Idelalisib is a delta isoform-specific, phosphoinositide 3-kinase (PI3-K) inhibitor. It has been used as a single agent for the treatment of relapsed or refractory small lymphocytic lymphoma (SLL), follicular non-Hodgkin’s lymphoma (NHL), and in combination with rituximab for patients with chronic lymphocytic leukemia (CLL). We present a case of a 77-year-old man diagnosed with SLL and was treated with multiple chemotherapeutic regimens in the past. Considering multiple relapses, he was started on idelalisib monotherapy almost 12 months ago. The treatment was stopped due to worsening neutropenia as well as mixed response on the scans. Almost, within one week of stopping the medication, he presented with complaints of altered mental status, hematuria, and worsening of generalized lymphadenopathy. This time, the patient was started on venetoclax (BCL-2 inhibitor) and rituximab which he is tolerating well without any complications. Idelalisib is one of the targeted therapies for refractory/relapsing small lymphocytic lymphoma (SLL). Idelalisib is an oral inhibitor of phosphoinositide 3-kinase (PI3K) delta that showed therapeutic activity in the initial studies of patients with non-Hodgkin's lymphoma (NHL). PI3K delta is integral to several signaling pathways in NHL cells including those that support survival proliferation and the retention of cells in lymphoid tissues. There are no clear guidelines for idelalisib discontinuation. Patients with a progression-free survival (PFS) less than the median expected for a treatment regimen are considered to have an early relapse. Abrupt discontinuation of idelalisib can also cause rapid disease progression resulting in complications .We present a 77-year-old male with a past medical history of NHL/SLL diagnosed almost 10 years ago, who presented to the hospital with abdominal swelling, altered mental status, and difficulty in urinating associated with hematuria. On physical examination, diffuse bulky lymphadenopathy was found in the cervical, axillary, and inguinal areas. Detailed oncologic history and treatment regimens that were taken by the patient have been well explained in Table Laboratory workup showed hypokalemia, hypophosphatemia, and elevated lactate dehydrogenase levels. During the hospitalization, computed tomography (CT) scan head was done that showed negative findings for any acute events. Due to altered mental status with underlying worsening of NHL and metabolic disturbances, the toxic and metabolic encephalopathy were the differentials under consideration. Metabolic derangements were corrected during hospitalization and that improved his mental status as well. Idelalisib treatment was discontinued abruptly a week prior to patient’s presentation to the hospital due to pancytopenia and a mixed response on the CT scan imaging. Upon admission, a repeat CT of the abdomen and pelvis showed diffuse bulky lymphadenopathy in the abdomen; one of the nodes in the anterior para-aortic region was measured about 5 cm × 5 cm × 8 cm Figure . BilaterBone marrow biopsy was done that showed a cluster of differentiation 5 (CD5) and CD23 positive B-cell population (37% of the lymphoid gate), lambda-restricted. Almost all of the B-cells showed immunophenotypic expression of CLL/SLL with lambda light chain restriction that was found in previous cases of this patient. Interestingly, a kappa light chain restricted population of monoclonal plasma cells co-expressed with CD56  is also identified. Urology was consulted for urinary complaints of difficult voiding and hematuria. However, the patient was further diagnosed with paraphimosis and scheduled to have an elective circumcision that alleviated his urinary complaints later. Idelalisib treatment for SLL/NHL that patient took for almost one year was stopped a week prior to current clinical symptoms. Based on progressive SLL/NHL, the patient was started on venetoclax  20 mg/day for seven days and 50 mg/day afterward. The patient did not develop any tumor lysis syndrome after starting the therapy and is tolerating the treatment well based on both imaging and clinical finding.Idelalisib is an oral inhibitor of phosphoinositide 3-kinase (PI3K) delta (δ) that showed therapeutic activity in initial studies of patients with NHL. P13K delta is integral to several signaling pathways in NHL cells including those that support survival proliferation and the retention of cells in lymphoid tissues. Idelalisib, the first Food and Drug Administration (FDA)-approved PI3Kδ inhibitor, is