A quantitative characterization of presynaptic Ca2+ signaling is critical for understanding synaptic transmission. However, this has remained challenging to establish at the required resolution. Here, we employ confocal and stimulated emission depletion (STED) microscopy to quantify the number (20–330) and arrangement (mostly linear 70 nm × 100–600 nm clusters) of Ca2+ channels at AZs of mouse cochlear inner hair cells (IHCs). Establishing STED Ca2+ imaging, we analyze presynaptic Ca2+ signals at the nanometer scale and find confined elongated Ca2+ domains at normal IHC AZs, whereas Ca2+ domains are spatially spread out at the AZs of bassoon-deficient IHCs. Performing 2D-STED fluorescence lifetime analysis, we arrive at estimates of the Ca2+ concentrations at stimulated IHC AZs of on average 25 µM. We propose that IHCs form bassoon-dependent presynaptic Ca2+-channel clusters of similar density but scalable length, thereby varying the number of Ca2+ channels amongst individual AZs.Ca 2+ channels in inner hair cells.Quantitatively studying components of the presynapse requires high resolution optical methods. Here the authors use confocal microscopy as well as 2D- and 3D-STED nanoscopy to quantify the number and activity of active zone Ca For a few decades, synaptic neuroscience has aimed to establish a quantitative understanding of AZ Ca2+ signaling10. Progress has been limited, however, by technical challenges such as the resolution limit of conventional light microscopy, which has precluded direct visualization of nanoscale Ca2+ domains. Therefore, indirect approaches analyzing Ca2+-dependent processes such as transmitter release and the gating of presynaptic Ca2+-activated potassium channels6 as well as mathematical modeling11 have been employed to elucidate spatiotemporal Ca2+-concentration profiles at the AZ. But understanding synaptic transmission requires information on the number, biophysical properties, and topography relative to vesicular release sites of the presynaptic Ca2+ channels that determine the AZ Ca2+ signaling. Indeed, localization and quantification of presynaptic Ca2+ channels by electron microscopy13 or cell-attached recordings15 has recently fueled the progress.Voltage-gated Ca2+ influx using AZs of sensory inner hair cells (IHCs) as a model system. IHCs are well-suited for studies of Ca2+ signaling at individual AZs because their AZs are relatively large and functionally separated due to µm-scale nearest-neighbor distance and strong Ca2+ buffering18. Moreover, voltage-gated Ca2+ influx in IHCs is almost exclusively mediated by a single type of Ca2+ channel (CaV1.3)19, which has also been characterized at the single-channel level20. Freeze-fracture electron microscopy21 and two-dimensional stimulated emission depletion (2D-STED) microscopy of immunolabeled Ca2+ channels22 suggest a stripe-like clustering of Ca2+ channels at the AZ, at least in fixed hair cells, similar to observations of the presynaptic protein bassoon at conventional synapses23. Based on analysis of the whole-cell Ca2+ current and the average number of AZs, the mean number of Ca2+ channels per AZ has been approximated to 80–10024. Ca2+ nanodomains with [Ca2+] up to hundreds of µM were postulated from modeling, readout of [Ca2+] by large conductance Ca2+-activated K+ channels3, or synaptic exocytosis24. Direct nanoscopic imaging of presynaptic [Ca2+], however, has yet to be established for AZs of IHCs or other presynaptic cells. Along with information on the number and topography of the Ca2+ channels and vesicular release sites14, such measurements will advance our understanding of stimulus–secretion coupling at the AZ. Moreover, a major heterogeneity of presynaptic Ca2+ signaling was observed among the AZs of a given cell17, which in IHCs likely contributes to the response diversity observed for postsynaptic neurons26. This calls for analysis beyond the average properties of AZs.Here, we studied the quantitative nanophysiology of presynaptic Ca2+ channels at individual synapses using innovative methods. We combine confocal Ca2+ imaging with selective inhibition of Ca2+ influx at individual AZs and optical fluctuation analysis for estimating the number of Ca2+ channels per AZ. Employing 3D-STED, we quantify the structure of AZ Ca2+-channel clusters in IHCs. We establish 2D-STED Ca2+ imaging and STED fluorescence lifetime measurements of AZ [Ca2+] and validate the method by mathematical modeling based on quantitative structural and functional characterization of presynaptic Ca2+ channels.Here, we make use of the good experimental accessibility of individual AZs in IHCs to analyze the number, distribution, and activity of Ca2+ channels located at IHC AZs using super-resolution STED microscopy. AZs were immunolabeled against both the CaV1.3α1 subunit of the CaV1.3 Ca2+ channel as well as against the presynaptic protein bassoon, which marks the presynaptic density at IHC AZs27. Using 2D-STED, we imaged AZs that were located at the basal membrane of an IHC, selecting those that seemed to lie parallel to the imaging plane  of ~70 nm . The apparent length  of the CaV1.3 channel clusters averaged 239 ± 20 nm  and varied between approximately 100 and 600 nm, likely reflecting differences in AZ size28. Similarly, bassoon clusters showed an average apparent length of 235 ± 31 nm . When relating the length of Ca2+-channel and bassoon clusters with the lower resolution estimate of the length of their corresponding bassoon and Ca2+-channel clusters (labeled with Abberior STAR580-conjugated secondary antibody), respectively, we found a high degree of correlation  scale with each other.For the most faithful 2D-STED measurements of the clusters, we used a secondary antibody tagged with the dye providing the highest resolution (Abberior STAR635P) for either Calar Fig. , with thxy) and no axial (z) super-resolution) would lead to an underestimation of the true cluster dimensions. We therefore turned to 3D-STED imaging29 to complement our 2D-STED data. While sacrificing some lateral resolution for increased axial resolution, we could obtain a near-isotropic effective point spread function (PSF), i.e., probing region. We acquired stacks of images -N,N,N',N'-tetraacetic acid (EGTA) to selectively reduce Ca2+ influx at a single AZ  by locally depleting free extracellular Ca2+ while maintaining the Ca2+ influx at the other AZs of the IHC. We employed fast confocal spinning-disk microscopy of patch-clamped mouse IHCs, loaded with the low affinity Ca2+-indicator Fluo-8FF (KD = 10 µM) and a fluorescently tagged peptide marking ribbon-type AZs30. This allowed us to simultaneously image Ca2+ signals at several AZs in the confocal section , we consider the observed Ca2+ signals to linearly relate to the voltage-gated Ca2+ influx.For the first approach, we used focal microiontophoresis of the Caion Fig.  and also−1). Selective manipulation of an AZ was verified by monitoring signals from nearby AZs , Fig. 2+ influx . We then converted this into a distribution of total Ca2+-channel number per AZ assuming a single-channel current of 0.29 pA31  and an open probability of 0.424.We aimed for selective manipulation of a single AZ by (i) targeting a sufficiently isolated AZ, (ii) close apposition of the iontophoresis pipette to the membrane, and (iii) vivid bath perfusion . We then used the mean slope of those 19 manipulated AZs (14.3 ± 1.4 pA) to estimate ICa,AZ for a set of 234 AZs representing most of the synapses of a large sample of 25 IHCs : 0.5). The resulting estimates of total Ca2+-channel number revealed major heterogeneity among the AZs: their Ca2+-channel complement ranged from 28 to 329 with a mean of 125 and a median of 118 —but does not change the single-channel current32. We argued that maxing out open probability would reduce potential errors in deriving the number of Ca2+ channels from the synaptic Ca2+ influx. We observed the expected doubling of ICa,AZ, but the slope of the relationship between ICa,AZ and ΔF/F0 was comparable . Importantly, the estimates of the number of Ca2+ channels per AZ in the presence and absence of BayK8644 did not differ for the strong synapses analyzed .In order to validate our approach, we repeated the experiment in the presence of the Ca2+ channels at single AZs by analyzing non-stationary fluctuations of Ca2+-indicator fluorescence  during the injection of deactivating Ca2+ currents , opening Ca2+ channels but not permitting Ca2+ influx, then elicited Ca2+-tail currents by hyperpolarization to −67 mV and recorded the accompanying transient increases in Fluo-4FF fluorescence at the center of the fluorescence hotspot , it remained to be elucidated whether an analysis of non-stationary fluctuations could work at AZs facing a large cytosolic volume, as is the case in IHCs. The analysis of channel-gating-related fluctuations of Ca2+-indicator fluorescence is limited by the relatively low recording bandwidth resulting from the kinetics of Ca2+ binding (0.412 µM−1 ms−1) and unbinding (4 ms−1)35 of Fluo-4FF. Moreover, the contribution of the Ca2+ channels to the fluorescence signal varies dependent on their position within the cluster relative to the PSF of the microscope, due to uneven excitation and detection. In order to better match the signal to the limited recording bandwidth, we slowed Ca2+-channel deactivation by the agonist BayK864424 (5 µM), which increases the channel’s open time32. We addressed the impact of the inhomogeneous contribution of Ca2+ channels by mathematical modeling, which indicated that the mean and variance of the fluorescence signal are both affected in a way causing the error in the estimation of the number of Ca2+ channels to be rather limited  by subtracting the detector noise and photon shot noise  per AZ (fluorescence) and per IHC (current) by binomial fitting to both data sets  was consistent with previous estimates24.We then isolated the ensemble variance  resulting from Caise Fig. . As expeise Fig. . Finallynts Fig. . We estiets Fig. . NCa val2+ channels per AZ (125 and 78), suggesting either an overestimation of the number of channels during iontophoresis , or an underestimation in the optical fluctuation analysis experiments , or a combination of both. Also, we cannot exclude a small contribution of an age dependence of the AZ Ca2+-channel complement, because the two approaches made use of mice of different age , for which we find subtle differences in the ΔF/F0,max of the Ca2+ indicator  were comparable to the one previously reported for the maximal synaptic Ca2+ influx (0.6517) and furthermore in good agreement with our data of the integrals of the 2D Gaussian fits to CaV1.3 immunofluorescence of Ca2+-channel clusters  with the count of synapses per IHC—typically 12 in the area of the organ of Corti used in our measurements—one arrives at a value between 950 and 1500. Relating this to the total number of Ca2+ channels per IHC  indicates that approximately 20–50% of all channels localize extrasynaptically.In summary, both experimental approaches reported somewhat different estimates for the mean number of Cas per AZ 25 and 782+] to reach ~3 µM at IHC AZs during depolarization17. This appears surprisingly low considering the large number of Ca2+ channels established here. Most likely, spatial averaging due to the microscope’s large PSF caused this low estimate, implying that the true [Ca2+] at the AZ might be far higher. We postulated that STED super-resolution imaging with its greatly reduced PSF size would provide much more accurate measurements of [Ca2+] at the AZ, allowing us to establish the concentrations occurring in vivo. First, we screened several Ca2+-indicator dyes for compatibility with STED nanoscopy and found Oregon Green BAPTA-5N (OGB-5N) to be the best-suited Ca2+ indicator with low affinity . In order to predict what [Ca2+] could be expected at the IHC AZ and whether super-resolution imaging could more accurately measure the local concentration, we performed reaction/diffusion simulations of the IHC AZ using the CalC modeling software37. Our model was based on the number and distribution of channels