When deformation only was induced, it resulted in an immediate elevation of [Ca2+]i which was localized to the probe periphery. Multiple consecutive local Ca2+ responses were induced by sequential application of low level forces, with characteristic recovery time of ~2 s. The duration of [Ca2+]i elevations was directly proportional to the tip-cell contact time. In contrast, cell micro-injury resulted in transient global elevations of [Ca2+]i, the magnitude of which was independent of the tip-cell contact time. Sequential micro-injury of the same cell did not induce Ca2+ response within 30 s of the first stimulation. Both local and global Ca2+elevations were blocked in Ca2+-free media or in the presence of stretch-activated channel blocker Gd3+. In addition, amount of Ca2+ released during global responses was significantly reduced in the presence of PLC inhibitor Et-18-OCH3. Thus, we found qualitative differences in calcium responses to mechanical forces inducing only membrane deformation or deformation leading to micro-injury.Micro-damage of bone tissue is known to regulate bone turnover. However, it is unknown if individual bone cells can differentiate between membrane deformation and micro-injury. We generated osteoblasts from mouse bone marrow or bone morphogenetic protein 2-transfected C2C12 cells. Single cells were mechanically stimulated by indentation with the atomic force microscopy probe with variable force load either resulting in membrane deformation only, or leading to membrane penetration and micro-injury. Changes in the cytosolic free calcium concentration ([Ca It was recently suggested that, in addition to deformation forces, microdamage plays an important role in regulating bone turnover and strength3. Bone is restructured through the coordinated action of bone cells, osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption. Cells of osteoblastic origin, including osteoblasts, osteocytes and bone lining cells are believed to act as mechanosensors in bone tissue4. Understanding how bone cells sense and react to mechanical forces is important for gaining insight into the mechanisms of bone adaptation to its mechanical environment.Mechanical stimulation of bone is well-known to regulate bone volume, structure and compositionin vivo, several models have been developed to understand the effects of mechanical stimulation on bone cellsin vitro5. These models include application of hydrostatic pressure, longitudinal substrate stretch and fluid shear. These studies have established that osteoblastic cells can sense mechanical stimulation through plasma membrane receptors, stretch activated ion channels, as well as focal adhesion sites6. Ca2+ signaling was shown to be the prominent first response of osteoblastic cells to any type of mechanical stimulation9. Ca2+ signaling induced by mechanical stimulation in turn influences numerous bone cell functions such as cytoskeletal reorganization7, gene expression10, proliferation and differentiation6. However, these studies also identified significant complex signaling interactions between multiple cells6, making it difficult to de-convolute the responses of single cells to mechanical stimulation. Moreover, these techniques do not allow exact control of forces applied to individual cells, nor do they report single cell micro-injury.Because of the complexity of the bone environment11, pipette suction13, and atomic force microscopy (AFM)15 have been used to study responses of individual osteoblasts to mechanical stimulation. From these techniques, only AFM allows application of precisely controlled forces in the nano-Newton scale, as well as provides readout of the extent of membrane deformation16. Moreover, AFM can be used with cantilever tips of different geometries, which allow additional control of the distribution of the force. Spherical tip allows creating high range of membrane strains17, while pyramidal tip allows reversible membrane penetration, which does not result in long-term cell damage19.Local indentation techniques allow characterization of the response to mechanical stimulation at the single-cell level. Pipette microinjection20. Mechanical forces of different magnitude were applied using AFM. To monitor cell responsiveness to mechanical forces, changes in cytosolic free Ca2+ concentration ([Ca2+]i) were assessed.The goal of this study was to examine how a single osteoblastic cell responds to forces inducing cell membrane deformation only, or membrane deformation resulting in micro-injury. We used either primary bone marrow cells cultured with ascorbic acid, or C2C12 cells stably transfected with bone morphogenic protein (BMP) 2. C2C12 cells have been shown to undergo osteoblastic differentiation when treated with BMP-22 asphyxiation followed by cervical dislocation. Femora and tibiae were isolated and separated from soft tissue. The bones were cut in two, placed in an Eppendorf tube, centrifuged twice at 103 rpm for 30 seconds. Cells (~2×107 cells/mouse) were re-suspended in Minimum Essential Medium (MEM (Eagle), from Invitrogen) supplemented with 2 mM of L-glutamine, 100 IU of penicillin, 100 μg/ml of streptomycin and 10% of fetal bovine serum (Wisent), and 5×106 cells were plated on round 25 mm No.1 glass coverslips (Matteck Corporation), and cultured with 50 μg/ml of ascorbic acid at 5% CO2, 37°C for 4–6 days to 50–70% of confluence, which allowed easy identification of individual cells. The media was replaced every third day. The osteoblastic phenotype was confirmed by alkaline phosphatase staining .All procedures were approved by McGill University’s Animal Care Committee (protocol number 2013-7332) and conformed to the ethical guidelines of the Canadian Council on Animal Care. Six week old male C57/BL6 mice (Charles River) were acclimatized for 1 week, fed ad libitum, and kept on a 12-hour light/dark cycle prior to euthanasia by CO4 cells on round 25 mm No.1 glass coverslips (Matteck corporation). Cells were cultured in Dulbecco's Modified Eagle Medium  supplemented with 2 mM of L-glutamine, 100 IU of penicillin and 100 μg/ml of streptomycin and 10% of fetal bovine serum at 5% CO2, 37°C to 50–70% of confluence. The media was changed every third day. The osteoblastic phenotype was confirmed by alkaline phosphatase staining.C2C12 cells stably transfected with BMP-2  were plated at 2.5×102+-sensitive dye fluo4–AM , added to 2 ml of culture media for 40 minutes at room temperature. The cells were washed twice with physiological solution 21, the coverslip was assembled onto the peek fluid chamber (Asylum Research) and physiological buffered solution (1.5 ml) was added after loading with fluo4-AM. Gd3+ (Sigma) was dissolved directly in buffer to 50 μM final concentration. 1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine  stock solution (5 mg/ml) was prepared in ethanol, which was used as a vehicle (0.07%) for corresponding experiments. Et-18-OCH3 was used at 5 μM final concentration. Cells were pretreated with inhibitors for 45 min at room temperature before mechanical stimulation. The cells were illuminated with 488 nm laser light and the emitted light was collected with the Cascade II camera. In each experiment a time-sequence of fluorescence images was acquired with one frame taken every 345 ± 10 ms with 250 ms exposure time. Traces were extracted from the video files using a code for Matlab 2011A provided in22. The fluorescence signal in cells not exposed to mechanical stimulation did not demonstrate associated changes and was used as a reference for signal correction for bleaching and cantilever reflection artifacts using the protocol for data processing for systematic signal recovery that we established and described previously22.The cells were loaded with 1.5 μl of CaThe experiments were conducted using an MFP-3D-BIO AFM  mounted on an Olympus IX-71 inverted optical microscope. The sample placed in the closed fluid cell was left undisturbed for 15 min to achieve thermal equilibrium at 37°C. A 60X oil immersion objective with 1.45 NA (Olympus) was put into contact with the coverslip allowing optical access from the bottom and AFM access on top of the sample. The region of interest was located and aligned with the cantilever tip using the bright field and fluorescence images.2 contact area with a focused ion beam microscope (FEI DB235). When sharp tips were used, the membrane rupture force event was not distinguishable in the force curve. Using Hooke’s law (F=k⋅d), the force exerted by the probe (F) was determined from the cantilever spring constant  and deflection (d). The net extension of the piezo element, the speed, the time of indentation, the time between indentations and the number of indentations were controlled using the Asylum MFP3D software in IgorPro 6.22 platform.The cantilever tip , was etched down to 1 μm23. Statistical differences were assessed using Fisher Exact Probability test for categorical data or Student’s t-test for continuous data and were accepted as significant at p < 0.05. Statistical analysis was performed in Microsoft Excel 2007.Data are presented as representative images, representative traces, means ± SE with n being the number of experiments analyzed. The normalized amplitude as a function of time, the amplitude decay rate as a function of recovery time and the response duration as a function of the contact time were analyzed using least squares regression and fitted a line or an exponential as appropriate. Categorical data were analyzed as described previouslyThe dataset consists of the following: Force-distance curves collected during AFM application using Asylum MFP3D software in IgorPro 6.22 platform, labeled as “Force curves” Video files of changes in fluorescence intensity of fluo-4 loaded cells during the indentation experiment labeled as “movie” Examples of traces of changes in fluorescence intensity extracted from the video files using a code for Matlab 2011 A available in reference 22 in the associated article, labeled as “Mean Ca Fluorescence intensity”Click here for additional data file.i) mouse bone marrow cells cultured in the presence of ascorbic acid (50 mg/ml) for 4–6 days (ii) C2C12 cells stably transfected with BMP-2 and cultured for 2–6 days (2 area tip (i) the maximum force was set below the membrane penetration force (at ∼400 nN), leading only to membrane deformation ; andii) the maximum force was set above the membrane penetration force (∼2800 nN), inducing membrane rupture and penetration .We employed two cell models to study osteoblast mechanosensitivity:–6 days  orii) C–6 days . Both mo–6 days  using a rea tip . Using Area tip . The mem2+]i elevations are known to be the most common first responses to mechanical stimulation24, we analyzed the changes in [Ca2+]i in response to different loading regimes in the osteoblasts loaded with [Ca2+]i fluorescent indicator fluo4-AM. In the low load indentation regime, where the force exerted on the cell induced membrane deformation  the region of interest was selected in the digital image; 2) the average fluorescence intensity data were extracted and normalized to the initial basal reading to correct for differences in dye loading; 3) the signal was corrected for bleaching and reflection artifacts as described in22; 4) the noise was determined as the standard deviation in a linear portion of the trace, and 5) the [Ca2+]i response was deemed positive when an increase in intensity exceeded four-fold the average noise. Cells exhibiting spontaneous [Ca2+]i fluctuations25 were excluded from the study.Since application of AFM may potentially interfere with fluorescence measurements, we have previously performed in depth analysis of the system performance2+]i transients in response to indentation performed at low and high speeds, we found that a