group were admitted to ICU for close monitoring and were further divided into two subgroups, the s-NOM included patients that treated successfully with conservative methods and the f-NOM included those who eventually required laparotomy according to the judgment of trauma surgeons after observation in ICU.Data are presented as percentages for categorical data, and means ± SE for ordinal and continuous data. Statistical analyses were performed using the chi-square test or Fisher’s exact test for discrete variables and the Mann Whitney U test for continuous variables. All differences at the p < 0.05 level were considered statistically significant.During the 3-year study period, 150 patients presented with high-grade BHSI, of whom 91 and 59 had BHI and BSI, respectively. The relationship between the severity of hemoperitoneum and CT grading is shown in Table The most common causes of high-grade BHI were motorcycle collision , motor vehicle collision , falls from greater height  or from own height , pedestrian struck n = 3, 3.3%), assaults n = 2, 2.2%), and bicycle collision . In high-grade BSI, motorcycle collisions were responsible for most injuries , while other causes included motor vehicle collision , assaults , falls from own height  and from greater height , bicycle collision , and pedestrian struck   on the second postoperative day, and 1 with grade IV BSI (ISS:38) and post angioembolization re-bleeding died of liver cirrhosis 6 days postoperatively.NOM was initially applied in 81.3% n = 122) of all patients with high-grade BHSI. Twenty-eight (18.7%) patients underwent emergency laparotomy. The incidences of initial NOM for high-grade BHSI were 88% (80/91) and 71% (42/59), respectively. The comparisons of characteristics of the initial NOM and immediate OP groups are presented in Table 2 of all Of the 122 patients initially treated with NOM, 116 were treated successfully (95%). Further analysis of the two subgroups of the initial NOM group is presented in Table In terms of operative treatment, comparisons between the s-NOM and patients receiving operations (immediate OP + f-NOM) are shown in Table NOM is currently the main treatment for patients with BHSI and has shown excellent results, 13–15. In present study, NOM was applied in 81.3% of high-grade BHSI patients with a failure rate of 3.7% and 7.1% for BHI and BSI, respectively, resulting in an overall failure rate of 4.9%. Our results are comparable to those of prior studies[Among previous organ-specific studies, those examining high-grade BHI, reported the use of initial NOM in 78%(63/81) of patients with a failure of 3.7%(3/81). In the Most prior studies concluded that the main reason for the failure of NOM is the hemodynamic instability, whereas this observation was contradicted by Mitsusada et al.. VariousAnother factor of NOM failure is a concomitant BHSI. In a study of Sharma et al. found a A potential drawback of NOM is that hollow viscus injuries are overlooked. Swaid et al. reportedMultiple studies have documented that successful NOM not only can increase organ salvage rates, but also can decrease blood transfusions requirements, hospital stays, nontherapeutic laparotomy rates, septic complications, and mortality rates, 5. StudOf the two mortality cases in f-NOM group, one ISS:34) died of concomitant severe lung contusion and the other one (ISS:38) died of coexistent liver cirrhosis coagulopathy. Fang et al. died of  suggesteThe present study had two limitations; one was we put discussions of BHI and BSI together and another was the lower number of cases included in the f-NOM group. Hence, it may not be an accurate reflection of the true results of the applicability of NOM to isolated hepatic or splenic injuries. Despite these limitations, our results provided valid information on the applicability of NOM to high-grade BHSI as the data of the study was collected prospectively with strict protocols.Parallel to the rapid growth of economics in Taiwan, motor vehicles accidents will continue to contribute significantly to the high-grade BHSI. Our study shows lower morbidities in successful NOM justify further attempts for NOM in high-grade BHSI in selected patients aiming at formulating a specific standardized diagnostic/management algorithm. With the incorporation of a protocol, 95% of hemodynamically stable patients with high-grade BHSI can be managed safely with NOM. This study can help emergency practitioners and trauma surgeons recognize and introduce the practice.