experimentally determined above  and non-fluorescent intracellular Ca2+ chelators .A previous study had estimated presynaptic [Ca2+] (Ca2+ not bound to Ca2+-indicator or non-fluorescent buffers) can reach up to 100–150 µM in close proximity (10–20 nm distance) to the channel mouth , we found concentrations of up to ~45 µM , and again calculated [Ca2+]. As expected, the spatial averaging  caused by the PSF leads to a marked underestimation of [Ca2+], with maximum values of ~10 µM  vs. 332 × 454 , indicating the potential benefits of STED Ca2+ imaging.Evaluation of the model showed that low Ca2+] Fig. . We therain Fig. , we deci µM Fig. . CompareBuilding on the above results, we tested the feasibility of combining patch-clamp with live STED imaging in HEK-293 cells and IHCs, with regard to both the stability for patch-clamping and nanoscale imaging, as well as to the physiological condition of the cell. During STED imaging, we observed neither strong mechanical drifts nor detrimental effects to the stability of the patch-clamp recording. Both HEK-293 and hair cells tolerated exposure to even the highest possible STED beam intensities for several seconds typically without showing signs of light-induced leak currents.2+ imaging, using physiological [Ca2+]e (1.3 mM) and 0.8 mM EGTA, 0.4 mM BAPTA, and 25 µM OGB-5N in the pipette solution. Aiming for AZs near the base of the cell , we depolarized the cell for 78 ms and imaged OGB-5N fluorescence hotspots in small (3 µm × 1.5 µm) xy-scans , in good agreement with the results of our modeling 38; these AZs typically lack the synaptic ribbon and hold fewer Ca2+ channels. Previous STED imaging of CaV1.3 immunofluorescence had suggested that these channels form small, spot-like clusters22, whereas live confocal Ca2+ imaging (with 2 mM intracellular EGTA and 5 mM [Ca2+]e) had reported that the width of the presynaptic Ca2+ domains at bassoon-mutant AZs was indistinguishable from wildtype AZs (FWHM of ~1 µm in both cases)22. Interestingly, we could clearly distinguish the bassoon-mutant AZs, which displayed significantly larger Ca2+ domains  than wildtype AZs. STED imaging did not reveal any further details within these large Ca2+ domains, suggesting that Ca2+ channels are more widely dispersed in the presynaptic membrane when tethering to the AZ by bassoon is not available. This leads us to suspect that the small spot-like appearance of CaV1.3 immunofluorescence22 was not physiological but artificial, e.g., resulting from the aggregation of untethered Ca2+ channels during precipitation by the fixative. Based on our above modeling, we assume that the inability of the previous study to visualize the size differences of the presynaptic Ca2+ hotspots in both genotypes is explained by the imaging conditions of that study, including a larger confocal PSF, high [Ca2+ indicator], and higher [Ca2+]e than [EGTA]. We further examined bassoon-deficient AZs in additional experiments using conditions designed to obtain a stronger signal (by enhancing Ca2+ influx (10 mM [Ca2+]e), utilizing higher [OGB-5N] (300 µM) and [EGTA] (10 mM); “intensified conditions”; Fig. 2+ domains in confocal or STED Ca2+ imaging. Interestingly, in wildtype AZs these “intensified conditions” (with the expected presynaptic [Ca2+] ≪ [OGB-5N]) evoked somewhat larger Ca2+ hotspots than observed under “physiological” conditions.We applied the same imaging protocol to observe IHC AZs in mice lacking functional bassoon  of OGB-5N fluorescence with STED resolution , which needs to be considered to avoid overestimating the free (fast) dye contribution during STED measurements  or completely de-populated , this was not the case when measuring inside living IHCs. Even when patching a cell with 60 mM [Ca2+], the contribution from the slow lifetime component never surpassed 60% .Furthermore, in contrast to the in vitro calibration, where the Cahes Fig. : (i) res2+-binding behavior of OGB-5N varied strongly depending on its immediate experimental environment. Initially, we performed in vitro calibrations of Ca2+ binding by OGB-5N in “minimalistic” solutions with well-known [Ca2+]  and found a KD of ~40 µM , we found a much higher effective KD (Keff) of ~195 µM  for all further calculations  covering an AZ  or Ca2+-bound OGB-5N (slow lifetime). After applying the aforementioned corrections, the ratio of fast and slow lifetime components allowed us to approximate the [Ca2+] at the AZ during depolarization  for reporting [Ca2+] close to the Ca2+-channel cluster. Clearly, however, the low resolution along the optical (z) axis of our 2D-STED microscope still caused significant spatial averaging, conflating the high [Ca2+] within the nanoscale space around the Ca2+ channels with the lower [Ca2+] further away from the AZ.We applied the TCSPC method to measure [Ca AZ Fig. . Taking 2+] was sampled. For this, we shifted the focus of the objective below the AZ while measuring [Ca2+], so that the majority of the volume illuminated by the microscope’s PSF was outside the cell and thus devoid of dye  will need to be clarified in further experiments.Our study corroborates the notion that bassoon and/or the synaptic ribbon organize the spatial arrangement of Caane Fig. . This diels Fig. . Whether9, and ideally their spatial relationship to synaptic vesicles tethered at the AZ. This will provide important input into biophysical modeling of how Ca2+ influx couples to exocytosis of synaptic vesicles. Biophysical experiments and modeling have so far indicated that, at the IHC AZ, exocytosis of a given readily releasable vesicle is under “Ca2+-nanodomain-like” control of few Ca2+ channels with a mean effective coupling distance between mouth of channel and Ca2+ sensor of 17 nm46, which we postulate to be common to IHC AZs regardless of their size. Using two independent methods to assess the number of Ca2+ channels at a single synapse, we found on average approximately 80–120 Ca2+ channels per AZ, whereby the counts varied dramatically between AZs (20–330). The wide distribution of lengths we measured for the long axis of CaV1.3 clusters by STED microscopy —considerably higher than typically found in central nervous system (CNS) neurons where it ranges from 500 to 150013—likely through a protein network organized by bassoon49 and including the synaptic ribbon50, RIM52, and RIM-binding protein54. Given this very high density it seems likely that the Ca2+ influx at IHC AZs serves additional roles besides triggering synaptic release, e.g., increasing [Ca2+] to facilitate vesicle resupply55. The distance between Ca2+ channels and synaptic vesicles appears to be comparable between IHC AZs and some CNS AZs58, which in some cases employ a placement of synaptic vesicles around the perimeter of Ca2+-channel clusters9. Thus, it seems possible that similar mechanisms are used in these presynaptic preparations, even though IHCs make use of a different set of proteins for the release of synaptic vesicles59. Our Ca2+-imaging data illustrates how disruption of bassoon disintegrates this sophisticated supramolecular machinery at the AZ in a manner reminiscent of what was seen at drosophila neuromuscular junctions upon mutation of the scaffold bruchpilot60. In addition, our study validated the previously assumed presence of extrasynaptic channels in IHCs24, which form a considerable fraction of 20–50%. In CNS AZs, a subdivision into smaller clusters has been described, with a 1:1 relationship between the number of Ca2+-channel clusters and the number of vesicular release sites13. Future analysis, e.g., employing MINFLUX microscopy61 or electron microscopy of immunolabeled SDS-replica9, will be required to test such a scenario for IHCs. Interestingly, despite the different complement of Ca2+ channels per AZ, the number of individual channels per release site appears comparable: nine channels/site reported in parallel fiber synapses13 vs. 80–120 channels for 10–15 release sites62 at IHC AZs.IHC ribbon-type AZs achieve the high average density of 4000 Ca2+] measurements, that insufficient spatial resolution causes artificially low estimates of AZ [Ca2+], because “spatial averaging” conflates the true [Ca2+] levels directly at the AZ with lower-[Ca2+] regions further from the Ca2+-influx sites. By establishing fluorescence lifetime Ca2+ imaging at confocal and 2D-STED resolution, we indeed saw higher (average) peak AZ [Ca2+] using 2D-STED (15–20 µM) compared to confocal microscopy (10–15 µM). We saw a further increase when additionally restricting the focal volume within the cell (by focusing slightly below the AZ membrane) up to 25 µM (average) with peak values up to 50 µM. Further refinement of the technique by application of 3D-STED microscopy can be expected to lead to more precise measurements of [Ca2+] in the immediate proximity of the Ca2+-channel cluster. We note, however, that functional 3D-STED imaging in living tissue faces challenges, such as the tissue-induced aberrations distorting both STED donuts in a different fashion, higher required laser powers, as well as the seemingly trivial problem of locating Ca2+-indicator hotspots with the reduced PSF size. Furthermore, we believe the comparatively modest increase in [Ca2+] measured with STED shows that our STED-efficiency correction is causing us to further underestimate [Ca2+]: the lower STED efficiency for the Ca2+-unbound dye means we are sampling the Ca2+-free dye from a much larger volume than the Ca2+-bound dye. So even if the focal volume of the bound dye is contained within the immediate AZ, the focal volume of the free dye might stretch considerably farther, encompassing more areas with lower [Ca2+] (and thus more free dye). Our STED-efficiency correction, however, is based on measurements within a homogeneous distribution of bound/unbound dye. This effect could be accounted for by either significantly increasing the spatial resolution or by considering the physical dimensions of the AZ when correcting.Our study also shows, through theoretical modeling and direct ) and further characterized using spot detection  with detection by the single-photon counting avalanche photo-diode at 2 kHz. To elicit Ca2+-tail currents, cells were depolarized to +53 mV for 5 ms  and then repolarized to −67 mV. After recording fluorescence and whole-cell currents during ensembles of K = 200 tail currents, trial-to-trial variance was calculated asCa63. After subtraction of baseline variance (shot noise and detector noise), variance of Ca2+-indicator fluorescence was plotted against mean fluorescence and fitted with a parabolic functionfs being the contribution by a single open Ca2+ channel (fluorescence increase or single-channel current), Smean being the average signal (mean fluorescence or current) of all trials at a single time point, and N being the number of Ca2+ channels at the synapse (fluorescence data) or in the entire cell (whole cell currents). Curve fitting used weighting according to the error-covariance of the variance 63. An equivalent analysis was performed on the whole-cell current to estimate the total number of Ca2+ channels per IHC, as described in ref. 22.Fluorescence data was subsampled to 667 Hz to avoid the impact of correlation among neighboring data points2+ and buffers at a simulated AZ were modeled with CalC software37 version 6.86 using the parameters given in Table 2+-bound and -free OGB-5N with Gaussian functions with FWHMs of 64 × 64 × 542 nm (STED) or 243 × 243 × 542 nm , corresponding to the PSFs of the microscope used in the experiments  and de-excited using a pulsed 595 nm STED beam with a donut-shaped beam profile . Laser power in the back aperture of the objective lens ranged typically from 1.5 to 15 µW for the excitation and between 10 and 35 mW for the de-excitation, with STED pulse durations between 200 and 300 ps, depending on the power used. When acquiring super-resolution images, the excitation laser intensity was typically increased to maintain comparable signal levels between confocal and STED.Super-resolution Ca2+ domains, we first needed to locate hotspots of Ca2+ influx, then bring them into the correct focal