higher percentage of C2C12-derived osteoblasts responded to the mechanical stimulation compared to primary osteoblasts . The [Ca2+]i elevations were further characterized by measuringi) the relative amplitude of the response ;ii) the duration of the response ; andiii) the amount of [Ca2+]i (duration multiplied by amplitude). The speed of indentation did not significantly affect the amplitude of calcium responses (2+]i (i) the extent of membrane deformation (the distance between the contact point and the maximum piezo extension) (ii) the energy spent to deform the cell (the area between the approach and retraction curves)  , andii)22). The [Ca2+]i signal was initiated when the tip came into contact with the cell and was maximum at the maximum deflection  when indentations with different contact times  were performed.We next assessed how the contact time (the net time the probe spent in contact with the cell during stimulation) affects calcium responses to low-load indentations performed at different speeds. We varied the contact time by increasing by 1 s the time that the tip spent in the maximally extended piezo position. The reflection of a cantilever on the calcium fluorescence recording allows achieving exact temporal overlay between the fluorescence intensity and the force-distance curve  of the best fit line for the responses. This slope is negative, representing a decrease in the amplitude, and when multiplied by 100, this slope is expressed as percentage decay. We have found that amplitude decay is significantly higher when cells are stimulated with higher frequencies , and reached their amplitude after the tip was released from cell contact . There was no significant correlation between the indentation speed and the amplitude (2+ released (When the maximum force was set at 2800 nN (three-fold above the membrane penetration force), the force-distance curves confirmed that membrane penetration occurred in all the cells tested. We have found that qualitatively similar responses were induced in primary and C2C12-derived osteoblasts , howevercontact . We asseplitude , duratioplitude  or amouneleased  during teleased , extent eleased , energy eleased  or membreleased .2+]i can be due to influx from the extracellular space as well as to release from intracellular stores, we next investigated the contribution of these pathways to membrane deformation and to micro-injury-induced [Ca2+]i responses. We analyzed changes in [Ca2+]i in response to low-load and high-load mechanical stimulations in C2C12 osteoblasts maintained in the control Ca2+-containing buffer or in Ca2+-free, EGTA (10 mM)-containing buffer (2+]i elevations (2+]i responses (3+ (50 μM)17. In the presence of Gd3+ both local [Ca2+]i transients in response to membrane deformation (2+]i responses (3) pathway leading to calcium release from intracellular stores, we used PLC inhibitor Et-18-OCH3 (5 μM)17. In the low-load regime, local [Ca2+]i transients  the extent of the horizontal involvement of the membrane,ii) the vertical deformation of the membrane andiii) the duration of the deformation. Mechanosensitive calcium channels were strongly implicated in generating Ca2+ responses to mechanical stimulations in previous studies25 and were confirmed to provide a critical contribution to both local and global Ca2+ responses in our study. Therefore, it is possible that local intracellular Ca2+ acts as an integrating signal that increases when open channels are more numerous or are activated longer, until it reaches the threshold Ca2+ concentration necessary to induce global Ca2+ response. Micro-injury then contributes to increasing local Ca2+ levels to threshold concentrations, inducing global response. Once global calcium elevation is triggered, it proceeds independently of the level of mechanical stimulation, as suggested by the lack of correlation between the magnitude of global Ca2+ response and mechanical stimulation in our study and that of Charras and Horton17. Our data demonstrate that calcium release from intracellular stores plays an important role in this process.In our model, micro-injury was required in order to achieve global elevations of [Ca2+ responses to fluid flow were reported to be in the order of 600–900 s26. The presence of refractory periods is of potential importance, since it was shown previously, that in order to induce potent osteogenic response, mechanical loading of bone should be performed as a series of repeated loading periods separated by the periods of rest28. Of interest, the rest period of 10 s was found to be sufficient to induce potent bone formation in response to low magnitude mechanical loading29.We identified the refractory periods during which the responses to subsequent mechanical stimulations were either absent or diminished in amplitude. This refractory period following membrane deformation only was relatively short, and the calcium response was fully reestablished after the recovery time of 10 s. In contrast, in experiments where micro-injury and global elevation of calcium were observed, the recovery period was longer than 30 s. Previously, the refractory periods for global CaTaken together, our study provides new insights into the complex dynamics of cellular responses to mechanical stimulations. In contrast to many previous studies of cell responses to mechanical forces, atomic force microscopy allows very precise control and monitoring of the physical parameters of the experiment, such as forces and deformations applied at a single cell level. Using a well-established readout of cellular response, calcium signaling, allowed us to identify novel correlates between mechanical stimulation and cell responses. Such knowledge is important for better understanding of the mechanisms of mechanical loading-induced bone formation, as well as micro-damage induced bone remodeling.http://dx.doi.org/10.6084/m9.figshare.109135630figshare: Data on the effects of local membrane deformation and micro-injury in osteoblasts, doi: et al. compares the calcium (Ca2+) response of osteoblasts to mechanical forces resulting from 2 types of well-defined micro-damages, namely a local membrane deformation and a micro-injury. An atomic force microscope is advantageously used to control and modulate these 2 types of stretching effects. In its present form, the article is already well structured, rich in new experimental results and easy to read. However, their message would be more complete if authors were considering the following points.The article by Lopez-AyonMajor commentsSince the probe calibration is very important for AFM measurements, authors should provide more details concerning this indispensable step. For instance, the method used to determine the spring constant of the probe is never mentioned.There is a non negligible hydro-drag force applied to the cantilever when it is moved at speed of 20 µm/s. If this aspect is not taken into account, the effective forces applied to cells and the energy spent to deform the membrane may be overestimated. A comment should be provided concerning this point.2+ concentration is based on the analysis of averaged intensity in a predetermined zone drawn on the images acquired by fluorescent microscopy. So, signal intensity could depend on the layer which is in focus. The increase in intensity might thus be to the fact that cell is locally compressed leading to the generation of a calcium flow toward the basal cell face which is in focus. This additional effect should be appropriately discussed.The method used to quantify the Ca Concerning methodological aspects: et al., 2009Wang). Clearly a tensed cytoskeleton plays a key role in such a distant phenomena and using, e.g., cytochalasin D, would have been of interest to prove that the distant local effects are related to the tensed cytoskeleton. Authors should at least comment on this interesting result revealed by the local membrane deformation test. Such an distant effect is not so evident in case of membrane rupture suggesting  important modifications for the cytoskeleton between the two types of stress studied. These distant effects could advantageously be analyzed and quantified.The authors write in page 4 that: "When consecutive low load indentation were performed at the same location on the same cell multiple local responses were induced". This results is indeed comforted by the calcium concentration images shown at 6 s in Fig 2B. Multiple calcium spots are visible at 6 s on the non magnified cellular image, suggesting that a local mechanical effect may induce multiple (and durable) distant yet local effects. Such a mechanically induced signaling at a distance has been largely described and commented by the group of Ning Wang presently working at University of Illinois at Urbana Champaign  or with some degree of confluence ... In the latter case, mechano-chemical interactions between cells could play a role in calcium signaling. This point should be clarified all along the paper.  Concerning Results and Discussion:Detailed commentsFigures 1A, 1B seem to show the culture at higher confluence than 50-70% as used for experiments.The linear fits in Fig. 5 A, B, C  don't represent the decay of peaks.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. at el. Overall, I think the authors have done a good job in characterizing qualitatively the differences in Ca2+ responses induced by local membrane deformation and micro-injury. I particularly liked the measurements involving cyclic membrane perturbation and inhibition of Ca2+ channels and internal store. I think the latter provide a good understanding on the different origin of the Ca2+ released when cells are probed or micro-injured. Nevertheless, i have a technical concern and some questions that i feel the authors could have discussed more in-depth in the manuscript:2+ release. Could the authors comment on that?Figure 4c and 4e(iv) show large deviations in the deflection of the cantilever when cells are indented for longer time periods. Which kind of feedback was used to control the AFM tip position? It appears as if no deflection feedback was really used, but rather the piezo was kept at constant z. It seems the system is either drifting, or the cell is changing its mechanical properties (softening) or its height (shortening) due to the Ca2+ released), the time required to recover baseline levels is constant. Would that be an expected behaviour, or is this the case only because the released Ca2+ levels are small and very localized compared to the whole volume cell?The fit obtained from fig 4d could be considered to have a slope of 1 (0.98), indicating an on-off response. More interestingly, the offset of 0.6s indicates that no matter the duration of the stimulation  of small indentations required to integrate enough Ca2+ signal to start a global Ca2+ response?In the discussion, the authors suggest that 'intracellular CaI've read with interest the manuscript by Lopez-Ayon I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Large-conductance calcium-activated K+ (BK-type) channels, abundantly distribute in excitable cells and often localize to the proximity of voltage-gated Ca2+ channels (VGCCs), spatially enabling the coupling of the intracellular Ca2+ signal to the channel gating to regulate membrane excitability and spike firing patterns. Here we utilized the sensitivity and dynamic range of BK to explore non-uniform Ca2+ local transients in the microdomain of VGCCs. Accordingly, we applied flash photolysis of caged Ca2+ to activate BK channels and determine their intrinsic sensitivity to Ca2+. We found that uncaging Ca2+ activated biphasic BK currents with fast and slow components (time constants being τf ≈ 0.2 ms and τs ≈ 10 ms), which can be accounted for by biphasic Ca2+ transients following