a valuable and safe management option for patients with relapsed SLL or chronic lymphocytic leukemia (CLL) and relapsed follicular lymphoma. Based on progression-free survival (PFS) significance, idelalisib joins ibrutinib-Bruton's tyrosine kinase (BTK) inhibitor as a FDA-approved oral agent for use in patients with relapsed NHLs . Acute tThe patient in the present case showed a mixed response and developed neutropenia which prompted the oncologist to discontinue idelalisib therapy immediately. A subsequent CT of the abdomen showed significant worsening in the size of the lymph nodes leading to complications including intractable abdominal pain, and hematuria with possible ureteral obstruction within one week of idelalisib discontinuation. Afterward, instead of re-starting the same regimen of idelalisib, treatment was switched to venetoclax plus rituximab combination. O'Brien et al. documented that single-agent treatment with ibrutinib was associated with a five-year PFS rate of 92% in CLL/SLL treatment-naïve (TN) older patients (≥65 years of age) and 44% in relapsed/refractory (R/R) patients. The onset of cytopenias was also reduced over five-year follow up of patients with a single treatment of ibrutinib [In summary, in a patient with refractory NHL being treated with idelalisib, we suggest performing the gradual discontinuation of idelalisib to prevent acute disease progression which may result in complications that can be life-threatening if not treated in a timely manner. A refractory case of SLL/NHL to multiple treatment regimens questions the understanding of underlying malignancy and also suggests evaluating alternate better treatment options.


In 1817, the London surgeon and pharmacist James Parkinson published a 66-page-long booklet entitled “An Essay on the Shaking Palsy”, which contains the first detailed, clinical description of the shaking palsy, or “paralysis agitans”, which is currently known as Parkinson’s disease (PD) . In factPD affects millions of people globally, but there is no cure, and its prevalence will double by 2030 -8. AlthoThe neuroscientific community has recently recognized that an increasing number of PD patients has died suddenly and unexpectedly, referred to as “sudden unexpected death in Parkinson’s disease” (SUDPAR) ,. SUDPARConsidering that SUDPAR is a rare phenomenon, difficult to diagnose, and only rarely reported, it is a phenomenon that has attracted the interest of the neuroscientific community since the late 1970s . More re2+ concentration ([Ca2+]c) and energy production in cardiomyocytes, such as Ca2+ channels, Ca2+-ATPases, the sarcoplasmic reticulum (SR), and mitochondria (MIT) (2+ homeostasis (PD-related cardiac dysfunction may manifest as ventricular arrhythmias due to the collapse of cardiac excitation-contraction coupling (CECC), primarily caused by persistent ionic deregulation in cardiomyocytes ,17,22,24ia (MIT) ,28. In mia (MIT) ,28. MIT eostasis ,28.2+ influx from the extracellular medium to the cytosol through L-type voltage-dependent Ca2+ channels (VDCCs) (2+ influx stimulates Ca2+-release (CICR) from the SR via ryanodine-sensitive Ca2+ channels (RyRs), generating a transient elevation in the [Ca2+]c and consecutive activation of the myosin-actin contractile myofilaments. This transient elevation in [Ca2+]c simultaneously increases the Ca2+ uptake by MIT and the Ca2+ concentration in the mitochondrial matrix ([Ca2+]m), which stimulates ATP production by the activation of the dehydrogenases in the tricarboxylic acid (TCA) cycle. To generate contractility for the ejection of blood from the heart, the activation of myosin by energy-stored ATP molecules is required to shift the head pulling on the actin filament and to shorten the sarcomere. The strength of myocardial contraction is directly related to the local Ca2+ concentration surrounding the myosin-actin myofilaments. Thus, the synchronization of [Ca2+]c transients throughout the myocardium is crucial for synchronous cardiac contraction  ,28. Thistraction ,28. Howehythmias ,28. In s2+ uptake by MIT is mainly mediated by the mitochondrial uniporter of Ca2+ (MUC), while its efflux is mainly mediated by the mitochondrial Na+/Ca2+-exchange channel (mNCE)  . Thus, t [Ca2+]m -30. Howeoduction ,27-29. TIt has been shown that mutations in genes causing PD, such as PINK1, parkin, DJ-1, alpha-synuclein, and LRRK2, cause mitochondrial dysfunctions, which is one of the reasons why they are called mitochondrial nigropathies . Mitocho2+ phosphate