plane, and finally image them before, during, and after depolarization. We found hotspots of Ca2+ influx by focusing at the basal pole of the patched cell and depolarizing the cell briefly to −14 mV for 78 ms while acquiring xy-frames of 150 × 150 pixels  without activation of the STED laser. After identification of a depolarization-evoked Ca2+ hotspot, an xz-scan  was performed through the center of the hotspot to determine the correct focal plane. IHCs were then depolarized 4× for 78 ms each  while acquiring frames of 150 × 75 pixels . Fluorescence was acquired with a Single Photon Avalanche Diode  using the TTL counting output of the detector. For the analysis of the spatial extent of the Ca2+ domains in xy-scans, the detector signal was electronically time-gated by diverting the first 450 ps  of the signal into a separate detection channel, thereby reducing the contribution of the unbound OGB-5N signal by 80–90%, while only sacrificing 15–20% of the Ca2+-bound OGB-5N signal. Images were acquired with Imspector software  and data were analyzed with Igor Pro 6 as follows: the frames acquired during depolarization were averaged and the average background signal was subtracted . The size of the Ca2+ domain was measured by fitting a 2D Gaussian function using a genetic fit algorithm65 to the subset of the frame containing the Ca2+ hotspot.In order to find and image depolarization-evoked synaptic Ca2+ concentration at the synapse by fluorescence lifetime, we established TCSPC. Fluorescence was acquired using the NIM timing output of the SPAD detector with 35 ps timing resolution. The timing of the fluorescence photons was correlated with the excitation pulse at a TCSPC Module  and sorted into a histogram of 25 ps time bins. The overall timing resolution of the setup was 110 ps FWHM, determined by measuring the instrument response function , 330 ms after the initial depolarization. In xz-scans , 176 ms after the initial depolarization.For the analysis of Caans Fig. , Ca2+ hoans Fig. , hotspot2+-bound OGB-5N (τfree = 0.23 ns and τbound = 3.24 ns) were determined in saturated ([Ca2+]=90 mM) and desaturated  Ca2+ conditions (see section “Determination of the lifetime of Ca2+-free and -bound OGB-5N” below) and kept fixed in the analysis of the Ca2+ hotspots. When analyzing lifetime measurements taken with STED, the fitting routine ignored the first 300 ps of the fluorescence decay (during which the STED pulse was active) to avoid artifacts caused by the STED-evoked quenching of the signal. To ensure that this “blanking” of the first 300 ps did not significantly alter the results of the routine, we fitted fluorescence decay traces recorded in confocal mode using both the full data range and the blanked data   onset of the fit was used). Then the photon count of the Ca2+-bound dye was downscaled by a brightness factor of b = 28.02  of the quenched, Ca2+-free dye with the short fluorescence lifetime. Less STED efficiency corresponds to a larger recorded focal volume, which would otherwise overestimate the Ca2+-free component. Therefore, the STED efficiency correction factor depends on the STED beam power and was determined by measuring the fluorescence depletion of the STED beam at different powers for OGB-5N dye solutions that were saturated with Ca2+ and Ca2+-free . Further corrections were necessary for recordings taken in live IHCs. As opposed to in vitro calibrations, where the percentage of Ca2+-bound/-free dye was measured to be <1% in a Ca2+-depleted/-saturated environment, in live IHCs the long-lifetime component of the signal  neither dropped below 2% for confocal and 4% for STED measurements (even in Ca2+-depleted cells that were patched with 10 mM EGTA in the pipette) nor did it rise above 60% .The analysis procedure of STED fluorescence lifetime data is summarized graphically in Fig. 2+-bound and -free states of OGB-5N, respectively, by first calculating the corrected fraction rB of Ca2+-bound and rU of Ca2+-free OGB-5N asFB and FU the unmodified photon count assigned to the Ca2+-bound and -free (unbound) dye, respectively, b the brightness factor of 28.02 (describing the higher extinction coefficient and quantum yield of the Ca2+-bound OGB-5N), and s the STED correction factor of 1  or 0.424–0.491 . We then further scaled these ratios according to the minimal and maximal fraction of Ca2+-bound OGB-5N  and rmax the minimum and maximum fractions of photons assigned to the Ca2+-bound dye in Ca2+-free and Ca2+-saturated conditions, respectively, , and R = rmax − rmin the dynamic range of the dye.with 2+-bound to free OGB-5N was then calculated asThe ratio of Ca2+ concentrations for each pixel were calculated usingKeff = 195 µM being the effective dissociation constant (KD) we had measured for OGB-5N in intracellular solution :Alternatively, if a CaFB,(stim/rest) and FU,(stim/rest) are the observed photon counts (during depolarization [stim] or at rest), which were assigned by the fitting routine to the slow (Ca2+-bound) and fast (Ca2+-free) states, respectively. Again, Keff is the effective KD of OGB-5N in intracellular solution, b is the brightness factor, s the STED correction factor, and R = rmax − rmin the dynamic range of the dye. Assuming a uniform [OGB-5N] and [Ca2+] inside the cell in resting condition, FU,rest was taken as the average of the 8 × 8 pixel frame for xy-raster scans to minimize noise. For xz-scans, where FU,rest decreases with z, no averaging was performed and individual pixel values were used. Comparison of this method with the first method of scaled lifetime ratios showed good agreement between the results  with various free [Ca2+]. We also verified the lifetime values in solutions that matched the used intracellular solutions for patch-clamping as close as possible.In order to determine the lifetime of OGB-5N in the CaWe recorded each solution at various excitation powers for durations in excess of 9000 ms, with pixel dwell times of 25 ps, for 1024 pixels in total, therefore spanning a time of 25.6 ns, although due to triggering requirements of the single-photon detection module the actual time during which signal was recorded amounted to 11.5 ns. The data was read into the Matlab fit routine, which fitted a single-exponential curve convolved with the IRF to the recorded data  was at around 12.2 ns and the recorded signal was fitted from 1 ns before onset of fluorescence until 9 ns after. The threshold for considering a pixel was set to 18 counts, the “shift” parameter, denoting the shift between the IRF and the recorded fluorescence signal, was typically left floating, but was consistently determined to be ~20 ps ± 15 ps (so the duration of one bin).2+-free and Ca2+-bound OGB-5N  and started about 100 ps before the excitation pulse arrived. During the 300 ps in which the STED beam de-excites the fluorophores, a temporary, albeit steep, drop in apparent fluorescence lifetime is visible, which ceases after the end of the STED pulse. In order to avoid misinterpreting this additional short lifetime component as unbound OGB-5N, we omitted (“blanked”) the first 300 ps from the fitting procedure and only included the subsequent bins in the fit  determined by the fit automatically. However, as the initial onset of fluorescence was omitted from our fitting range, the algorithm had difficulty determining the shift accurately. Therefore, the shift parameter was first automatically determined in the standard range (from bins 450–850), then locked down as a fixed parameter for the actual fit of the STED data with the aforementioned truncated boundaries.2+-indicator OGB-5N exists in two distinct states: Ca2+-bound or Ca2+-free, which display different STED efficiencies (or: saturation intensities). This is mainly due to the fact that the lifetime of the unbound state is extremely short, of the same order of magnitude as the STED pulse itself. Therefore, the STED beam has little time to effectively de-excite the dye before it spontaneously fluoresces. Different STED efficiencies correspond to different effective focal areas achieved using the same STED beam power. More STED-efficient dyes are switched off better and show a much smaller focal area and thus higher spatial resolution. Since the resolution along the optical axis is unchanged by the 2D-STED donut, this corresponds to a smaller “effective focal volume”, meaning that in the same recording, we will be sampling a higher volume of Ca2+-free dye than of Ca2+-bound dye. For quantitative measurements, we need to compare the amount of Ca2+-bound to Ca2+-free dye, so we need to correct for any differences in sampling volume.The CaAssuming a homogenous dye distribution , we can say that the number of fluorophores in solution is proportional to the (sampled) volume. Assuming linear responses from each dye component, the number of recorded photons will depend on the number of fluorophores in the sampled volume and in consequence on the volume and focal area.2+-bound or Ca2+-free, for different STED powers, including zero STED power, i.e., a pure confocal recording. For each STED power, we counted the number of photons emitted from the dye (keeping the excitation power and dye concentration constant) and plotted the ratio of deactivated dye against the STED power. Note the much steeper descent of the Ca2+-bound dye as opposed to relatively gentle decline of the Ca2+-free dye  channel by the factor of effective focal volumes.To correct for different STED efficiencies, we measured OGB-5N dye solutions, either Ca2+] using the lifetime method at different STED powers. Without applying the STED efficiency correction factor, we got discordant results. When correcting with the factor, however, the resulting ratio of bound dye matches for all the used STED powers, as well as for the confocal powers . This held true for the range of bound dye (<40%), as encountered in the IHC experiments. When confronted with higher fractions of bound dye, additional considerations during evaluation need to be taken to correctly discern the diminishing amount of photons in the fast (unbound) channel amidst the increasing number of photons emitted from the bound dye.To validate this correction factor, we measured  was calculated using the software Patcher’s Power Tools in Igor pro.The measured dissociation constant Keff by plotting the photon counts Fslow in the slow channel (as determined by the fitting routine) against the calculated free [Ca2+] in the solution .Significance testing was performed using two-tailed tests, either Student’s 2+ reaction/diffusion modeling at IHC AZs can be obtained from www.innerearlab.uni-goettingen.de/materials.html. The Igor Pro and Matlab code is available from the corresponding authors upon request.The data supporting the findings of this study are available from the corresponding authors upon request. The CALC scripts of the CaSupplementary InformationPeer Review File