light photolysis. We estimated the Ca2+-binding rate constant kb (≈1.8 × 108 M−1s−1) for mSlo1 and further developed a model in which BK channels act as a calcium sensor capable of quantitatively predicting local microdomain Ca2+ transients in the vicinity of VGCCs during action potentials.Ca For example, fast release of synaptic vesicles from presynaptic terminals is triggered by Ca2+ influx via voltage-gated Ca2+ channels during action potentials, and the local high concentration of Ca2+ transients at the active zones is critical for gating synchronized fusion of synaptic vesicles (SV) and transmitter release. Experimental measurements of global Ca2+ concentrations with Ca2+ indicators and mathematical modeling extrapolate that the peak Ca2+ concentration seen by the Ca2+ sensor on SVs briefly reach tens and even hundreds of micromole levels. Although tremendous progress in recent years has been made with developing novel fluorescent chemical or protein Ca2+ sensors, limited spatiotemporal resolution of these sensors presents a major challenge to directly read out local Ca2+ transients in real-time.Ca2+, abundantly distributed in the excitable cells, regulate the membrane excitability and electrical signals in response to the Ca2+-influx from the Ca2+-permeable channels1+ (RCK) domains of the caboxy-terminal region32+ rise. However, the affinity of these binding sites is primarily determined under the circumstance of Ca2+ uniformly sojourning to its binding sites at equilibrium with very little consideration of dynamics of Ca2+ influx or release. Although elaborate Markov models containing multiple parallel open and closed states have been developed to simulate both voltage- and Ca2+ dependent gating kinetics of BK channels well562+ (kb) remains unknown, making model parameters too unconstrained to meaningfully profile local Ca2+ dynamics.Large-conductance Ca-activated potassium channels (BK channels), uniquely sensitive to both membrane potential and intracellular Cab by ultrafast Ca2+ concentration jumps via a piezoelectric stepper of two barrel theta pipette82+ (~10 ms) to reach the inner face of the membrane patch where the RCK domain of BK channels situatesPrevious experiments in inside-out patch configuration have attempted to directly measure k2+ dynamics using BK channels as a sensor, it is therefore necessary to develop a superfast approach of Ca2+ delivery mimicking calcium influxes via calcium channels induced by action potentials, and precisely measure kb in order to quantitatively describe the kinetics of BK channels to such fast Ca2+ transients. In this study, we have applied laser flash photolysis technique of the caged-Ca2+ compound (e.g. NP-EGTA) to achieve instantaneous Ca2+ rises, which has been widely used for studying Ca2+-dependent processes such as the secretory responses2+ rise with peak concentrations up to tens of micromole from the basal [Ca2+]i of ~10–200 nM in sub-milliseconds and a slow uniformly steady-state elevation of global [Ca2+]i11122+ compound to examine both voltage- and calcium-dependent gating behavior, and determined the Ca2+ forward binding rate kb for BK. Our results demonstrate that BK channels have higher calcium-sensitivity capable to follow up to tens of μM transient Ca2+ changes 0.1–0.2 ms, and established a quantitative model for its utility as the fast local Ca2+ sensor to profile the local Ca2+ transients during action potential firing.To extrapolate the local Ca2+-sensitivity of BK-type channels, laser UV flashes were used to release Ca2+ from caged compound NP-EGTA (10 mM) and activate the currents of mSlo1 and several mutants (BK-type) at various voltages. 2+ to increase the intracellular Ca2+ concentration([Ca2+]i) to ~10 μM in less than 1 ms (blue line). The mSlo1 current was further enlarged by the uncaged Ca2+ and exhibited a biphasic activation process, presumably as a result of uncaging that produced an early instantaneous transient followed by a plateau increase of intracellular Ca2+ concentrations in a spatially uniform manner2+-release from internal store because preloading cells with thapsigargin (1 μM TG) in recording pipettes for at least 5 min to deplete internal Ca2+ stores before flash photolysis has no effect . Strikingly, a hooked current appeared at the end of the fast phase. The boxed trace (right) showed typical biphasic activation with the fast on-time (τf or τf-on), off-time (τf-off) and slow time (τs) constants of ∼0.2, 2.4 and 10 ms, respectively. As an approximation of relative distribution of two components at any given flash, we define the fast and slow proportion of the current as Rf = h1/h and Rs = h2/h, respectively (f + Rs = 1.To directly investigate the Ca effect s. Strikinf(5D5N) is much smaller than Rf(D362A/D367A) (2+ after UV flash (2+ did not cause a Ca2+-dependent recruitment of BK channels from intracellular pool to membrane surface. This was particularly evident for the triple mutant (D362A/D367A/5D5N) in which a lack of the channel responsiveness to uncaged Ca2+ should have not precluded its trafficking presumably derived from Ca2+ dependent fusion of vesicles in HEK293 cells. f from mSlo1, aforementioned mutants and several others . The Rf < 1 of all the mSlo1 and mutants, albeit there is a big difference among their values of Rf, indicates that they all respond to uncaged Ca2+ in a biphasic manner. This may imply a lack of direct relationship between the Ca2+ binding sites and biphasic waveform of BK currents, as each of Ca2+ binding sites produces both phases. Additionally, we found the distinct difference in Rf between the mutants L312A and G311I in sFig. 1B,C, i.e., Rf (L312A) ≫ Rf (G311I), possibly due to their extremely different gating propertiesTo examine whether different calcium binding site produces different component, we next made a series of constructs including mutant 5D5N  and the mutant D362A/D367A . Even though both of the 5D5N and D362A/D367A show similar biphasic currents, RA/D367A) . The triUV flash , indicatf is voltage dependent, we stimulated the currents of mSlo1, 5D5N, D362A/D367A, D369G and D369A/5D5N by flash at various voltage, measured their values of Rf and then plotted their Rf-V curves  and found that their values of V0.5 and slope factors increase by degrees according to the following rank oder: D369G, D369G/5D5N, D362A/D367A, mSlo1, D369A/5D5N, 5D5N, basically in parallel to their corresponding G-V curves, implying that the Rf values can mirror differences in the calcium sensitivity (or Ca2+ forward binding rate) of BK-type channels and their mutants in response to instantaneous Ca2+ rise.To determine whether the RV curves . The sha2+-dependence of Rf is not apparent, based on the measured [Ca2+]i during flash experiments . All the data suggest that the uncaged Ca2+ transients in HEK cells under our experimental conditions contain both fast and slow components, consistent with those described in artificial conditions in vitro112+]i can only be made during slow plateau phase  is fixed to constrain relative changes in the open (ko) and closed (kc) dissociation equilibrium constants, which hold the ko/kc constant. To acquire the physical Ca2+ binding rate kb, we only consider a simplest two-state 2+) and receptor (BK) to describe the BK currents evoked by the uncaged Ca2+ release, which can be seen as an unit model of the MWC model of BK. Here the C is a closed state, and the O an open state. The forward rate between C and O is Ca2+-dependent. Based on the C-O model, we have the calcium binding rate constant kb = 1/(τ * ([Ca2+]a+kd)) in μM−1 ms−1 , we define the [Ca2+]a as an averaged [Ca2+]i over the fast rising time of uncaged Ca2+ pulse  for mSlo1, 0.057 ± 0.003 (n = 5) for 5D5N, 0.014 ± 0.01 (n = 4) for D362AD367A and 0.26 ± 0.02 (n = 5) for D369G, respectively, as indicated in sTable 2 and b value of mSlo1 is near to the typical limitation of calcium binding rates 108 * s−1M−1 b, we now for the first time able to constrain the BK model with physiologically meaningful parameters  and yield optimal match between all the fits and the current traces of mSlo1 channels to drive subsequent non-stationary calculation of local Ca2+ transients.In previous models for BK-type channels in10-state gating schemems−1 eq. , where k2+ pulse . During odel eq. , the  is the intracellular calcium concentration, KC the closed Ca2+ dissociation constant, KO the open Ca2+ dissociation constant, F Faraday’s constant, R the universal gas constant, T temperature, V voltage, Q the equivalent gating charge associated with the closed-to-open conformational change, and L(0) the open-to-closed equilibrium constant in the absence of bound Ca2+ at 0 mV.where, a the averaged intracellular [Ca2+]i.where I(t) can be depicted as a function of membrane voltage V(t) and calcium concentration [Ca2+]i(t),Kinetic modeling is an important method for better understanding the electrophysiological characteristics of ion channels. In kinetic modeling, the membrane current G represents the whole-cell conductance determined by both the voltage and calcium concentration and E the reversal potential of channels. Unlike the Hodgkin-Huxley model, the total conductance is described as follows:where n is the number of ion channels on the membrane, ĝ the single-channel conductance, and Po the open probability, which can be solved from the kinetic models of channels with numerical method of differential equation (Runge-Kutta or Q-Matrix).where Combining eq. I, n, ĝ, V, E and Po, the instantaneous [Ca2+]i can be determined. However, unlike I, V, E, and ĝ, Po, n is hardly to be obtained directly through existing experimental techniques. In this paper, a novel three-step method is proposed to obtain the instantaneous [Ca2+]i value. Firstly, Po is determined based on the Monod-Wyman-Changeux (MWC) model. Then, n is got through the steady-state calculation of eq. Ca2+] can be obtained through eq. In eq. Po.1. Step one: establishing the MWC model for BK-type channels and calculating its open probability 2+-dependent open probability ). Firstly, a set of activated and deactivated data at different [Ca2+]i level (fits) were fitted to a MWC model to determine the eleven unknown parameters with the software CeLPo.Models of BK-type channels are an allosteric MWC model of 10-state C5-O5 with 11-parameters, which reflects the voltage- and Can in eq. (7)(2) Step two: determining the channel number Isteady(∞) can be depicted asThis step is performed at steady state. From the eq. Isteady, [Ca2+]steadyi-, V and E were measured from experiment. Po was determined from the model in step 1. Therefore, we got n from the eq. where Ca2+]i calculation3. Step three: Instantaneous [Ca2+]i can be determined based on the eq. Ca2+]i is difficult to obtain because Po is in a form of complex differential equation system.After step 1 and 2, the instantaneous [Ca2+]i calculation is treated as a numeric optimization problem, where [Ca2+(t)]i is the only parameter to be optimizes. Here the Q-Matrix method is used to calculate the values of Po(t) and I(t) at the time t, and then to minimize the error between the calculated I(t) and the actual I(t) by an optimization algorithm of Evolution Strategy (ES).Here, instantaneous i measured at time 0 in experiment is set as the initial steady-state [Ca2+]i. Then [Ca2+(t)]i at next time step can be got through ES optimization. This process goes step by step until [Ca2+(t)]i at all time step is gotten.When calculation begins, [It is obviously that the method in step 2 used to determine the channel number in the UV-flash case completely differs from the variance methodHow to cite this article: Hou, P. et al. Extrapolating microdomain Ca2+ dynamics using BK channels as a Ca2+ sensor. Sci. Rep.6, 17343; doi: 10.1038/srep17343 (2016).