crystals that severely compromise the functional integrity of MIT . Hmyocytes  32). Si. Si2+ ov2+ channel, thereby reducing the influx of Ca2+ into MIT  also prevented cardiac arrhythmias and hemodynamic dysfunctions in laboratory animals exposed to cardiac IR injury . It has into MIT ,28,32,33myocytes ,28,32,33myocytes ,28,32,33Several studies suggested that myocardial dysfunctions similar to those induced by cardiac IR can be involved in SUDPAR ,17,22,24Scorza FA and Caricati-Neto A contributed to the design, and manuscript writing and editing. Menezes-Rodrigues FS, Errante PR and Tavares JGP contributed to the acquisition, analysis and interpretation of experimental data. Scorza CA, Ferraz HB, Finsterer J and Olszewer E contributed to the critical review of the manuscript.


Despite long-standing issues still holding back the field, an increasingly large repertoire of genes and mechanisms has now been described. The unanticipated complexity revealed by genomics is giving way to a renaissance in our understanding, and the characterization of novel molecular mechanisms. The reviews in this special issue bring together research focused on specific structures, including mitochondria, pollen tubes and guard cells, as well as on the processes of ion homeostasis and salt stress tolerance, and nodulation.Ca2+ signaling researcher, to signify the importance and relevance of calcium (Ca2+) as the most versatile signaling second messenger, involved in practically all aspects of cell and developmental biology from egg activation to cell apoptosis. Eventually the French pronouncement made it into the title of an essay about the nature and mechanisms behind Ca2+ waves in plants  and the CBL–CIPK  pairs. Some ‘usual suspects’, such as animal homologs of calmodulin and the CMLs  were also confirmed as playing important roles  even when the same molecular mechanisms are present, they may result from convergent evolution with adaptation to the very different contexts of plant physiology, and thus (ii) the same function may be achieved through different associations and regulatory mechanisms. 2+-permeable channels, the ones for which there are more data available are the Glutamate Receptor-Like (GLRs) and the Cyclic Nucleotide Gated channels (CNGCs).Of all the gene families documented as coding for CaGLRs made it to center stage directly from their genomic identification during the assembly of the Arabidopsis genome . Multipl2+ signatures in the nuclei of Medicago root cells during Rhizobium infection  have distinct peptide signals that target these organelles, and the authors were pioneers in showing that to be the case and so implicating them in Ca2+ homeostasis , with critical comparisons from the leading group in the world in this area.More profoundly the team has been at the forefront in characterizing the mitochondrial channel uniporter (MCU) in plants  of other downstream proteins. 2+ oscillation either on a standing gradient (the pollen tube) or spatially distributed (guard cells/stomata) to infer common patterns and different properties that could help explain the network of interactions, feedback loops and pattern-generation mechanisms. Both systems have been extensively used for Ca2+-signaling research, but the meaning of their Ca2+ signatures remains elusive.The hallmark of Ca2+ gradients of any cell at their growing tip, and when germinated in vitro display oscillations in many species. However, this is not always the case, and there are no sound data showing that they exist in vivo  constitutes a formidable challenge such that all combinations are tested under specific screens. Nevertheless, the prospect that some of these combinations might bear the right kinetics and affinities to make them ‘the’ specific sensor for a certain Ca2+-signaling field is experiencing. In addition, it shows the fragmentation that has occurred into each specialist area, which calls for a more systems-oriented perspective to integrate these different parts. The reviews in this issue provide challenging perspectives on ways to reach this goal, but achieving it would lay the ground for the next steps where the formation of waves and the decoding of specific signatures still lack defined molecular mechanisms.The representation of novel molecular mechanisms provided in 2+ signaling in the plant cell, with different types of organization color coded by quadrant of the ‘textbook’ diagram. Moving clockwise: (i) structures—mitochondria (Unified representation of Cachondria .