The electrophysiological remodeling associated with this pathology alters both the action potential and the Ca2+ dynamics, leading to a defective excitation-contraction coupling that ends in mechanical dysfunction. The importance of maintaining a correct intracellular Ca2+ concentration requires a better understanding of its regulation by ionic mechanisms. To study the electrical activity and ionic homeostasis of failing myocytes, a modified version of the O’Hara et al. human action potential model was used, including electrophysiological remodeling. The impact of the main ionic transport mechanisms was analyzed using single-parameter sensitivity analyses, the first of which explored the modulation of electrophysiological characteristics related to Ca2+ exerted by the remodeled parameters. The second sensitivity analysis compared the potential consequences of modulating individual channel conductivities, as one of the main effects of potential drugs, on Ca2+ dynamic properties under both normal conditions and in heart failure. The first analysis revealed the important contribution of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) dysfunction to the altered Ca2+ homeostasis, with the Na+/Ca2+ exchanger (NCX) and other Ca2+ cycling proteins also playing a significant role. Our results highlight the importance of improving the SR uptake function to increase Ca2+ content and restore Ca2+ homeostasis and contractility. The second sensitivity analysis highlights the different response of the failing myocyte versus the healthy myocyte to potential pharmacological actions on single channels. The result of modifying the conductances of the remodeled proteins such as SERCA and NCX in heart failure has less impact on Ca2+ modulation. These differences should be taken into account when designing drug therapies.Abnormal intracellular Ca AP duration (APD) was measured as the time from the upstroke to 30% and 90% repolarization (APD30 and APD90), as well as the [Na+]i peak.All the simulations in this study were carried out in endocardial cells paced at 1 Hz. Quantitative indicators characterizing CaNaL, the transient outward K+ current (Ito), and the inward rectifier K+ current (IK1), to the time constant of inactivation of the INaL (τhL), to the maximal fluxes of the Na+/ K+ ATPase current (INaK), the Na+/Ca2+ exchange current (INCX), the Ca2+ uptake via SERCA pump (JSERCA), and the SR Ca2+ leak (Jleak), to the fraction of active binding sites of the Ca+2 calmodulin-dependent protein kinase II (CaMKa), and to the SR Ca2+-dependence of the steady-state activation of ryanodine receptor (RyR) release . Further details of these variables can be found in the Supplementary Material  and sensitivities  were calculated as follows:p,x being the magnitude of the characteristic “c” when parameter “p” undergoes a change with respect to the basic HF model (x = 1: without HF and x = 2: 150% HF), and cbasic the value of the same property in the basic HF model; Δa is the total interval of change of parameter p. The evaluated EP characteristics are the indicators specified in the simulation protocol section and were obtained from the steady-state APs and CaTs , the rapid delayed rectifier K+ current (IKr), the slow delayed rectifier K+ current (IKs), IK1, INCX, INaK, JSERCA, the SR Ca2+ release flux via RyR (Jrel), Jleak, and the Na+ background current (INab). Since we wanted to study and compare the modulation in normal and failing conditions, these changes were applied to both models separately, obtaining two sensitivity analyses. When the HF model was used, modifications were done by maintaining the electrical remodeling affecting ionic parameters. Once the simulations had been performed, the EP characteristics were measured from the steady-state APs and CaTs  and sensitivities  were calculated similarly to those of the first sensitivity analysis  is shown in each row to indicate the total impact of the parameters on a particular characteristic. In addition to sensitivity values, it is also important to find the dependency directions between the variables. For this, positive and negative signs indicate whether the change of the ionic current and the HF characteristic follow the same tendency or an inverse tendency, respectively.Firstly, we analyzed the individual effects of the variability on the different ionic parameters remodeled in HF on Cators see , to findNaL and INaK are the most important contributors to APD variations. The enhancement of both currents prolongs APD, but the former has a greater effect on APD90 and the latter on APD30. τhL and INCX also have a mild effect. 90 is modulated by these parameters.From this sensitivity analysis, it can be deduced that ISERCA is the major contributor to most of the EP properties related to Ca2+ dynamics  has a positive dependence and could help to reduce the duration, the sensitivity value is small. Thus, restoring JSERCA to its normal value would restore the CaTD value of non-failing myocytes (around 400 ms). The CaTD30 sensitivities show a similar tendency to the CaTD80 sensitivities in all the parameters but the values are higher.Interestingly, Jdynamics . It can 2+ dynamics is the rise time (t10-90). The SERCA pump is the strongest modulator of t10-90. As shown in leak and Krel,Ca also affect t10-90, although to a lesser extent than SERCA. Both parameters are related with Ca2+ extrusion from the SR, in the cytosol and the subspace, respectively. Indeed Krel,Ca is the sensitivity of Jrel to [Ca2+]JSR, meaning that a reduction of Krel,Ca leads to a higher release through RyRs. In general, HF conditions slow down the CaT rise, but reduced Krel,Ca helps to accelerate it  contributes to a slow rise in CaT, although to a lesser extent.Another important characteristic of Ca2+] in all cell compartments is strongly modulated by the reduced JSERCA in HF. Specifically, systolic [Ca2+]i values decrease when SERCA activity is reduced (NCX (dotted blue line) enhancement is also important and contributes to a general reduction of [Ca2+]i. Reduced INaK (dashed red line) also leads to higher systolic Ca2+. Diastolic values are also sensitive to changes in Jleak and Krel,Ca . In the subspace, the sensitivity of [Ca2+] to ionic parameters is similar to the sensitivity in the cytoplasm, but with higher sensitivity values of the systolic Ca2+ peak  remains the most important factor in modulating SR Ca2+ load, followed by Jleak, Krel,Ca, and INCX . Furthermore, APD30 shows a negative dependency on ICaL (dashed brown line) and JSERCA (dashed green line) when these are reduced, which is not seen in normal conditions . Thus, under HF conditions, the INaL block should be more effective in reducing APD90 than in normal conditions, and unexpectedly, an ICaL block or a reduction in JSERCA could have negative effects and increase APD30.First, APD is highly sensitive to ISERCA seems to be the major contributor . However, the enhancement of Ca2+ uptake reduces CaTD similarly in both conditions. Blocking NCX (dotted blue line) shortens CaTD, but the influence of ICaL (dashed brown line) is reduced in HF in such a way that an enhancement of this current does not improve CaTD. On the other hand, it is to be noted that the effect of changes in IKr on CaTD in the HF model becomes important (Kr (dotted red line) block in HF prolongs CaTD, whereas this effect is non-existent in normal conditions. Thus, IKr block in HF could have negative effects on CaTD, in addition to the prolongation of APD.With respect to CaT characteristics in normal conditions, again J10-90), a JSERCA block in normal conditions significantly increases this property, acting as the most influential parameter. On the other hand, when it is enhanced, a very slight reduction of t10-90 is observed . ICaL and Jleak also modulate the rise time, especially in HF.Regarding CaT rise time  and JSERCA (dashed green line) produce smaller variations than those in normal conditions  has a relevant effect, enhancing diastolic levels and worsening HF changes  to shed light on the role of some ionic current characteristics and reveal other mechanisms that may take part in Ca2+ cycling during HF. The first sensitivity analysis highlights the strong effect of JSERCA, among other parameters remodeled in HF, on the modulation of Ca2+ indicators. Under HF conditions, depressed SERCA activity worsens all the EP characteristics related to Ca2+, which are the hallmark of HF: CaT rise time, systolic and diastolic [Ca2+], CaT duration and SR Ca2+ content. Enhancing the SERCA uptake function to its normal activity improves all these Ca2+ characteristics and could restore contraction.In this study Ca2+ sensitivities to drug-induced alterations in the main ionic currents in normal and HF conditions reveals some parameters that modulate Ca2+ dynamics in a different way. As blocking IKr has a marked influence in prolonging CaTD in HF in comparison to its effect in normal conditions, IKr blocking drugs should be avoided in HF for their impact not only on APD but also on contractility. Blocking INCX could be beneficial in HF, as it improves CaT rise time, however it could also increase diastolic [Ca2+]i, which worsens contraction. These opposing effects should be taken into account when considering blocking NCX. SERCA enhancement in HF is highly effective in reducing t10-90, but its depressed activity requires extreme enhancement to restore systolic [Ca2+]i.The comparison of Ca2+ dynamics i, slow the rate of CaT decay, and reduce the SR Ca2+ load. SR Ca2+ store depletion indirectly reduces Ca2+ release and systolic function. In fact, after Ca2+ uptake function inhibition, healthy myocytes showed changes in CaT and SR Ca2+ content, for example using the use of thapsigargin or TBQ in rats i accumulation, increasing Ca2+ influx. At the same time, the upregulated forward mode increases Ca2+ efflux, resulting in cellular Ca2+ loss and a reduced SR Ca2+ load. Therefore, most of the consequences of remodeled NCX worsen Ca2+ dynamics and the possible beneficial effect of NCX inhibition has been analyzed in several studies. SEA0400, a NCX inhibitor, did not produce significant changes either in the shape or in the magnitude of the CaT in normal guinea pig or canine myocytes  in different cellular compartments. With this approach, the modification of each parameter is quantified, while this cannot always be done accurately in experiments.Unlike experimental studies, sensitivity analyses are a systematic methodology that allows many parameters to be varied either independently (one at a time) or at the same time, which avoids the difficulty of replicating conditions and allows the control of all the sources of variability that affect experimental results. The selection of the animal species when analyzing cellular electrophysiology is one of the most influential factors. Due to the need for human data and the scarcity of human hearts for experimental purposes, the use of a detailed human model such as the ORd model is extremely useful. Modeling and simulation also allow the assessment of EP characteristics, which cannot be measured experimentally, such as i to INaK and the increase in [Na+]i due to the dysfunction of this pump in HF extruding less Na+ out of the cell were also as expected [+]i accumulation is related to APD shortening when the electrogenic Na+/K+ pump is inhibited because it increases the NCX reverse mode (outward current) and contributes to cell repolarization i , while enhancing SERCA always contributes to improving Ca2+ dynamics. These predictions would of course need to be validated using experimental results. The strength of systematic sensitivity analyses is their predictive power and the indications they provide for the design of the required experiments thus contributing to reducing the time and costs involved in drug design.The use of sensitivity analyses has been widely extended to study physiological processes providing valuable predictions ,17. Othenditions –50. The expected ,52. Thisrization ,17,51,532+ levels. Additionally, in some situations, the model does not represent all the ionic mechanisms that are commonly described in the literature as is the case of Jleak which, in the ORd model, was formulated only as a backflux of the SERCA pump. However, in other models [leak is considered as a secondary efflux through the RyR channels, sometimes coexisting with a reverse SERCA flux [leak and Jrel should be taken with caution, i.e. accordingly to the model formulation. Also, in our study, JSERCA was considered separately from the Jleak term, both of them included in the original ORd Jup formulation. Finally, the electrical remodeling associated with HF is not unique [The limitations of the present study are linked, on the one hand, to the use of a mathematical model for the action potential. Despite its validation using human data, there are still some quantitative differences in intracellular Car models ,54,55, JRCA flux . For thit unique ,56–58, aThe scarcity of available human data has hampered the validation of our results, thus affecting their predictive value, and the wide variations in the existing experimental studies on different species and treatments, also make the validation of the simulations difficult.S1 FileExtended Methods and Steady-state conditions.(DOCX)Click here for additional data file.