In this regard, distortion of the DNA methylation setting and the presence of mutations in epigenetic modifier genes can also be molecular drivers of the disease. In recent years, somatic missense mutations of the DNA methyltransferase 3A (DNMT3A) have been reported in ~20% of AML patients; however, no obvious critical downstream gene has been identified that could explain the role of DNMT3A in the natural history of AML. Herein, using whole-genome bisulfite sequencing and DNA methylation microarrays, we have identified a key gene undergoing promoter hypomethylation-associated transcriptional reactivation in DNMT3 mutant patients, the leukemogenic HOX cofactor MEIS1. Our results indicate that, in the absence of mixed lineage leukemia fusions, an alternative pathway for engaging an oncogenic MEIS1-dependent transcriptional program can be mediated by DNMT3A mutations.Close to half of  In recent years, distortion of the DNA methylation setting and the presence of mutations in epigenetic modifier genes, such as Tet methylcytosine dioxygenase 2 and isocitrate dehydrogenase 1/2, have been directly implicated in the pathogenesis of AML.2 In this regard, somatic missense mutations of the DNA methyltransferase 3A (DNMT3A) have also been reported in ~20% of AML patients, in whom they are usually associated with an unfavorable prognosis.6Acute myeloid leukemia (AML) comprises a group of hematopoietic malignancies derived from myeloid precursors that have a highly heterogeneous clinical course and response to therapy. AML is characterized by greater proliferation and lower differentiation of the hematopoietic progenitor cells. Non-random cytogenetic aberrations are the single most important prognostic factor of the disease, but close to half of de novo DNA methyltransferase that catalyzes the transfer of a methyl group onto the 5′-position of cytosine of CpG dinucleotides. Most of the DNMT3A mutations present in AMLs are heterozygous, with a great predominance of missense alterations in the R882 residue located in the catalytic domain.6 R882H DNMT3A has recently been shown to act as a dominant negative that inhibits wild-type DNMT3A.7 In this context, AML samples carrying DNMT3A mutations have been found to be associated with DNA methylation changes.9 However, no clear and common epigenetic signature has so far emerged and, most importantly, no obvious critical downstream gene has been identified that could explain the role of DNMT3A in the natural history of AML.DNMT3A is a 10 and a widely used DNMT3A wild-type AML cell line (AML5). Using whole-genome bisulfite sequencing quencing , we gene6 To find specific target genes affected by the DNA hypomethylation events noted in the AML cells harboring the DNMT3A mutation, we searched for particular differentially methylated regions (DMRs) between the two AML cell lines. These were defined as consecutively and consistently differentially methylated loci located beyond the 95% confidence interval (CI) of the smoothed methylation profiles. Using these criteria, we identified 182 800 DMRs between OCI-AML3 and AML5 cells. The most common DMR change was the presence of a methylated sequence in AML5 that was unmethylated in OCI-AML3: 156 919 hypomethylated events that represented 86% of the identified DMRs (http://www.cancerrxgene.org/downloads/). This approach yielded 292 genes with transcriptional activation associated with promoter hypomethylation in DNMT3A mutant cells relative to wild-type cells (We observed that DNMT3A mutant AML cells had a 9% (66.1% vs 75.1%) decrease in average DNA methylation level and fewer methylated CpG dinucleotides than did the DNMT3A wild-type cells . The dimied DMRs . We focupe cells .6 consisting of 13 R882 mutations  and 1 S525C mutation. DNMT3A mutations were enriched in the AML cases that showed no cytogenetic abnormalities . None of our AML cases had mixed lineage leukemia (MLL) translocations. AML patients with DNMT3A mutations had a shorter 5-year overall survival (OS) .We next examined how the profile of genes with hypomethylation-associated expression derived from the DNMT3A AML cell line models translated to primary samples obtained from AML patients. To this end, we screened sixty-eight AML patients   (n=194),2 in which the described hypomethylated CpG sites were enriched in DNMT3A mutant patient samples   . Interes03–3.60) . The DNA12–3.18) . We furt12–3.18) . These tMEIS gene at the protein level in the DNMT3A mutant cell line context was also confirmed   . MEIS1 i15Our results suggest that, in the absence of MLL fusions, as in our cases, an alternative pathway for engaging a leukemogenic MEIS1-dependent transcriptional program can be mediated by DNMT3A mutations. Under these circumstances, those AML patients carrying the alteration in the DNA methyltransferase would undergo a hypomethylation event at the MEIS1 promoter that would lead to the overexpression of this key oncogene in leukemia.


Occluding tumor blood supply by delivering the extracellular domain of coagulation-inducing protein tissue factor  to tumor vasculature has enormous potential to eliminate solid tumors. Yet few of the delivery technologies are moved into clinical practice due to their non-specific tissue biodistribution and rapid clearance by the reticuloendothelial system. Here we introduced a novel tTF delivery method by generating a fusion protein (tTF-pHLIP) consisting of tTF fused with a peptide with a low pH-induced transmembrane structure (pHLIP). This protein targets the acidic tumor vascular endothelium and effectively induces local blood coagulation. tTF-pHLIP, wherein pHLIP is cleverly designed to mimic the natural tissue factor transmembrane domain, triggered thrombogenic activity of the tTF by locating it to the endothelial cell surface, as demonstrated by coagulation assays and confocal microscopy. Systemic administration of tTF-pHLIP into tumor-bearing mice selectively induced thrombotic occlusion of tumor vessels, reducing tumor perfusion and impairing tumor growth without overt side effects. Our work introduces a promising strategy for using tTF as an anti-cancer drug, which has great potential value for clinical applications. Over the past two decades, approaches to targeted coagulation therapy  markers –7. Free  markers –11; howe markers , 13.The pH (low) membrane insertion peptide (pHLIP) can insert into cell membranes by forming an inducible transmembrane α-helix under acidic conditions –20, and,We have generated the tTF-pHLIP fusion protein in which the N-terminus of pHLIP was fused to the C-terminal region of tTF, and found that tTF-pHLIP can localize to the acidic tumor endothelium of blood vessels in tumor-bearing mice and induce local intravascular thrombosis Figure , resultir values of approximate 32, 000 Da for tTF-pHLIP, 28, 000 Da for tTF and 32, 000 Da for tTF-K-pHLIP . We also generated free tTF and tTF-K-pHLIP as controls. K-pHLIP is a mutant of pHLIP where the residues Asp 13 and Asp 24 in the transmembrane segment of pHLIP are replaced by Lys residues. This leads to the loss of the pH-dependent membrane insertion function , 27. SDSP Figure . WesternP Figure .We first tested whether tTF-pHLIP has the ability to insert into the lipid bilayer by using circular dichroism (CD) spectra. The CD spectral signal has been used to monitor the conformational changes of pHLIP in a free state at neutral pH to α-helix formation when inserted into bilayers at lower pH . Our datNext, we evaluated the interaction of tTF-pHLIP with the cell membrane at neutral and acidic pH values by confocal microscopy and flow cytometry. In the confocal microscopy assays, HUVEC cells were incubated with Alexa 594-labeled tTF-pHLIP (1.24 μM) for 30 min at 37°C, at either pH 7.4 or 6.5. Our results demonstrated that bare fluorescence was found on the tTF-pHLIP-treated cell membrane at pH 7.4 Figure . In cont3 orthotopic MDA-MB-231 human breast xenograft tumors. A 20 μg/mouse  dose of tTF-pHLIP was injected into the mice via the tail vein, with saline as a negative control. Note that this dose was sufficiently safe as demonstrated in a dose escalation study -carboxyfluorescein)-labeled tTF-pHLIP, staining of tumor sections with antibody against vascular endothelial cell marker CD34 showed the binding of tTF-pHLIP to the blood vessel walls as verified by colocalization with CD34 Figure .2) and hemoglobin (Hb). Our results showed that in contrast to the pre-injection state, the HbO2 signal declined sharply while the Hb signal increased considerably 6 hours after tTF-pHLIP treatment  or tTF-pHLIP by the tail vein 4 times at intervals of 6 days. After the second injection, a reduction in tumor volume was observed in mice treated with tTF-pHLIP compared with the saline group, and this became statistically significant from day 13 onwards. In contrast, the tumors continued to grow in all other treatment groups, and none of these groups were significantly different from the saline control. After the fourth injection, tTF-pHLIP-treated mice had a mean tumor volume of 552 vs 2443 mm3 for saline, 2109 mm3 for tTF-K-pHLIP and 2289 mm3 for the tTF/pHLIP group  at the indicated time intervals as mentioned above. This indicates that tTF-pHLIP was not overtly toxic to the animals. Histological analysis also showed no visible thrombosis in the major organs  of the MDA-MB-231 tumor-bearing mice after single or multiple (3 times) tTF-pHLIP administrations , indicatTo investigate whether tTF-pHLIP induces an innate immune response, we assessed IL-6, IP-10, TNF-α and IFN-α serum concentrations of non-tumor-bearing C57BL/6 mice before and after tTF-pHLIP treatment. No significant changes in concentration of any of the cytokines were found after single or multiple (3 times) injections of tTF-pHLIP , revealiA number of methods for tTF delivery have previously been described by fusing tTF to peptide or antibody ligands that target various overexpressed tumor vascular markers –6. Howevin vivo [In contrast, we have found in this study