We evidence crystalline heterogeneities in BaTiO3 nanocrystals manifested by a centrosymmetric shell around the tetragonal core of the crystals, corroborating hypotheses from previous ensemble structural investigations. This study shows that in contrast to bulk materials, nanocrystals exhibit a complex composition, which is seen to be reproducible among nanocrystals. P-SHG appears furthermore as a powerful methodology that reports structural behaviors in nanoscale dielectrics materials, at the individual nanoparticle scale.Ferroelectric nanocrystals have considerable interest for applications in nanophotonics, optical memories and bio-imaging. Their crystalline nature at the nanoscale remains however poorly known, mostly because structural investigation tools on single nanocrystals are lacking. In this work we apply polarization resolved second harmonic generation (P-SHG) imaging on isolated Barium Titanate (BaTiO BaTiO3 nanocrystals are thus efficient non-centrosymmetric second harmonic generation (SHG) emitters, which signal is detectable down to 20 nm sizes1, making them suitable biomarkers for nonlinear background-free imaging in cells or tissues3. Recently the SHG efficiency of BaTiO3 nanocrystals has been even further improved by plasmonic enhancement4 or benefiting from Mie resonances5. BaTiO3 nanocrystals are thus promising candidates for nonlinear imaging, along with other oxides and dielectric crystals8.Understanding the optical and physical processes occurring at the nanoscale in nanomaterials is fundamental for various applications that cover their use as nano-emitters in imaging, as ferroelectric nano-objects in non-volatile memories and piezo-electric devices, but also for electro-optics and more generally nanophotonics. One of the most attractive property of dielectric and ferroelectric nanocrystals is their ability to generate efficient nonlinear optical interactions even though their size scale down below hundreds of nanometers, which opens to an extensive range of functions that rely on optical frequency mixing. Barium Titanate  imaging, a process that relies on the sensitivity of SHG to the incident polarization at each measured point of the nanocrystal.As a consequence, there is today a poor knowledge on how the structure of such nanocrystals influences their optical functions, which are averaged over the scale of optical diffraction limit (e.g. a few hundreds of nanometers). This knowledge is yet a key element for the design of future nanophotonics devices. In this work, we address the question of the structure and crystalline heterogeneity of BaTiO17. The principle of P-SHG imaging, schematically depicted in Fig. 21, this method thus expands spatially the monitoring of SHG polarized responses. By adding an extra degree of freedom (polarization) to spatial scanning, P-SHG allows to probe the structure of nano-objects in their spatial dimension. This method has recently permitted to map nonlinear vectorial coupling properties at the surface of metallic nanoparticles of complex shapes, which plasmonic modes where spatially extended and strongly anisotropic22. In the present work, the sample is made of isolated dielectric nanocrystals deposited on a microscope cover slip from a diluted solution. Mapping spatial heterogeneities in dielectric nanoparticles is complexified by their unknown orientations, in contrast to pre-defined metallic nanostructures fabricated by nanolithography. Nevertheless we show in this work that the rich nature of polarized SHG added with imaging capabilities is capable of extracting structural information even in such situation. The samples used in this work are either made of KTiOPO4 (KTP) of 150 nm size used as a reference23, or BaTiO3 (named BTO in what follows) of 100 nm size, unless otherwise mentioned (see Methods).The light-matter coupling process at the origin of SHG nonlinear radiation in crystals is intrinsically vectorial. It strongly depends on the orientation of the crystal, the symmetry of its crystalline unit cell, and the polarization direction of the incident electromagnetic fieldα), called P-SHG response, which is the result of the coupling between the rotating incident polarization and the nanocrystal portion illuminated by the beam parked at the pixel position . For each nanocrystal, a stack of SHG images is formed that contains