The simultaneous Occurrence of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) has been rarely reported. Most of these cases have been occurring more frequently as a secondary event in patients receiving chemotherapeutic agents for CLL.9/1, PLT 143× 109/1. The peripheral blood (PB) smear examination showed circulating blast cells (20 %) cells and 50 % lymphocytes, with smudge cells.We describe a case of a 77-year-old man who presented with fatigue, pallor and lower limb pain and weakness. Initial laboratory studies showed Hb 7.7 g/dl, WBC 279.6 × 10A bone marrow examination showed infiltration by two discrete abnormal cell populations, one represents the leukemic blast cells (60 %) and the other represents small mature lymphocytes (30 %). The immunologic phenotype of blasts was characterized by the co-expression of CD13, CD33, CD14, CD4, CD15, CD64, HLA-DR, CD11c. Lymphocytes were characterized by a typical CLL immunophenotype: CD19+, CD5+, CD23+, CD20+ (dim) and negative for FMC7, CD34, CD10 and TdT. Cytogenetic studies were negative for CLL and AML panels. PCR assays for AML specific genetic abnormalities were negative. Immunoglobulin gene analysis established the clonal nature of the B-cell expansion. A final diagnosis of concomitant CLL and AML(FAB: M5) was made.We have reported a case in which there was simultaneous presentation of AML and CLL. Both forms of leukemia were well documented by morphology, cytometric analysis and molecular studies. Our findings support the idea that this rare concurrence of AML and untreated CLL may represent two separate disease processes. The association of different malignancies with chronic lymphocytic leukemia (CLL) such as lung and skin cancer has been reported –5. The cWe report a case of concomitant CLL and AML (FAB: M5) without previous exposure to a cytotoxic agent or irradiation. Morphologic features, flow cytometric analysis, molecular and cytogenetic findings of peripheral blood and Bone marrow samples are discussed.A 77 year-old man presented with 3 weeks history of fatigue, pallor and lower limb pain and weakness with no history of previous exposure to a cytotoxic agent, irradiation or other medications except for hypertension. There was no history of fever, night sweats, weight loss, anorexia, chest pain, headache, blurring of vision or other relevant symptoms.Physical examination was significant for enlarged spleen , but no lymphadenopathy, hepatomegaly, jaundice or gum hypertrophy. Chest X-ray was normaland a computerized tomography (CT) scan revealed no lymphoadenopathies.9/1, platelets 45 × 109/1) with two distinct abnormal populations, the small cells were corresponding to mature lymphocytes (60 %) and large cells which corresponded to blast cells (20 %), and smear cells were also present  279.6 × 10The immunophenotypic analysis, performed on PB and bone marrow (BM) aspirate samples confirmed the morphological findings of the presence of two abnormal malignant populations. The diagnosis of CLL was confirmed by demonstration of expression of mature B-cell markers , the co-expression of CD5 and CD23 and the absence of immaturemarkers, such as CD34, Terminal deoxynucleotidyltransferase (TdT) and CD10. Similarly, the diagnosis of AML (FAB-M5) was confirmed by demonstration of expression of myeloid markers  and monocytic markers  and positive for CD38 and CD56 but negative for CD34 and CD117 , Hydroxyurea was subsequently discontinued.On the basis of clinical features and Later on, the kidney function tests started to deteriorate and allopurinol was changed to Rasburicase. He developed acute on top of chronic kidney disease and he was started on peritoneal dialysis.FLT3 gene mutations (ITD and D835) using published primers . Fluorescence in situ hybridization (FISH) analysis was performed for common abnormalities associated with CLL and AML according to the manufacturer's instructions. FISH analysis for CLL was performed using the following probes panel: TP53/CEP17, D13S319/13q34, IGH/CCND1 , IGH/BCL2  whereas FISH analysis for AML was performed using the following probes panel: AML-ETO /t, PML-RARA /t, CBFB /inv(16), MLL /11q23 and CEP8/D8Z2. FISH results were negative for both CLL and AML panel- specific abnormalities. RNA isolated from the BM sample according to the manufacturer's instructions was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using a nested PCR reaction, to ascertain the presence or absence of AML specific abnormalities mentioned before. Similarly, DND was extracted according to the manufacturer’s instruction to detect both  primers  and repoDue to old age, comorbid conditions and his performance status, the patient received palliative therapy with no curative treatment of his hematological malignancies. The patient died on the tenth day of hospitalization due to complications of both metabolic functions and infection.The association of different malignancies with chronic lymphocytic leukemia has been reported. The most common second malignancies are solid tumors, especially lung and skin cancers . The assIn the present case study, the PB and BM morphologic findings are of AML with monoblastic features. FCM and IHC data are also consistent with AML-M5 but in addition, flow cytometric studies detect the presence of a lymphoproliferative process, CLL (CD5+/CD19+). The association of AML and CLL has been previously described –6. CytogThe history of the patient in the present study did not reveal any exposure to known leukemogenic agents or chemotherapy for CLL. The diagnoses of both AML and CLL were simultaneous, although we cannot rule out a previous asymptomatic indolent course of CLL. Although the simultaneous occurrence of AML and CLL is rare, it should not be overlooked as a possible cause of lymphocytosis in AML patients, as it has been reported that a reactive polyclonal lymphocytosis may occur in isolated cases of AML or, more commonly, in myelodysplastic syndrome patients .Multiple theories behind the development of simultaneous malignancies in patients with CLL have been proposed . The firIn summary, we have reported a case in which there was simultaneous presentation of AML and CLL. Both forms of leukemia were well documented by morphology, cytometric analysis and molecular studies. This increases the number of cases in which concomitant CLL and AML have been found without previous exposure to a cytotoxic agent or irradiation. Our findings support the idea that this rare concurrence of AML and untreated CLL may represent two separate disease processes.


Besides disease-modeling, such a system can be used to clarify basic aspects of human skeletal muscle development. In the present study, we focus on the development of the excitation-contraction (E-C) coupling, a process that is essential both in muscle physiology and as a tool to differentiate between the skeletal and cardiac muscle. The occurrence and maturation of E-C coupling structures (Sarcoplasmic Reticulum-Transverse Tubule (SR-TT) junctions), key molecular components, and Ca+-myogenic progenitors were differentiated in culture, and developmental changes were examined from a few days up to several weeks. Ion channels directly involved in the skeletal muscle E-C coupling (RyR1 and Cav1.1 voltage-gated Ca2+ channels) were labeled using indirect immunofluorescence. Ultrastructural changes of differentiating cells were visualized by transmission electron microscopy. On the functional side, depolarization-induced intracellular Ca2+ transients mediating E-C coupling were recorded using Fura-2 ratiometric Ca2+ imaging, and myocyte contraction was captured by digital photomicrography.Pax72+ transients become effective in triggering myocyte contraction after 1 week of differentiation, when nascent myofibrils show alternate A-I bands. At later stages, myofibrils become fully organized into adult-like sarcomeres but SR-TT junctions do not reach their triadic structure and typical A-I location. This is mirrored by the absence of cross-striated distribution pattern of both RyR1 and Cav1.1 channels.We show that the E-C coupling machinery occurs and operates within a few days post-differentiation, as soon as the myofilaments align. However, CaThe E-C coupling machinery occurs and operates within the first week of muscle cells differentiation. However, while early development of SR-TT junctions is coordinated with that of nascent myofibrils, their respective maturation is not. Formation of typical triads requires other factors/conditions, and this should be taken into account when using in-vitro models to explore skeletal muscle diseases, especially those affecting E-C coupling.The online version of this article (10.1186/s13395-017-0147-5) contains supplementary material, which is available to authorized users. Notice that the mean values depicted in Fig. 2+ signal decay phase were made after normalization of the signal to enable comparison between different datasets.The signal obtained for each individual cell was integrated over the first 30 s of membrane depolarization using Origin Pro software (OriginLab Corporation). This provided the integrated fluorescence (area under the curve), which reflects the total amount of Ca2+ imaging). Quantitative data are given as mean ± SEM. Unpaired, two-tailed Student’s t test was used for comparing two groups. One-way ANOVA was used to detect differences between intracellular Ca2+ signals obtained at different stages of myocytes differentiation. A post hoc Tukey’s test was subsequently used to compare data obtained at each differentiation time point with those obtained at late stages (> 3 weeks). Analyses were made using STATISTICA software (StatSoft Inc.).At least three independent experiments were carried out for each time point regardless of the method used  when rapid changes occur, an intermediate time point (day 7) when most of the skeletal muscle landmarks are settled, and a late period that extends from 3 to 8 weeks post-differentiation, during which skeletal myocytes have reached steady maturation with no variation in their differentiation state at least regarding the features investigated herein. These cells will be referred to as mature cells.Changes in cell morphology during differentiation were first noticed by phase contrast microscopy in routine cell culture observation  in the proliferation medium (high-serum media) and ultimately form a tight layer of flat cells with round prominent nucleus , which reached ~ 22% of the total cell population . Dense structures of irregular shape, presumably Z-bodies, appeared along these bundles and co-existed now and then with nascent Z-bands. Although recurrent, these structures did not delineate a regular sarcomeric pattern; A and I bands could not be clearly identified, and H-band and M-line were unseen. Nuclei had various shapes from oblate to clearly flattened. Appositions between SR and T-tubule-like structures were encountered at this stage , but the junctional cleft was generally irregular and RyR feet could not be clearly discerned. On day 5 post-differentiation, α-actinin labeling was found in a higher proportion (~ 47%) of cells . At the ultrastructural level, both Z-bodies and winding discontinuous Z-bands were found; however, there was generally no clear-cut A-I banding pattern apart from a few exceptions . We also noticed that the most organized cytoskeleton pattern was preferentially located in the center, while nascent Z-bodies were scattered at the periphery of the cell .On day 4, the number of elongated cells increased . SR-TT junctions with unambiguous rows of RyR feet were found in different areas of the cells . These structures were relatively large and were composed of extended SR cisternae and T-tubules with a wide lumen, often surrounded and/or fused to caveolae. Diads, inverted triads as well as classical triads were encountered, but none followed an ordered distribution pattern in register with the