that pHLIP technology can be used to selectively deliver tTF to tumor blood vessels based on the ability of pHLIP to undergo membrane insertion in acidic tumor vascular compartments . pHLIP cin vivo . We genein vivo –6, but din vivo demonstrated no overt immune stimulation, its possible impact on the endotoxin levels will need to be further characterized.We have demonstrated that systemic administration of tTF-pHLIP can specifically induce thrombotic occlusion of tumor vessels and impairs tumor growth in two mouse tumor models: MDA-MB-231 human breast orthotopic xenograft tumor and B16-F10 melanoma. Thrombosis or other abnormalities were not observed in normal organs of the mice. The selective binding of tTF-pHLIP fusion protein to tumor endothelium is in accordance with the prior findings of pHLIP targeting of acidic tumors –25. FurtIn conclusion, we have shown that the fusion protein tTF-pHLIP is an effective antitumor molecule by targeting tumor vessels and inducing vascular infarction. This study, we believe, represents the first time pHLIP technology has been introduced into cancer therapy and this new anticancer treatment approach is uniquely attractive for moving into clinical practice.de novo. The cDNAs were cloned into Nde I and Xho I sites of the expression vector pET-30a(+). The mutant control tTF-K-pHLIP was produced by replacing pHLIP in the tTF-pHLIP with K-pHILP (changes underlined)  using the same approach. A cDNA encoding tTF containing a hexahistidine-tag at the 5′-end was amplified by polymerase chain reaction (PCR) using the primer pair 5′- ATTATATGCTGCCACAGTATTTGTAGTGCCTGA CATATGCC-3′ (5′-primer) and 5′- GGATTCAGAGA AGAGCACCACCACCACCACCACTGAGAGCTCC-3′ (3′-primer), and subcloned into the expression vector pET-30(+)a at the Nde I and Xho I sites. The vectors were introduced into competent E. coli cells (BL21DE3) according to the manufacturer's protocol . After stimulating with IPTG (0.5 mM) for 4 hours, the cells were harvested and homogenized by sonication in washing buffer . To solubilize inclusion bodies, 5 to 10 mL of urea buffer  was added and the samples were incubated overnight at room temperature (RT). The suspension was centrifuged at 5,000 × g for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter and loaded onto a nickel-nitrilotriacetic acid column . Purification and refolding were carried out using a His Bind Buffer Kit  according to the manufacturer's instructions. To remove the salt, the protein solution was dialyzed in a Slide-A-LyzerR10 K dialysis cassette  against Tris-buffered saline (TBS) . Subsequently, tTF and tTF-pHILP were analyzed under denaturing conditions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using mouse monoclonal anti-human tissue factor antibody .The cDNAs coding for tTF (amino acids 1 to 218 of human TF) and tTF-pHLIP in which the pHLIP polypeptide  was linked to the C-terminus of tTF, were synthesized et al. [The ability of tTF, tTF-K-pHLIP and tTF-pHLIP to facilitate the specific proteolytic activation of factor X by factor VIIa was assessed as described by Ruf et al. . In brieCircular dichroism (CD) measurements were carried out on a Bio-Logic Modular Optical System 450 (MOS-450) at 25°C in phosphate buffer. Briefly, POPC liposomes (700 μM) and tTF-pHLIP (10 μM) were mixed in 10 mM phosphate buffer. HCl was added to lower the pH from 8.0 to 6.5, and CD spectra were recorded. tTF itself was used as the base line.3) were seeded into confocal dishes (2 cm diameter) and maintained at 37°C and 5% CO2 in DMEM supplemented with 10% FBS. Twenty four hours later, the medium was removed, cells were washed twice with PBS and resuspended in PBS at a pH of 7.4 or 6.5 using phosphate buffers made by mixing appropriate proportions of Na2HPO4 and NaH2PO4. Alexa 594-labeled tTF-pHLIP (40 μg) was added to the cells. After 30 or 60 min, the cells were washed twice with PBS and Hoechst 33342 was added to stain cell nuclei. Images were analyzed with a FV1000-IX81 confocal laser scanning biological microscope .For confocal microscopy studies, HUVECs  at 37°C for 1 h. After washing the cells to remove free tTF-pHLIP, they were resuspended in 500 μL PBS (pH 7.4) and analyzed by flow cytometry .6) were mixed with 50 μL of Matrigel and injected into the mammary fat pads of the mice. For C57BL/6 or BALB/c nude models of melanoma, B16-F10 cells (1.0 × 106) were injected subcutaneously into the right flank. Tumors were allowed to grow to a volume of approximately 100 to 200 mm3 . The mice were then used for in vivo imaging and therapeutic experiments.All animal experiments carried out in this study were approved by the Committee on the Ethics of Animal Experiments of the Health Science Center of Peking University . Five week old BALB/c nude mice weighing 20–23 g were purchased from Beijing Vital River Laboratories. Human MDA-MB-231 breast cancer and B16-F10 melanoma cells (American Type Culture Collection) were grown as attached monolayer cultures in DMEM, and were afterwards dissociated in trypsin, washed and resuspended in PBS. MDA-MB-231 cells (2.0 × 102) and hemoglobin (Hb) signals. MDA-MB-231 tumor-bearing mice were anesthetized with isoflurane and placed in a supine position in an animal holder. Cross-sectional multispectral optoacoustic image datasets of the tumors were acquired at different wavelengths in the near-infrared window , where the absorbance wavelengths of HbO2 and Hb differ widely and can thus be used to easily distinguish these two forms.A real-time multispectral optoacoustic tomography (MSOT) scanner  was utilized to determine tumor blood perfusion by detecting oxyhemoglobin  with repeated measures followed by Tukey's HSD test. Cumulative survival curves between groups were compared using Kaplan-Meier analysis followed by the Log rank (Mantel-Cox) test. Statistics were calculated using SPSS 18.0. P-values less than .05 were considered statistically significant.For the study of coagulation activity


Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca2+ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca2+ affinities and accelerated kinetics by weakening the Ca2+-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca2+ decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca2+ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca2+ at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca2+ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.Faithful reporting of temporal patterns of intracellular Ca Monitoring fast or high frequency Ca2+ transients, characteristic of excitable cells, requires rapid kinetics with millisecond time resolution. Genetically encoded fluorescent Ca2+ indicators (GECI) are being developed to fulfil these roles and have become important tools for activity studies using targeted expression in cultured cells and tissues and in live transgenic animalsVisualising Ca2+ binding, which causes the Ca2+.CaM.RS20 complex to form, stabilizing the highly fluorescent, deprotonated form of GCaMP by blocking solvent access to the chromophore4Green fluorescent protein - calmodulin protein - (GCaMP)-type GECI are based on a circularly permutated EGFP molecule (cpEGFP)2+ binding by shifting the equilibrium towards the deprotonated state and/or increasing the brightness of the deprotonated form. These mutations, however, left unchanged the high affinity (equilibrium dissociation constant Kd in the range of hundreds of nM) and concomitant slow response kinetics of the probes to Ca2+. There has been a lack of insight into the onset kinetics and slow fluorescence decay rates of 1–5 s−1 have constrained tracking rapid processes. In excitable cells monitoring fast Ca2+ fluxes requires millisecond time resolution and thus fluorescence decay and rise rates of up to 1000 s−1. These issues have begun to be addressed172+ transients associated with synaptic transmission and activation of skeletal and cardiac muscle.Several generations of GCaMP-type GECI obtained by mutagenesis have presented improvements in brightness, signal-to-noise ratio and photostability67891011121314152+ response kinetics on the millisecond time scale and to understand the kinetic mechanisms by which GCaMPs respond to Ca2+ concentration changes. Using both excitable and non-excitable cellular model systems we compared the response kinetics of the newly developed probes with those of the chemical dye Fura-2, GCaMP3 and GCaMP6f. Imaging with the newly developed probe GCaMP3fast revealed significantly faster Ca2+ response kinetics than previously seen with GECI.Our aim was to generate fast derivatives of GCaMP3 by rational design with Ca2+ (Kd of 100 - 300 nM8112+.CaM.RS20 peptide complex. We have previously shown that the mutation of the Trp residue to Tyr in a RS20-related peptide increases the Kd of the complex for Ca2+.CaM192+ binding sites of CaM by single point mutations (Asp → Ala) in the first loop residue of each EF-hand increased the Kd of CaM for Ca2+212+ dissociation from complexes of Ca2+-bound EF-hand mutated CaM with a peptide derived from αCaMKII (CaMKIIP) was significantly accelerated. Based on these observations our hypothesis was that fast Ca2+ response rates can be achieved for GCaMPs by weakening the Ca2+.CaM.RS20 interaction by the peptide and EF-hand mutations described above.The high affinity of GCaMPs for Ca2+ binding sites in the CaM sequence in GCaMP3-derived proteins, and to investigate the effect of Trp → Tyr mutation in the RS20 target peptide sequence, alone, and in combination with the EF-hand mutations : EF-1, EF-2, EF-3, EF-4, EF-1:2, EF-1:3, EF-1:4, EF-2:3, EF-2:4, EF-3:4. The Trp → Tyr peptide mutant was termed RS-1 and the peptide mutation combined with mutation of the first EF-hand gave RS-1 EF-1 and so on, resulting all together in 21 GCaMP3 mutants (mGCaMP3). The same EF-hand mutagenesis strategy that was applied to GCaMP3 was also tested on two of its derivatives developed by Zhao et al.Triple EF-hand mutations in all cases resulted in a loss of fluorescence response to Cafast (mGCaMP3 RS-1 EF-3) with the fastest Ca2+ response times is promising for monitoring high-frequency Ca2+ transients and GCaMP3bright (mGCaMP3 EF-4) with the highest fluorescence dynamic range. Other mutants presented in Kd (mM range) that are potentially useful for monitoring Ca2+ in organelles. Indicator responses occurred on the millisecond time scale and thus Ca2+ response kinetics were assessed by stopped-flow. A kinetic model proposed to explain the Ca2+ response mechanism of GCaMP3 and mGCaMP3 is illustrated on GCaMP3fast and GCaMP3bright (without the red star at P2 that signifies the RS-1 mutation)  . Fitted resented .fast was stable at 37 °C for four hours, in the absence and presence of Ca2+  moderately reduced the fluorescence dynamic range  obtained by Ca2+ titration were increased from 330 nM for GCaMP3 to 1–3 μM for GCaMP3fast and GCaMP3bright. Cooperativity of Ca2+ binding, characterised by a Hill coefficient (n) of 3.2 for GCaMP3 was higher, 4.3 and 4.7, for GCaMP3fast and GCaMP3bright, making their switch-like Ca2+ response steeper than that of GCaMP3  with the GCaMP3fast probe ) was observed for all GECI and mGECI generated in this study. For GCaMP3 at physiological ionic strength and 20 °C the rate saturated at 20 s−1 (t1/2) of 35 ms. The GCaMP3fast and GCaMP3bright mutations increased the limiting rate to ~140 s−1 (20 °C). Second, the observed rates showed a steep dependence on [Ca2+]. Consistent with their increased cooperativity for Ca2+ compared to GCaMP3, the observed rates of the two mutants switched to a limiting maximum over a narrow range of [Ca2+] around their dissociation constants , GCaMP6f (68 ms) and GCaMP3fast (39 ms) showed that GCaMP3fast was ~1.5-fold faster than either of the other two probes (fast (71 ms) were even more markedly improved, GCaMP3fast was 3-fold faster than GCaMP3 and twice as fast as GCaMP6f  with GCaMP3fast compared to the other two probes (rise-times 44 ms for GCaMP3 and 43 ms for GCaMP6f compared to 33 ms for GCaMP3fast and decay-times of 245 ms for GCaMP3 and 168 ms for GCaMP6f compared to 95 ms for GCaMP3fast)  . While Δing cell , the fluaches ~1 .fast and GCaMP3bright in the cellular environment of human umbilical vein endothelial cells (HUVEC) and HEK293T cells revealed no detectable damage to cell viability. Fluorescence dynamic ranges (ΔF/F0) of mGCaMP3 expressed in the cytosol of HUVEC during acute exposure to ionomycin remained high with values of 3.4 and 5.6 for GCaMP3fast and GCaMP3bright respectively, comparable to GCaMP3  or two-photon absorption represents the absorption coefficient for the simultaneous absorption of two photons for the excitation of a fluorophore. Although the depth of penetration is limited by the wavelength, the signal strength and signal-to-noise ratio will depend on the absolute value of σ2. Hence we determined the σ2 values the novel mGECI in the presence and absence of Ca2+ relative to fluorescein−50 cm4.s.photon−1 value for fluorescein, GCaMP3 had the lowest σ2 value in the absence of Ca2+ (0.06 × 10−50 cm4.s.photon−1). In the presence of Ca2+ the σ2 value increased to 0.76 × 10−50 cm4.s.photon−1. The GCaMP3fast probe had a ~5-fold increase upon Ca2+ binding. The brightest probe with the greatest fluorescence dynamic range under two-photon excitation was GCaMP3bright with a ~10-fold increase and higher σ2 values, 0.22 × 10−50 cm4.s.photon−1 and 2.0 × 10−50 cm4.s.photon−1, in the presence and absence of Ca2+, respectively. It is envisaged that the probes developed here will be useful to study fast signalling processes, such as the Ca2+ changes associated with neuronal activity in tissues and in vivo.Because animal tissue is transparent to infrared light, two-photon excitation microscopy has become a crucial tool for tissue and 2+ signalling events. Disabling the function of individual Ca2+ binding sites by single point mutations in the CaM portion of GCaMPs was expected to lower the Ca2+ affinity and accelerate Ca2+ kinetics2+.CaM - peptide interaction by mutation of the RS20 peptide was expected to further lower Ca2+ affinity as substitution of the Trp residue with Tyr was known to increase the Kd for peptide binding to Ca2+.CaM19Kd for Ca2+ binding from 330 nM to 1–3 μM. Fura-2 and GCaMP3 with sub-μM Kd for Ca2+ showed slow and complex Ca2+ response kinetics in ATP-stimulated in HUVEC and HEK293T cells, indicating probe saturation. GCaMP3fast and GCaMP3bright revealed that the hormone-induced Ca2+ transients were faster than when reported either by Fura-2 or GCaMP3. Our data thus show that the amplitude and kinetics of hormone-evoked intracellular Ca2+ transients are better matched by Ca2+ probes with a μM working range, like GCaMP3fastand GCaMP3bright, than by those with Kd values for Ca2+ of 200–300 nM.Based on previous studies192+dynamics has been its slow Ca2+ dissociation (t1/2 of 131 ms) at 37 °C. Recently reported probes GCaMP6 ‘fast’ (GCaMP6f)t1/2 of 66 ms (37 °C) ), the RS09 GCaMP3 mutantt1/2 of 25 ms, (37 °C)) and fast-GCaMP6f-RS09 (t1/2 of 20 ms (37 °C))fast probe with a t1/2 of 3.3 ms for fluorescence decay at 37 °C is the fastest currently available GECI.A major limitation of GCaMP3 in faithfully tracking Ca2+ transient measurements with the fast linear-response fluorescent dye Furaptra of 36 ms in patch-clamped cardiac myocytes3-evoked Ca2+ release3-evoked Ca2+ releasefastare not limiting the detection of fast Ca2+ transients in cells. The RS-1 and EF-hand mutations resulted in up to 40-fold higher rates compared to GCaMP3, taking the response times of GCaMP3fast down to just under 1 ms at 37 °C.The maximal fluorescence onset rates determine the time resolution at which fluorescence changes are detected by the probes. Response times of >50 ms for GCaMP6f, fast-GCaMP6f-RS06 and fast-GCaMP6f-RS09 were measured in mammalian brain slices. When compared to times to peak of Cafast to GCaMP3 and GCaMP6f in a fast response physiological system. In neonatal cardiac myocytes Ca2+ rise and decays times were significantly faster during spontaneous beating, after caffeine stimulation and in ICa-gated Ca2+ release when recorded using GCaMP3fast. In contrast to GCaMP3 and GCaMP6f, GCaMP3fast revealed entirely discrete Ca2+ oscillations in spontaneously beating cardiac myocytes with a 2.5-fold shorter duration (~210 ms) compared to GCaMP6f (~535 ms) for each oscillatory calcium spike. GCaMP3fast thus shows greater time resolution and is more suitable for tracking fast Ca2+ transients than GCaMP6f. In cardiac myocytes following 50 ms depolarisation, Furaptra had 29 ms time to peak rise and 66 ms decay timesfast in cardiac myocytes following 100 ms depolarisation were a comparable 33 ms and 96 ms, respectively  fast (time to peak 0.8–2.7 s) transients were seen with acetylcholine with decay times of 1.6–9.1 s. GCaMP3fast responded with a faster 0.22 ± 0.08 s rise and a comparable 3.2 ± 0.4 s decay time in ATP stimulated HUVEC kon = 4 × 108 M−1 s−1, koff = 100 s−1, Kd = 260 nM, n = 1)2+ response kinetics in that they have a limiting rate of onset. Faster response kinetics by GCaMP3fast and GCaMP3bright were achieved by an increase in both the limiting rate of onset and the rate of decay. Secondly, having low Kd-s may often result in saturation, e.g. when the Ca2+ stores are fully released. Fast kinetics are achieved by increasing the Kd and consequently also the off-rate. However, when the Kd does not match the signal, e.g. Kd ≫ [Ca2+] peak, then the fluorescence dynamic range is reduced. Thirdly, the genetically engineered dyes can be targeted to specific proteins and therefore be used to probe micro-domains of Ca2+ that are of relevance for privileged communications between subcellular organelles. In this case speed and low affinity are both required 2+ concentration may increase to tens or hundreds of μM, but do so only briefly before being dissipated by diffusion.The basic considerations that go into choosing a Ca2+] concentration ranges roughly fall into three groups: 100 nM to 1 μM, 1 μM to 20 μM (cytosolic) and 100 μM to mM (subcellular organelles and extracellular matrix). Useful indicators (example probes in Kd that is slightly higher than the range of interest (with a moderate dynamic range) or have a high enough Kd ([Ca2+] ~ × 10) (example probes in Kd. This is illustrated well in cardiac myocytes where GCaMP3fast and GCaMP3bright (Kd-s of 1–3 μM) indicate 0.5–1 μM [Ca2+] with the greatest fidelity when compared with the high affinity GCaMP3, GCaMP6f and Fura-2 which distort the time course of Ca2+ signals due to saturation and slow equilibration Apparently greater degrees of cooperativity beyond that predicted by the number of Cabright show no temperature dependence in the 20 °C to 37 °C range. The GCaMP6f probe behaves the same way . The lack of temperature dependence suggests a diffusion limited binding process. As the Ca2+ binding rates are unlikely to be limiting, it may be that peptide binding within the GCaMP construct is sterically constrained, thus the limiting rate is set by the low frequency of collision. In contrast, GCaMP3fast displayed a 5-fold increase of the saturating rate over the same temperature range. By contrast, dissociation rates of all the probes were greater at 37 °C than at 20 °C, an advantageous property for in vivo applications.Interestingly, the limiting rates for GCaMP3 and GCaMP32+ association and dissociation kinetics, we developed a kinetic model to quantitatively account for the observed behaviour of GCaMP3fast  by peptide binding to the P1 CaM binding site; a second intermediate (with relative fluorescence F2) formed by completing Ca2+ binding to the C-lobe site and peptide at the P2 site too. Postulation of two fluorescent conformers was based on the observation of biphasic Ca2+ association and dissociation kinetics for some mGCaMP and the requirement for functional N-lobe Ca2+ binding sites in CaM (EF-1 and EF-2) for fluorescence to develop.Taking advantage of our detailed measurements of CaaMP3fast . The mod−1 