typically 18 images, corresponding to polarization angles α ranging regularly from 0°  to 162° Fig. . In ordeion Fig. . Averagiα) obtained at each pixel of the drift-corrected, averaged nanocrystal image are then decomposed into a Fourier series. Since SHG responses depend on the fourth power of the in-plane incident field E(α), I(α) can be decomposed onto circular functions of second and fourth orders22:0 is the total intensity, resulting from the sum over all incident polarization angles. Here the incident field polarization is assumed to be rotating in 2D with coordinates in the sample frame 24. This approximation is discussed below. The  coefficients are retrieved experimentally by projection on circular functions, following an approach previously described22. They can be grouped into amplitude and phase terms which reflect the second and fourth orders of the α-dependence of the P-SHG response:φ4 given modulo π/2. 4 such that the fourth order response lies either along the second order (I4 > 0) response or π/4 phase shifted (I4 < 0)25.P-SHG intensities I and  are direct signatures of the shape  of the P-SHG modulation response present at each pixel position  on the other side represent the in-plane orientations of these responses, e.g. the mean direction of the anisotropy (φ2) and fourth order response (φ4).  are thus directly linked to the orientation of the unit-cell structure probed at the specific pixel position. The P-SHG analysis results are rendered as oversampled images, using a pixel size of 50 nm  direction, and which color is encoded with the I2 (resp. I4) values. The signal level used for P-SHG analysis is thresholded with a lower limit that ensures standard deviations lower than 0.01 on the  parameters, accounting moreover for a pre-calibrated bias that occurs at low signal to noise conditions22.Altogether,  map recorded for the KTP nanocrystal  map  nanocrystal of about 200 nm size, which has been imaged in parallel by scanning electron microscopy (SEM)  convolved by the size of the nanocrystal  values are not uniformly distributed over the size of the P-SHG image, in contrast to what is observed in KTP nanocrystals. Second, φ2 and φ4 values, represented by  sticks orientations, exhibit strong differences between the center of the map, where these angles are uniform, and the border, where they are radial-like . The shapes of nanocrystals are not perfectly spherical, as depicted in the SEM image  Fig.   cancels this radial pattern and leads to homogeneous  and  maps that behave now similarly to KTP nanocrystals 28, thus BTO exhibits a higher surface index mismatch in air and is more likely to create surface induced responses from a stronger local normal asymmetry29. This surface response has been identified in early works on SHG in isotropic media31, and has be shown to be non-negligible in centrosymmetric media, in particular crystals35. It can be described as an effective χ(2) susceptibility tensor which includes dipolar and multipolar contributions arising from both the structural discontinuity that breaks the centrosymmetry at the crystal interface and the strong local fields gradients31. The associated tensor exhibits three independent components: n is the surface normal direction and t denotes directions parallel to the surface36. The diagonal element 34. Additionally in BaTiO3 nanoparticles, the incident field locally scattered by the nanoparticle has a specific geometry. Finite element method simulations5 show that at 800 nm illumination, the local field exhibits strong gradients at the interface with a preferred polarization along the normal direction , the longitudinal field amount is about 33% of its in-plane components (on average over its point spread function), which leads to bias of P-SHG results by no more than 20% in BTO  positioned on a grid every 40 nm steps (see Supplementary Information). χ(2) is defined inside the core of the nanocrystal by the BTO crystalline tetragonal symmetry 40, in the shell by a cubic symmetry for which χ(2) = 0, and at the surface by a unique coefficient n the surface normal direction. The relative efficiencies between the core and surface nonlinearities is expressed by a ratio θ, φ, ψ)  map illustrated in Fig. θ is varied, since  involve only a rotation of the P-SHG maps rather than a pattern change, due to the symmetry of the nanocrystal  