myofibrils.On day 7 post-differentiation, the large majority of the cells was elongated, had several long or oval nuclei predominantly at their edge, and their cross striations were readily visible in phase contrast microscopy ; H zones with central M-lines were readily detectable in the middle of the A-band , similar to what is usually observed in adult skeletal muscle  were examined at short post-differentiation intervals  during the first week when changes occur rapidly, then from 3 to 8 weeks, when the muscle cells have largely reached steady maturation. The development of intracellular Ca2+ signals induced by membrane depolarization with high external [K+], together with their main characteristics is depicted in Fig. 2+ signaling on day 3 post-differentiation, followed on day 4 by a shallow, unsynchronized but consistent increase in intracellular Ca2+ level in elongated cells. Day 5 was a critical time point marked by the occurrence of a typical Ca2+ transient with a sharp upstroke and slow decay. From this stage onward, Ca2+ transients were present, and their intensity and kinetics changed with time until stabilization at mature stages. Changes over time of three different characteristics of the Ca2+ transient, i.e., the integrated fluorescence over the first 30 s of membrane depolarization, the maximum stimulation and the signal time to peak , Ca2+ decay followed a single exponential function with a comparable time constant . In contrast, at late stages, the best fitting required two exponential functions in 70% of the cells: a fast component similar to that found at early stages (τfast = 9.1 ± 0.5) and a slow component that appeared with cell maturation (τslow = 53.4 ± 3.6 s)  relative to the baseline), which is insignificant and most likely due to small fluctuations of the baseline. On day 4, there was a slight increase in integrated fluorescence from the baseline confirming the occurrence of an early form of CaThe relative peak to the baseline or maximum stimulation increased rapidly between days 4 and 5 and then more gradually to the end of the first week. This characteristic did not change significantly during the maturation period Fig. .2+ transients with a single peak, and hence was not applicable to unsynchronized Ca2+ signals recorded on day 4. Time to peak did not change significantly with time, and mean values obtained at different stages of development were all comparable . It is worth noting that in a normal culture medium, the first spontaneous contractions can be noticed between 7 and 10 days post-differentiation and last throughout the maturation period  and L-Type Ca2+ channels (Cav1.1) by fluorescence immunolabeling. However, at this early stage, the junctional space between SR and T-tubule membranes is not clearly delineated, and RyRs feet are hard to identify in the cleft. This observation is reminiscent of the first event in the developmental sequence of SR-TT junctions in mouse skeletal muscle [2+ signaling is present but its small size and slow kinetics both suggest an immature coupling process leading to the release of small amounts of Ca2+ ions in a desynchronized fashion. In the adult skeletal muscle, SR-TT junctions are highly ordered; RyR1 and Cav1.1 are organized into arrays composed of four Cav1.1 which associate with every other homotetrameric RyR1, forming alternate tetrads [2+ signaling. Nonetheless, molecular rearrangement might rapidly occur at the SR-TT junctions as suggested by the tremendous increase in size and rate of Ca2+ release on day 5 and the occurrence of a Ca2+ transient reminiscent of that found in the skeletal muscle. The idea of a rapid rearrangement of RyR1 and Cav1.1 is supported by the clear appearance of RyRs feet and a well-delineated SR-TT cleft in electron micrographs at this time. Interestingly, the presence of this early Ca2+ transient was not accompanied by cell contraction. This could be due to immature contractile apparatus and/or to the insufficient amount of Ca2+ released into the cytosol. The first explanation is, in our view, the most plausible and is supported by studies showing that Z-bodies or nascent Z-bands, similar to those observed on day 5 in our system, do not support muscle contraction [2+ signals produce muscle contractions, and spontaneous contractions are also present under normal culture conditions.In a previous study , we showl muscle . On the  tetrads . In addi tetrads , 24, 25.traction . At thistraction . Further2+ transient peak and by the occurrence of a slow component in the decay phase. The former change can be explained by an increased expression of RyR1 and Cav1.1 channels, while the latter may be related to maturation of the Ca2+ buffering process that follows E-C coupling [2+ signals would generate a rise in intracellular Ca2+ that peaks and lasts long enough to enable strong myocyte contraction.After the first week of differentiation, the maturation process of the iPSC-derived muscle cells is characterized by further organization of the myofilaments as well as changes in SR-TT junctions, but these two structures do not ultimately reach the same degree of development. Indeed, by the end of the second week of differentiation, many myofibrils show a full banding pattern including the H-band and M-line. Thus, their contractile apparatus has reached a degree of organization that should sustain contraction in the most efficient manner. In parallel, SR cisternae and T-tubules become smaller, and the junctions seem better delineated and more central, i.e., closer to the myofibrils. Such changes are paralleled by a reduction in the size of RyR1 immunolabeling particles that plausibly reflects the spatial restriction of the SR cisternae in which they are expressed. Nevertheless, the occurrence of typical triads as well as their position at the A-I junction remains seldom in the present model. SR and T-tubule membranes seem to meet and form junctions at more or less random locations, and although more centrally localized at mature stages, SR-TT junctions remain irregularly distributed even after the myofibrils have reached their full organization. Thus, the orderly sarcomeric distribution of the SR-TT junctions typical of adult skeletal muscle  is not ecoupling , 29. Thu.Interestingly, a similar timeframe is shared by muscle innervation which reaches its mature mono-synaptic form about 2 weeks after birth in rodents [According to a number of developmental studies, the spatial positioning of SR-TT junctions is a slow and final process that occurs about 2 weeks after birth in rodents , 30–33.  rodents , 36 and  rodents . A possi rodents .2+ transients typical of skeletal muscle. Such signals become effective in triggering muscle contraction as soon as the A-I banding pattern is in place. At later stages of differentiation, myofibrils are fully organized while SR-TT junctions are not, suggesting that their respective maturation is regulated by different signals. The final organization of the E-C coupling apparatus may depend on factors/conditions that are not provided or reproduced under these in vitro conditions, among which motor innervation represents an appealing candidate.SR-TT junctions occur in iPSC-derived skeletal muscle cells soon after the switch to differentiation conditions in vitro, and, although structurally immature, they are able to generate depolarization-induced intracellular CaAdditional file 1: Figure S1.A Proliferating myogenic progenitors. B–F Cells photographed at different times  following the switch to differentiation conditions, . Notice the rapid elongation of the cells within the first week post-differentiation and the high density of mature skeletal myocytes on day 22. (TIFF 3627 kb)Bright field photomicrographs of human iPSC-derived skeletal myocytes differentiating in culture. Additional file 2: Figure S2.A Immunofluorescence labeling of α-actinin in one of the few elongated cells at this very early stage of differentiation. Note the punctuate labeling along the periphery of the cell. B1–3 Electron micrographs shown at increasing magnification to disclose structural details of the early cytoskeleton remodeling. The area occupied by nascent myofilaments is relatively limited and mainly localized at the periphery of the cell. Note the presence of many undifferentiated cells surrounding this early myocyte. (TIFF 3844 kb)Alpha-actinin immunofluorescence and ultrastructure of human iPSC-derived skeletal myocytes on day 3 post-differentiation in culture. Additional file 3: Figure S3.Developing myofibrils in a 5-day-old human iPSC-derived skeletal myocyte. Nascent sarcomeres found at the center of the cell appear relatively more organized than those located at the periphery. Electron microphotographs were taken at low (upper image) and high (lower image) magnification from two different skeletal myocytes, 5 days after the switch to differentiation conditions. (TIFF 7644 kb)Additional file 4: Video S1.+ in an extracellular medium devoid of Ca2+ ions. A typical Fura2 image sequence is first presented as 340/380 fluorescence ratio to reflect the increase in intracellular Ca2+ (from blue to red color) then as raw images captured using 380 nm excitation wavelength from which myocyte contractions can be more readily followed. To obtain a video file, Fura2 images  were first converted into universal Tiff format using a screen capture software (Gadwin Systems Ltd). Tiff images sequence was then converted into a video file (.avi) with ImageJ software (NIH). (AVI 38635 kb)Depolarization-induced contraction in mature human iPSC-derived skeletal myocytes. Membrane depolarization was triggered by 50 mM KAdditional file 5: Video S2. Spontaneous contractions of 10-day-old human iPSC-derived skeletal myocytes. Live cell imaging on day 10 post-differentiation using time-sequence microphotography . Images were acquired with ×20 lens objective on a phase contrast TE2000 Nikon inverted microscope. Cells were bathed in standard culture medium, and images were captured at room temperature (~ 21 °C). (MP4 1740 kb)


The evolution of polarization-dependent anisotropy patterns proves that there is a linear relationship between the rotation angle of second harmonic generation anisotropy patterns and the polarization angle of BaTiO3 single crystals. It is a direct evidence illustrating that the polarization of BaTiO3 crystal can be qualitatively identified in 0°–180° by second harmonic generation technology. This work gives a glance at improving a nonintrusive and convenient method to identify the polarization of perovskite ferroelectric materials.For ferroelectric materials, where the polar state breaks the inversion symmetry, second harmonic generation is a useful tool to prove their ferroelectric properties. However, the correlation between the anisotropy patterns and the polarization orientation of the ferroelectric domains has not been clarified yet. In this work, we systematically investigated this correlation in a typical perovskite oxide ferroelectric, Barium Titanate (BaTiO Probing their ferroelectric properties non-destructively is crucial for further both electrical and optical tests, yet there is none well-established technique hitherto. The most promising method to do so should be optical second harmonic generation (SHG)4, which is one of the nonlinear processes occurring in non-centrosymmetric crystals via the second-order nonlinear susceptibility χ(2) that relates the induced second-order polarization with the applied fundamental optical field5. As a noninvasive technique, SHG can be applied under various conditions, such as magnetic or electric field, high-temperature or hypothermal environment and pressure7. Since it is highly associated with the symmetry of the object, SHG can give a visual information on different structures with the special resolution of hundred microns and down to 1 micron, mainly depending on whether involving objective lens or