from the Ca2+.CaM.peptide complex with an RS20 analogue peptide (Trp peptide)2+ dissociation rates from the Ca2+.CaM.cpEGFP.RS20 mutant complexes . This further suggests a strategy for improving the fluorescence and kinetic properties of GCaMP-type GECI. N-lobe CaM mutations are detrimental whereas C-lobe mutations improve or are neutral to 2+ kinetics are most markedly affected by combining mutations of the RS20 peptide and CaM EF-hands. The specific mutations carried out on GCaMP3 and derived GECI are applicable to the new generations of GECI e.g. GCaMP5, GCaMP6 and R-CaMP213152+ probes, a major hurdle has been overcome.The new data and interpretations presented here show that we have made a systematic and successful effort to improve the kinetics of biologically engineered calmodulin-based fluorescent peptide Ca2-BAPTA was purchased from Life Technologies.GCaMP6f, G-GECO 1.0 and GEM-GECO plasmids were purchased from Addgene . pET41a and pEGFP-N1 vectors were obtained from Novagen and Clontech, respectively. XL10-Gold and BL21 (DE3) Gold cells were purchased from Invitrogen. Fluo-3 was purchased from Molecular Probes and EZ-Run protein ladder from Fisher Bioreagents. Restriction enzymes were obtained from New England Biolabs and T4 DNA ligase from Fermentas. Brgcamp3 gene was subcloned by restriction-ligation from pUC57 into pET41a using BglII and NotI restriction enzymes and T4 DNA ligase following manufacturers’ protocols. The ggeco and gemgeco genes contained in pTorPE vectorsThe A series of DNA mutations were performed on pET41a-GCaMP3, pET41a-G-GECO and pET41a-GEM-GECO. Site-directed mutagenesis was carried out following the QuickChange II XL protocol (Agilent Technologies) to mutate the CaM EF-hands using the following primers (5′→3′): EF-1, GCTTTCTCCCTATTTGCCAAGGACGGGGATGGG; EF-2, CATGATCAATGAAGTAGCTGCCGACGGTAATGGC; EF-3, GCGTTCCGTGTGTTTGCTAAGGATGGCAATGGC; EF-4, GATCAGGGAAGCAGCCATCGATGGGGATGG; for the Trp →Tyr substitution in the RS20 peptide sequence, primer RS-1 (5′ →3′), CGACTCATCACGTCGTAAGTACAATAAGACAGGTCACGCAG was used. Mutations were confirmed by DNA sequencing (Beckman Coulter Genomics).E. coli BL21(DE3) Gold cells. Cells were grown at 37 °C and expression was induced overnight at 20 °C in the presence of 0.5 mM isopropyl thio-β-D-galactoside (IPTG). Cells were resuspended in 50 mM Na+-HEPES, 200 mM NaCl, pH 7.5 containing one tablet of Complete protease inhibitor cocktail . Cell lysis was performed by sonication on ice  and clarified lysates were purified by a single-step GST-chromatography  at 4 °C. The column was equilibrated with 50 mM Na+-HEPES, 200 mM NaCl, pH 7.5. The purified protein was eluted in 50 mM Na+-HEPES, 200 mM NaCl, 10 mM reduced glutathione, pH 7.5 and aliquoted fractions were stored at −80 °C. Purity of the eluted fractions was determined by SDS-PAGE (gradient of 6.4%–20% acrylamide/bisacrylamide) and the identity of the GCaMP3, G-GECO and GEM-GECO proteins was confirmed by MALDI mass spectroscopy using sinapinic acid as a matrix .GCaMP3, G-GECO and GEM-GECO proteins were overexpressed in εo) at 280 nm calculated from the amino acid compositiono(280) were verified by parallel measurements using the Bradford assay280/A400 was affected by the mutations introduced. From the εo(280) values and the A280/A400 ratios, the εo(400) values were calculated for each probe in a folded state at physiological pH rather than following protein denaturationMutant GCaMP3 proteins were highly purified , allowinfast was tested at 37 °C by measurement of fluorescence emission over 4 h. Excitation wavelength was 492 nm.The stability of GCaMP3Kd for Ca2+ of GST-GCaMP3 was 0.33 ± 0.01 μM compared to 0.42 ± 0.06 μM of GCaMP3 from which the GST-tag had been removed on the column by thrombin digestion  of 326 ± 12 nM. Similar dissociation constant (Kd = 325 nM) has been reported by Molecular Probes. Fluo-3 was used as a reference compound to verify the calculated [Ca2+] of the solutions made for the stopped-flow experiments of association kinetics . Fluo-3 or GECI proteins at 50–100 nM concentration  were titrated with 325 mM CaCl2 at a 10 μL/min flow rate in a stirred 3 mL cuvette. Fluorescence was measured at 492 nm excitation and 512 nm emission wavelengths using a Fluorolog3 spectrofluorimeter (Horiba Scientific). For mGCaMP3 RS-1 EF-hand mutants, Br2-BAPTA was used for Ca2+ buffering instead of EGTA. Fluorescence records were corrected for dilution and photobleaching. Data were normalised and expressed as bound fraction and Ca2+ dissociation constant (Kd) and cooperativity (n) were obtained by fitting the data to the Hill equation using Prism GraphPad 6 software. All titrations were performed at least in triplicates and expressed as mean ± s.e.m.Ca2+ association and dissociation kinetic experiments of GCaMP3, G-GECO and GEM-GECO proteins and of Fluo-3 were carried out on a Hi-Tech Scientific SF-61DX2 stopped-flow system at 20 °C, unless specified otherwise. Fluorescence excitation was set to 492 nm for mGCaMP3 and mG-GECO, or 392 nm for mGEM-GECO. Fluorescence emission was collected using a 530 nm cut-off filter. At least 3 replicates were averaged for analysis. Data were fitted to either a single or a double exponential to obtain the rise or decay rate using KinetAsyst software (TgK Scientific). As the “dead-time” of the instrument (time between the mixing and the point of observation) is ~1.5 ms, rates above 500 s−1 (t1/2 of 1.4 ms) cannot be accurately measured. To overcome this limitation, kinetics were measured at lower temperatures and the rates obtained by extrapolation from Arrhenius plots.Ca+-HEPES, 100 mM KCl, 2 mM MgCl2, 10 mM EGTA pH 7.5 was rapidly mixed (1:1) with 50 mM K+-HEPES, 100 mM KCl, 2 mM MgCl2, 10 mM EGTA pH 7.5 containing increasing [Ca2+] concentrations (concentrations in the mixing chamber). For the determination of temperature dependence of Ca2+ association rates, protein samples at 1 μM concentration were mixed as above to give a 20 μM final [Ca2+] in the mixing chamber. The Ca2+ association rates to Fluo-3 were too fast to measure.The solution containing 1 μM protein in 50 mM K+-HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.5 with saturating Ca2+ between 0.1–0.5 mM was rapidly mixed (1:1) with 50 mM K+-HEPES, 100 mM KCl, 2 mM MgCl2, 10 mM EGTA pH 7.5 (concentrations in the mixing chamber). The Ca2+ dissociation rate from Fluo-3 was measured using the same conditions.The solution containing 1 μM protein in 50 mM K2 with either 5 mM EGTA or 1 mM CaCl2) and the fluorescence spectra were recorded on a Fluorolog3 (Horiba Scientific). GCaMP3 quantum yield measured in Ca2+-saturated buffer was used as a reference (Φ+Ca2+ = 0.44protein = ΦGCaMP3 × (Sprotein/SGCaMP3).The concentration of GCaMP3 proteins was adjusted such that the absorbance at the excitation wavelength (490 nm) was between 0.001 and 0.1. A series of dilutions was prepared in a buffered solution . The pH titrations were performed by recording fluorescence spectra in Ca2+-free  or Ca2+-saturated  using 1 μM protein in 0.5 pH unit intervals . BAPTA was chosen as Ca2+ chelator because of its stable affinity for Ca2+ over the pH range.To determine the apparent pgfp initially present in the pEGFP-N1 vector was replaced by the gcamp3, ggeco or gem-geco gene.For expression in eukaryotic cells, the GCaMP3, G-GECO and GEM-GECO mutant DNAs were subcloned from pET41a into pEGFP-N1 vectors by restriction-ligation using BglII and NotI restriction enzymes and T4 DNA ligase following manufacturer’s recommendations. During this process, the fast under the control of the CMV promoter were obtained from Vector Biolabs (USA). Titer was estimated to be 5 × 1010 PFU/mL.Adenoviruses (Ad5 backbone) expressing GCaMP3, GCaMP6f and GCaMP33 that were incubated in HBSS with trypsin (50 μg/mL) for 14–16 h at 4 °C. The digestion was then arrested by exposure to trypsin inhibitor (200 μg/mL) for 20 min in 37 °C. Thereafter collagenase (100 U/mL) was used for 30 min to isolate single rN-CM, which were filtered through a cell strainer and centrifuged at 1000 rpm for 3 min. Cells were re-suspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) with 1% penicillin/streptomycin and 1% non-essential amino acids, plated on 100 mm dishes and placed in the incubator for 1–2 h to eliminate fibroblasts. Isolated single rN-CM were plated onto non-treated glass cover slips and used for adenovirus infection with Ad-GCaMP3, Ad-GCaMP6f or Ad-GCaMP3fast. After adenoviral infection of neonatal cardiomyocytes, the level of expression changes several-fold during 2–5 days of experimentation without significant changes in the time course of the measured fluorescence transients. Cells were used for confocal imaging and electrophysiology 48–72 h after viral infection. Although contraction artefacts are not particularly bothersome in short time cultured neonatal cardiomyocytes, they were suppressed by treating the cells with 10 μM blebistatin.Rat neonatal CMs were isolated using an isolation kit from Worthington Biochemical Corporation . One to three day-old neonatal rats were decapitated and the beating hearts were surgically removed and placed in chilled Hank’s Balanced Salt Solution (HBSS). The main vessels and atria were removed and the ventricles were minced with a razor blade to pieces <1 mm+-free solutions to probe the cellular Ca2+ stores or spontaneous Ca2+ release from sarcoplasmic reticulum.Rapid applications of 3 mM caffeine were used in the external KCa using a Dagan amplifier and pClamp software (Clampex 10.2). In most experiments the membrane under the patch pipette was ruptured and the cells dialyzed with Ca2+-buffered pipette solutions containing 110 mM Cs+-aspartate, 5 mM NaCl, 20 mM TEA-Cl, 5 mM Mg2+-ATP, 0.1 mM EGTA 0.1 mM CaCl2, 10 mM glucose and 10 mM Cs+-HEPES pH 7.2. In some experiments the cells were examined with the perforated patch technique using pipette solutions containing 145 mM Cs+-glutamate, 9 mM NaCl, 1 mM MgCl2, 10 mM Cs+-HEPES pH 7.2 and 0.69 mg/mL amphotericin B (Fisher Scientific). The extracellular solution used during experiments contained 137 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose and 10 mM Na+-HEPES pH 7.4.Cells were voltage clamped in the whole-cell configuration at −50 mV to measure voltage dependent I2+, [Ca2+]i, was measured simultaneously with the patch clamp or independently using a Noran Odyssey XL rapid two-dimensional laser scanning confocal microscopy system  attached to a Zeiss Axiovert TV135 inverted microscope fitted with an ×63 water-immersion objective lens. The excitation wavelength of the argon ion laser was set to 488 nm, and fluorescence emission was measured at wavelengths >515 nm. Cells were imaged confocally at 30–120 frames/s depending on the experimental requirement. To reduce photobleaching, the laser beam was electronically shuttered and triggered to open on command by the pClamp program only during acquisition of data. Images were filtered by 2 × 2 pixel-averaging. Results are presented as whole-cell measurements by integrating the fluorescence response from an entire cell .Cytosolic Caire cell , or as l2, 10 mM glucose, 10 mM Na+-HEPES pH 7.4) and placed on the stage of an inverted Olympus IX71 fluorescence microscope. The microscope was equipped with an Olympus UPLSAPO ×100 1.40 NA objective and an OptoLED light source  for fast epifluorescence illumination of Fura-2 (355 ± 10 nm and 380 ± 11 nm) and EGFP (470 ± 20 nm) fluorescence. Excitation light was directed to the specimen via a 500DCXR dichroic mirror  and emitted light collected through a 535 ± 30 nm emission filter onto an Ixon3 EMCCD camera  operated in frame transfer mode at full gain and cooled to −68 °C. Images were acquired at 30 frames/s using Winfluor software (http://spider.science.strath.ac.uk).HUVEC were grown and transfected as previously described372+-dependent cell output, of fluorescent secretory granules occurred37fast-expressing cells with low expression levels (low intrinsic levels of resting fluorescence) were chosen to minimise the effect of buffering, and the time courses for GCaMPfast signals were similar with or without simultaneous Fura-2 loading. Arbitrary units of fluorescence were used as Fura-2 calibration is not meaningful due to the saturating levels of [Ca2+] rise.Fura-2 loading was performed under conditions where it had previously been established that no perturbation of stimulated exocytosis, a particularly sensitive Ca2+] to high levels. For physiological studies cells were stimulated with the extracellular hormone ATP (2 μM). Drug application was by pressure injection from a glass pipette  at ~2 p.s.i. positioned close to the cell as previously describedTo determine the dynamic range of mGECI, cells were stimulated using ionomycin (2 μM) to increase intracellular free [Ca2+]i, as previously describedTo determine the time course for cytoplasmic Rura-2 and/or GCaMP fluorescence changes during ATP stimulation a rectangular region of interest (ROI) was placed over a peripheral region of each cell, excluding the nucleus and immediate perinuclear region. The mean background fluorescence was measured by ROI with the same dimensions placed at a point at which no cell was present. GCaMP fluorescence changes were obtained from the background-subtracted fluorescence at 470 nm excitation within the WinFluor program. Fura-2 ratio changes were calculated by dividing the background-subtracted fluorescence at 355 nm by that at 380 nm in WinFluor. The ratio of the resulting background-subtracted fluorescence signals at 355 nm and 380 nm were taken to represent the change in  by Fura-2 and GCaMP3 or mGCaMP3 by employing alternating 380/25 and 488/20 nm wavelengths, respectivelyTransfected HEK293T cells were loaded with 1 μM Fura-2 AM  for 20 minutes at 37 °C, and then incubated in dye-free solution for at least 20 minutes before beginning experiments to allow for the de-esterification. Cells were mounted on the stage of an inverted Nikon microscope with a ×40 oil-immersion objective and perfused with a Tyrode’s solution at 37 ± 2 °C  is the absorption coefficient for the simultaneous absorption of two photons for the excitation of a fluorophore. TPA becomes relevant at high light intensities and as it is generated by the sum of the energies of two photons, it occurs at approximately double of the wavelength at which one photon excitation would have occurred. The two-photon fluorescence cross-sections of GCaMP3, G-GECO, GEM-GECO and their mutants were typically measured at 940 nm (unless specified otherwise) in 50 mM K+-HEPES, 200 mM NaCl, pH 7.5, 10 mM reduced glutathione at high Ca2+ (200 μM) or low Ca2+ (2 mM EGTA). Measurements were made in a 50 μL quartz cuvette with 3 mm path length. Excitation was with femtosecond-pulsed light from a tuneable TiS laser  focused into the cuvette with a 50 mm f.l. achromatic doublet after 3× expansion. Average power was monitored after the cuvette. Fluorescence was measured normal to the excitation path through a band pass filter (Chroma ET525/50-2P) with a photodiode and I/V amplifier. A reference spectrum for fluorescein (20 μM in 10 mM Na+-borate buffer pH 11) was determined in each run. After subtraction of background, fluorescence cross-sections were calculated for Ca2+-saturated and Ca2+-free protein with reference to the published values for fluoresceinTwo-photon absorption (TPA) or two-photon cross-section  software. Cardiac myocytes, HUVEC and HEK293T cell experiments were carried out on three independent preparations each. The total number of cells analysed in each condition is given in the figure legends. Significance level was obtained using the one-way ANOVA test with multiple comparisons. P values in the figures are represented by stars . Kinetic modelling was performed using DynaFit4 software according to the scheme presented in How to cite this article: Helassa, N. et al. Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics. Sci. Rep.5, 15978; doi: 10.1038/srep15978 (2015).


Heart failure with preserved ejection fraction (HFpEF) accounts for about 50% of heart failure cases. It has features of incomplete relaxation and increased stiffness of the left ventricle. Studies from clinical electrophysiology and animal experiments have found that HFpEF is associated with impaired calcium homeostasis, ion channel remodeling and concentric left ventricle hypertrophy (LVH). However, it is still unclear how the abnormal calcium homeostasis, ion channel and structural remodeling affect the electro-mechanical dynamics of the ventricles. In this study we have developed multiscale models of the human left ventricle from single cells to the 3D organ, which take into consideration HFpEF-induced changes in calcium handling, ion channel remodeling and concentric LVH. Our simulation results suggest that at the cellular level, HFpEF reduces the systolic calcium level resulting in a reduced systolic contractile force, but elevates the diastolic calcium level resulting in an abnormal residual diastolic force. In our simulations, these abnormal electro-mechanical features of the ventricular cells became more pronounced with the increase of the heart rate. However, at the 3D organ level, the ejection fraction of the left ventricle was maintained due to the concentric LVH. The simulation results of this study mirror clinically observed features of HFpEF and provide new insights toward the understanding of the cellular bases of impaired cardiac electromechanical functions in heart failure. Heart failure (HF) is often categorized into two major types   and a decreased Ca2+ release from ryanodine receptors , the background Ca2+ channel and the Na+/Ca2+ exchanger  to develop a novel HFpEF electromechanical single cell model based on extant experimental data; (2) to investigate the cellular mechanisms influencing myocardial calcium homeostasis in HFpEF using an electromechanical single cell model; and (3) to evaluate the functional impacts of impaired Ca2+ handling, ion channel remodeling and degrees of concentric LV hypertrophy on the electro-mechanical activity of the heart.The functional impacts of HFpEF-related abnormal Ca2+ vs. voltage-dependent inactivation of L-type Ca2+ current and Ca2+/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca2+ cycling. For simulating cellular mechanics properties, we used the Tran et al. myofilament (MM) model , we utilized the O'Hara-Rudy (ORd) human ventricular single cell model  is the sarcolemmal Ca2+ pump current, CabI (μA/μF) is the Ca2+ background current, NaCa,iI (μA/μF) is the myoplasmic component of Na+/Ca2+ exchange current, capA (cm2) is capacitive area, F (coul/mol) is the Faraday constant, myov (μL) is the volume of the myoplasmic compartment, nsrv (μL)is the volume of the network sarcoplasmic reticulum compartment, ssv (μL) is the volume of the subspace compartment, upJ (mM/ms) is the total Ca2+ uptake flux, via SERCA pump from myoplasm to the network sarcoplasmic reticulum, diff,CaJ (mM/ms) is the flux of the diffusion of Ca2+ from the subspace to the myoplasm and TropJ (μM/ms) is the flux of Ca2+ binding to troponin calculated via the MM model. βCai is formulated as:2+ buffer in the myoplasm and m,CMDNK is the half-saturation concentration of calmodulin.The intracellular calcium concentration from the EP model was used as the coupling link to the MM model. i level, a reduction in SR Ca2+ content and reduced SR Ca2+ release, a reduction in SR Ca2+ sequestration, an increase in resting tension, incomplete relaxation, reduced systolic stress and prolonged stress and strain durations. These mechanisms suggest that in HFpEF patients, impaired Ca2+ handling principally caused by reduction in NCX activity is a dominant abnormality in the condition that explains the mechanisms for impaired cardiac electro-mechanics in HFpEF.We have developed a novel, biophysically detailed model of HFpEF and used it to investigate the cellular mechanisms underlying myocardial CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.