not. Based on the high sensitivity of SHG on symmetry changing, second-harmonic microscopy system has been developed and employed in many impressing explorations in physics and biology, such as nonlinear edge resonance of MoS2 monolayer8, domain structures in thin films9, and quantitative analysis of collagen fibrillar structure10.Perovskite oxide ferroelectrics have attracted great interests as a candidate class of materials used in visible-light-absorbing and photovoltaic devices11. A set of papers from the group of Venkatraman Gopalan have used SHG to reveal the domain variety of perovskite ferroelectrics and their nonlinear properties13. Other researchers, such as H. Yokota and Morgan Trassin et al., have proved the shapes of SHG anisotropy patterns are different in various polarization directions of CaTiO3 and BiFeO3 samples14. However, the correlation between SHG anisotropy patterns and the polarization orientation of ferroelectric domains has not been systematically clarified yet, which hinders SHG to be a more convincing and reliable technique in sufficiently illustrating the ferroelectric information of solid state materials.The mostly used SHG technology to verify the symmetry of a sample is the SHG anisotropy pattern, which is also called SHG polarization diagram. Previous researches have mainly related its variation to structural changing and even determined domain structures and symmetries of samples by mapping their SHG anisotropy patterns3, BTO) has outstanding ferroelectric and piezoelectric properties at room temperature, thus has continuously attracted much attention for its potentially applications in nonvolatile memory storage devices, optical switches, high-density capacitors and electro-optical devices16. As BaTiO3 is a carefully studied ferroelectric material, which has been widely used as a model system for the explanation of ferroelectric phenomena17, in this paper we explore the azimuth angle-resolved SHG anisotropy patterns of this classic ferroelectric material, tetragonal BTO perovskite single crystal19, and illuminate their evolution. The samples measured are 10 × 10 × 0.5 mm3 (100)-cut BTO crystals with tetragonal symmetry in room temperature and ferroelectric curie temperature TC = 125 °C20. The band width of crystal BTO in room temperature is around 3.2 eV, which means there is no absorption for light at 800 nm or 400 nm. Together with determining the polarization direction of the samples by piezoresponse force microscopy (PFM), it is demonstrated that there is a linear relationship between the rotating angle of SHG anisotropy pattern and the net polarization orientation of a ferroelectric material.Barium Titanate (BaTiO21 (see Methods). It can be clearly seen that these patterns evolve with azimuth changing, where azimuth is the rotation angle of sample relative to its initial position.The SHG anisotropy patterns are collected by a typical SHG transmission setup as shown in Fig. As can be seen in Fig. As to parallel configuration, shown in Fig. y = x”. Obviously, the line and the experimental data fit very well, indicating that there is a linear correlation between the patterns and the azimuths. Since the incident light is along sample normal, the non-centrosymmetry out-plane contributes little to the second harmonic (SH) signal. Namely, the SH signal mostly generated from the net polarization in-plane, which rotates synchronously with the samples. Thus, it proves that the polarization orientation of samples has a direct and linear relationship with the SHG anisotropy pattern.In order to illustrate the correlation more directly, the dependence of the rotation angle of major axis in the patterns upon the azimuth of samples is shown in Fig. 22, it means that the crystal axis z is in y-z plane of laboratory coordinate shown in Fig. θ is defined as the angle between the z axis of crystal and laboratory coordinate. Please note that the θ here is not the same definition as the azimuth mentioned before. From the theoretical analysis, it can be derived that the SHG anisotropy pattern would have a four-valve structure in the cases that θ = 0° and 180° for perpendicular and θ = 90° and 270° for parallel configuration. Therefore, it can be further concluded that the SHG anisotropy pattern with fourfold rotational symmetry would be obtained in the cases when the net polarization of samples is perpendicular to the polarization direction of analyzer.As mentioned above, the SH signals are mostly generated from the polarization in-plane. For 100)-cut tetragonal BTO crystals (point group 4 mm)00-cut tey-axis at the initial position, namely, azimuth = 0°, θ = 90°. To further prove this, in-plane PFM scan is engaged to characterize the ferroelectric polarization characteristic of the sample. As shown in Fig. y-axis, which proves the correctness of our deduction. We also investigated the PFM responses of several other areas around in the light spot and found they are all consistent, with the main polarization along y-axis.Consequently, we deduce that the net polarization of the measured BTO sample is along 23. The solid lines in polar plots in Fig. θ = 90° and 0° respectively. For parallel configuration, shown as Fig. θ = 90° and 0° respectively. As can be seen, the theoretical results have a good agreement to the experimental data.Figure Along with the anisotropy pattern evolution in Fig. In this work, direct evidence of correlation between SHG anisotropy patterns and the polarization orientations of BTO single crystals has been systematically investigated by SHG and PFM techniques. The evolution of polarization-dependent anisotropy patterns proves that there is a linear relationship between the rotation angle of the SHG anisotropy patterns and the polarization angle of BTO single crystals. Since theoretically the intensity of polarization is closely related to SHG intensity, this work indicates that SHG can be a promising technique to exactly probe both the orientation and the quantification of net polarization for the perovskite ferroelectric materials. Further application approaching some devices for detecting the polarization orientations of materials based on the present results is highly expected.–x direction. The initial polarization of the incident light is set along z direction and rotated by a 1/2 wave plate. Single photon counting technique is conducted to count second-harmonic photons, indicating the intensity of second-harmonic signal generated from samples. An image magnification system and multidimensional translation stage are engaged to ensure the incident light focuses on the same area while rotating samples, which guarantees that the net polarization orientation of light spot rotates synchronously with the sample azimuth. Because of the configuration we employed, which includes strong light filed and up to 0.5 mm sample thickness in the straight light path, we believe that the photorefractive effect in BTO crystals contributes little to the measured SH signal.We got the SHG anisotropy patterns with a typical SHG transmission setup as shown in Fig. The PFM is on a commercial atomic force microscope  and the PFM images were collected and recorded using a Ti/Ir-coated Si cantilever (Olympus Electrilever) with a nominal ~2 N/m spring constant and a free air resonance frequency of ~73 kHz. The size of measured area shown in this paper as Fig. 22, the optical second-order susceptibility tensor has a form of 16.θ as the angle between the z axis of crystal and laboratory coordinate. Using coordinate transformation, the effective optical second-order susceptibility tensor under the laboratory coordinate takes the form of5, the sum of photons S detected in the two configurations areΩ, 5, and α denotes for the angle between directions of the incident light polarization and the y-axis, as shown in Fig. For a crystal BTO in its tetragonal phase (point group 4 mm)


The key anti-apoptotic protein, Bcl-2, has been reported to regulate the movement of Ca2+ across the ER membrane, but the exact effect of Bcl-2 on Ca2+ levels remains controversial. Store-operated Ca2+ entry (SOCE), a major mode of Ca2+ uptake in non-excitable cells, is activated by depletion of Ca2+ in the ER. Depletion of Ca2+ in the ER causes translocation of the SOC channel activator, STIM1, to the plasma membrane. Thereafter, STIM1 binds to Orai1 or/and TRPC1 channels, forcing them to open and thereby allow Ca2+ entry. In addition, several anti-cancer drugs have been reported to induce apoptosis of cancer cells via the SOCE pathway. However, the detailed mechanism underlying the regulation of SOCE by Bcl-2 is not well understood. In this study, a three-amino acid mutation within the Bcl-2 BH1 domain was generated to verify the role of Bcl-2 in Ca2+ handling during ER stress. The subcellular localization of the Bcl-2 mutant (mt) is similar to that in the wild-type Bcl-2 (WT) in the ER and mitochondria. We found that mt enhanced thapsigargin and tunicamycin-induced apoptosis through ER stress-mediated apoptosis but not through the death receptor- and mitochondria-dependent apoptosis, while WT prevented thapsigargin- and tunicamycin-induced apoptosis. In addition, mt depleted Ca2+ in the ER lumen and also increased the expression of SOCE-related molecules. Therefore, a massive Ca2+ influx via SOCE contributed to caspase activation and apoptosis. Furthermore, inhibiting SOCE or chelating either extracellular or intracellular Ca2+ inhibited mt-mediated apoptosis. In brief, our results explored the critical role of Bcl-2 in Ca2+ homeostasis and the modulation of ER stress.Ca The first identified apoptotic regulator was Bcl-2. The Bcl-2 family of proteins decide the fate of cells with response to survival and death. The proteins of the Bcl-2 family are characterized by homology domains BH1–4 (for Bcl-2 homology domain 1–4). The family can be subdivided in two major groups: the anti-apoptotic subgroup  and the pro-apoptotic subgroup comprising Bax-like proteins , which contain the BH1–3 domains, and the BH3-only proteins 2. Bcl-2 plays an important role in mitochondria and endoplasmic reticulum (ER)6. Most of the Bcl-2 family proteins contain a hydrophobic C-terminal domain, required for their specific localization to different subcellular compartments, such as the ER, mitochondria, and perinuclear membranes8. In the ER, Bcl-2 interferes with the induction of apoptosis by Bax9, ceramides, ionizing radiation10, serum withdrawal, and c-myc expression11. Recently, the focus of researchers has shifted toward finding the possible association between the effects of the Bcl-2 family on Ca2+ homeostasis and their role in the control of apoptosis13. In addition, the specific localization of Bcl-2 in the ER membrane indicates that Bcl-2 regulates filling of ER intracellular Ca2+ store15, suggesting that Ca2+ signaling might be a target of the Bcl-2 oncoprotein.Deregulation of apoptosis can lead to cancer and to autoimmune and degenerative diseases2+ level in the ER and mitochondria. However, determining whether Bcl-2 increases or decreases the ER luminal Ca2+ will reveal its true role in the ER Ca2+ handling16. One hypothesis states that Bcl-2 decreases the Ca2+ concentration within the ER such that less Ca2+ is available for release into the cytosol, thereby leading to a more modest mitochondrial Ca2+ uptake. Many studies show that Bcl-2 can protect cells from stress-induced Ca2+ release from ER and lead to subsequent cell death by lowering the loading of Ca2+ in the ER18. In contrast, it has been argued that Bcl-2 does not diminish the content of Ca2+ pool, instead it inhibits the opening of inositol 1,4,5-trisphosphate receptors (IP3Rs) on the ER, thereby reducing the extent of Ca2+ mobilization for a given magnitude of cell stimulation19. He et al. reported that Bcl-2 mediated Ca2+ uptake and preserved the Ca2+ pool of the ER to prevent depletion of the pool14. Furthermore, Bcl-2 overexpression is associated with the reduction in the transient elevation of cytosolic Ca2+ induced by thapsigargin (TG)20. A proposal common to many of these studies is the proposal that Bcl-2 functions to reduce the magnitude of increase in cytosolic Ca2+ concentration in response to apoptotic stimuli. However, the two different functions of ER-resident Bcl-2 may possibly depend on different cell contexts; this relation needs to be clarified in detail.The anti-apoptotic activity of Bcl-2 is mediated by its regulation of handling Ca2+ can serve as an apoptotic signaling factor when delivered at the wrong time and to the wrong place22. Disruption of intracellular Ca2+ homeostasis by influx of extracellular Ca2+ is always lethal to cells23. It was believed that Ca2+-related cell death could be triggered by large, sustained increases in cytosolic Ca2+. More specifically, both persistent Ca2+ release from the ER and store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ channels are apoptogenic25. Several anti-cancer drugs that are used to induce cancer cell apoptosis function not only through the dysregulation of Ca2+ signaling but also via the activation of other apoptotic modulators27, such as death receptor- and/or mitochondria-dependent pathways29. Ca2+ is consdered to be a link between ER stress and mitochondrial apoptotic pathways31. SOCE, by definition, is activated by Ca2+ efflux from the internal store. Stromal-interaction molecule 1 (STIM1) is a Ca2+ sensor in the ER that triggers SOCE pathway activation. Once Ca2+ is depleted in the ER, STIM1 proteins aggregate into multiple puncta and translocate to regions with close proximity to the plasma membranes32. Activated STIM1 binds to Orai1  or/and TRPC1 , two Ca2+ channels, located in the plasma membrane, which allow Ca2+ entry and are therefore involved in SOCE35. TG, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor that induces SOCE drastically following the depletion of Ca2+ stores of the ER, has been shown to induce apoptosis36. However, the underlying mechanism of Bcl-2-mediated regulation of TG-induced apoptosis has not been clearly elucidated.Ca37. The α5-helix within the BH1 domain of Bcl-2 is essential for its cytoprotective function. Bcl-2, which lacks a BH1 domain (Bcl-2/ΔBH1) or bears alanine as a substitute for glycine 145 in the BH1 domain (Bcl-2/G145A), fails to interact with either Bax or Bak, accelerating Bax- or Bak-induced apoptosis. Bcl-2/ΔBH1 or Bcl-2/G145A acts as a dominant-negative mutation of endogenous anti-apoptotic proteins such as Bcl-2 and Bcl-xL38. Furthermore, Bcl-2, but not the inactive point mutant Bcl-2/G145A, undergoes a conformational change in response to apoptotic agonists39. Bcl-2/G145A completely abrogated the death-repressor activity of Bcl-2 and disrupted its heterodimerization with Bax. Therefore, the structure of this region of Bcl-2 is important for its biological function40. In this study, in order to focus specifically on the effect of Bcl-2 on Ca2+-mediated apoptosis, we chose the apoptosis-inducing agent TG as an ER stress inducer to disrupt Ca2+ homeostasis in the ER41. We observed that Bcl-2 downregulated the basal Ca2+ levels in cytosol and mitochondria and prohibited Ca2+ elevation under apoptotic stimulation. We also observed that SOCE contributed to the TG-induced Ca2+ elevation and cytotoxicity when the α5-helix of Bcl-2 was mutated.Interaction of Bcl-2 with Bax appears to be important for its activity, and the BH1 and BH2 domains of Bcl-2 are required for the inhibition of apoptosis and heterodimerization with Bax144WGR146 to 144AAA146) was generated within the α5-helix of the Bcl-2 BH1 domain , an ER-resident chaperone protein and a hallmark of ER stress, can prevent cell death by protecting against many apoptotic stimulations. The results revealed that after TG treatment, Grp78 was upregulated in the cells that overexpressed WT, and downregulated in the cells that overexpressed mt to that in MDCK cells (C) Fig. . Cleavag2+ levels by Ca2+ efflux from the Ca2+ pool in the ER, and influx of extracellular Ca2+ across the plasma membrane42. As shown in Fig. Immunoprecipitation results revealed no interaction between Bcl-2 and Bax under TG treatment, regardless of the overexpression of WT or mt Fig. . Convers2+ in cytosol or organelles may cause stress and Ca2+ cytotoxicity, leading to cell death. At first, we evaluated Ca2+ levels in the Ca2+ pool of ER stores by passive TG-induced Ca2+ leakage and active ATP-induced Ca2+ release from the ER into the cytosol. We found that overexpression of WT and mt increased and decreased the Ca2+ concentration within the ER, respectively , fura-2/acetoxymethyl ester (fura-2/AM), and rhod-2/acetoxymethyl ester (rhod-2/AM), were used to identify ER ([Ca2+]ER), cytosolic ([Ca2+]i), and mitochondrial ([Ca2+]mito) Ca2+ levels in the resting state, respectively. Cells that overexpressed mt showed a lower Ca2+ level in the ER but a higher Ca2+ level in the cytosol and mitochondria . In addition, changes in the levels of μ-calpain expression also corresponded with the calpain activity as shown by cleavage of the calpain substrate, α-spectrin .Depletion or overload of Caely Fig. . IP3Rs a2+-ATPase inhibition allows Ca2+ to flow from the ER lumen into the cytoplasm, producing a transient elevation of cytosolic Ca2+ level, and triggering a sustained elevation of cytosolic Ca2+ level through SOCE. The genetically encoded FRET-based fluorescent protein, cameleon, and the ratiometric chemical dye, fura-2/AM, were used as indicators in Ca2+ measurement , the level of Grp78 expression in the cells that overexpressed WT was higher than that in cells that overexpressed mt , extracellular Ca2+ chelator (EGTA), and SOCE inhibitor (2-APB) attenuated TG-induced apoptosis  Most cells that overexpressed mt rounded up with the appearance of apoptotic bodies after TG treatment Fig. , which w mt Fig. . Our datsis Fig. . These r37. Therefore, mutation of the Bcl-2 α5-helix within the BH1 domain may disrupt the interaction between Bcl-2 and Bax. Herein, overexpression of WT led to the downregulation of the pro-apoptotic Bcl-2 proteins, Bax and Bak, and increased the anti-apoptotic Bcl-2 activity, which was confirmed using anti-active Bcl-2 antibody. In contrast, overexpression of mt increased Bax activity, which was confirmed using anti-active Bax antibody . Immunoprecipitation and HA14-1 pretreatment showed that the interaction between Bcl-2 and Bax is not involved in TG and TUN-induced apoptosis  overexpression of the WT gene; (ii) overexpression of the control vector (C); and (iii) overexpression of the mt gene. These cells were maintained in DMEM supplemented with 10% FBS under 5% CObcl-2 cDNA sequences inserted into pCΔj vector (pCΔj-bcl-2) were expressed by simian virus 40 (SV40) enhancer/promoter regulatory elements54. A DNA construct without bcl-2 cDNA sequences (pCAj-SV2) was used as a control. Transfection of cells was done using Lipofectamine™ 3000 Reagent . Foty-eight hours after transfection, cells were passaged by 1:10 dilution into G418 (500 μg/ml) selective medium. The IPTG-inducible expression of Bcl-2 by the lac operator/repressor system was established55. The stable transfection of a constitutively expressed lacI gene, encoding lac repressor, and the human bcl-2 gene that had been inserted downstream of a SV40 promoter containing the lac operator sequence in MDCK cells. The expression of the bcl-2 gene could be specifically activated by administration of the lactose analog IPTG.The vector pCΔj contained G418 resistance gene and EBV-derived replication origin. The human TM), and the lysates were analyzed by Western blotting using antibodies against Bcl-2 , Bax, Bak, calnexin, SERCA2, β-actin , porin , caspase-8, caspase-9, caspase-12, SERCA3, pSer70-Bcl-2, pThr167-Bax , Grp78, STIM1, STIM2, IP3R3 , Orai1, Orai2, Orai3, and TRPC1 .Cell lysates were harvested in RIPA buffer  on ice. The homogenate was centrifuged at a low-speed of 1000×Cells were stained with 500 nM tetramethyl rhodamine methyl ester , an indicator of mitochondrial membrane potential. The TMRM-stained cells were incubated in the dark at room temperature for 30 min and subsequently analyzed by flow cytometry  with excitation wavelength at 543 nm. In order to analyze the apoptotic ratio, the cells were fixed in 70% alcohol, followed by treatment with RNAse (100 mg/ml), and stained with PI (40 mg/ml) . The PI-stained cells were incubated in the dark at room temperature for 30 min and analyzed by flow cytometry  with excitation wavelength at 543 nm. The apoptotic ratio was assessed from the hypodiploid DNA peak of apoptotic cells (sub G0/G1 phase) using Cell Quest software.2+ was measured at 37 °C using the fura-2 fluorescence ratio method on a single cell fluorimeter. Cells were loaded with 2 μM fura-2/AM in DMEM culture medium at 37 °C for 30 min. The excitation wavelength was alternated between 340 nm (I340) and 380 nm (I380) using the Polychrome IV monochromator . The fluorescence intensity was monitored at 510 nm, stored digitally, and analyzed by the program of TILLvisION 4.0 (Till Photonics).Cytosolic Ca2+, in DMEM culture medium at 37 °C for 30 min. Fluorescence intensity of fura-2/AM and mag-fura-2/AM were observed under a single cell fluorimeter  and presented with pseudocolor images. Fluorescence images of rhod-2/AM were captured using a confocal imaging system .The cells were loaded with 2 μM fura-2/AM, 2 μM mag-fura-2/AM, or 2 μM rhod-2/AM, a direct probe for the cytosolic, ER, or mitochondrial CaWe performed the stable transfection of the cells with the cameleon plasmid, followed by live-cell imaging using a confocal imaging system . An optional 440 nm LD laser was used to directly excite the enhanced cyan fluorescent protein (ECFP) without exciting the enhanced yellow fluorescent protein (EYFP). Conversely, emission spectra of ECFP and EYFP were 470–500 and 535–565 nm, respectively. The analysis program in FV-1000 imaging system allowed ratio calculation and ratio image acquisition after subtracting the background fluorescence intensity.Cells were fixed with 4% buffered paraformaldehyde, permeabilized using 0.5% Triton X-100 for 15 min, and stained with mouse anti-Bcl-2 antibody , mouse anti-STIM1 antibody , or rabbit anti-calreticulin antibody  for 12 h at 4 °C. In addition, cells were stained with goat anti-mouse IgG conjugated with Alexa 488 or goat anti-rabbit IgG conjugated with Alexa 594  for 1 h. The fluorophore was excited by laser at 488 or 543 nm, respectively, and detected using a scanning confocal microscope .t-Boc-LM-CMAC 56. Cells were pretreated with 10 μM t-Boc-LM-CMAC for 30 min. Intracellular fluorescence was sequentially imaged under a confocal imaging system  and the mean fluorescence intensity (excitation by 405 nm LD laser) of individual cells was quantitatively analyzed.The μ-calpain activity was assessed by generation of the fluorescent product, 7-amino-4-methoxy coumarin (AMC), from hydrolysis of an artificial μ-calpain fluorescent substrate Activation of caspase-3 and caspase-9 was measured by a fluorimetric assay based on the specific hydrolysis of DEVD-AFC and LEHD-AFC , respectively. The cells were resuspended in chilled cell-lysis buffer kept on ice for 10 min followed by the addition of reaction buffer. Finally, 50 μM of DEVD- and LEHD-AFC was added, and the mixture was incubated at 37 °C for 2 h. The cleavage of DEVD- and LEHD-AFC was analyzed by measuring the release of AFC from DEVD- or LEHD-containing peptides. Fluorescence emission of AFC (excitation by 405 nm LD laser) was measured at 505 nm using a Fluoroskan AsCent FL fluorometer .t-test was performed for statistical analysis. A p value < 0.05 was considered significantly different.All data were represented as mean ± SEM (standard error of the mean) and Student’s Supplementary informationSupplementary Video S1Supplementary Video S2