Fig. 25. The ss r Fig. . For eacmentclass2pt{minimtal Fig. . Note thtal Fig. .I2, φ2) maps appear dominantly in the presence of a shell whatever the nanocrystal orientation, size or η ratio . By introducing a centrosymmetric shell in the structure, the radial surface response originating from the nanocrystal surface discontinuity is visibly emphasized, evidencing both the presence of the shell and the surface response. Figure Interestingly, modelling a pure tetragonal symmetry surrounded by a surface response Fig. , left leθ angles in the presence of a shell, the  maps depart from a pure radial shape and take a more unidirectional feature  tends to induce purely radial-like patterns whatever the θ value with a vanishing I2 at the center  patterns can differ in  responses. Consequently, to discriminate the presence or not of a shell and the interface nonlinear strength contribution, both patterns need to be compared to models.At low see Fig. , which isee Figs  to S12. I2, φ2) and  responses measured in part of the BTO nanocrystals. This population corresponds to 25% of the 60 measured nanocrystals. Comparing qualitatively the experimental patterns with theoretical responses for both second and fourth orders in 100 nm size nanocrystals permits to deduce that they exhibit a cubic shell of core-to-size ratio around r = 0.4 and a non-negligible surface contribution of η = 0.5. A typical range of η that leads to a reasonably good agreement with the experimental observations is [0.2–0.7]. Note that we do not expect an exact quantitative correspondence between experiment and theory, since the nanocrystal is simulated in 2D, using a simplified structural model, and the incident field is supposed spatially homogeneous without any longitudinal contribution, which is a first order approximation. Nevertheless, this comparison permits to reveal the structural heterogeneity of such nanocrystals, to give order of magnitudes of its spatial extent, and to estimate its 3D orientation. In particular, Fig. θ lies between 0° and 45° (a good agreement is found for θ ~ 40°), while Fig. θ higher than 45°. ψ does not have a major effect on the P-SHG patterns, due to the tetragonal symmetry structure, while φ governs the in-plane orientation of the P-SHG pattern only for θ < 45°.Figure I2, I4) values in the central part of the patterns are not vanishing. This case is found to correspond to a larger nanocrystal size of about 300 nm diameter, with a core-to-size ratio r = 0.4 and η = 0.5  patterns, it is necessary to investigate more closely the fourth order symmetry  response of such nanocrystals  pattern is non-vanishing in the presence of the shell while with no shell, it completely vanishes  patterns, we conclude that the observations of Fig. θ ~ 0°). Interestingly, the anisotropy of this pattern permits moreover to identify the azimuthal in-plane orientation of the nanocrystal, as illustrated in Fig. I2 and I4 values such as in Fig. Among the rest of the BTO nanocrystals measured, features appear that differ slightly from a pure radial response Fig. . Such resee Fig. . In partsee Fig. . From a 3 nanocrystal, which size is of the order of 40% of its total diameter, representing tens of nanometers. This nano-scale structural information is obtained by the analysis of spatial polarization modulation dependences, together with an a priori knowledge of the nanocrystal structure. In this respect, P-SHG is different from a super resolution imaging method, since it does not directly reconstruct a crystalline nano-scale picture. Nevertheless, SHG polarization responses from known nanocrystals can serve as markers for super resolution imaging41.Finally, the different cases investigated by P-SHG evidence the presence of a centro-symmetric shell around the tetragonal core of BaTiOφ orientation angle has an effect on the in-plane orientation of P-SHG patterns, the out-of-plane θ orientation has an effect on the shape of this pattern. A complementary read-out of this angle can be done via the measured P-SHG amplitudes I2 and I4, which are directly related to the unit-cell crystal symmetry and orientation. Assuming a bulk BTO crystal of tetragonal symmetry, the  values follow a specific dependence that is unique to such symmetry with variations due to the θ orientation of the unit cell relative to the longitudinal axis  dependence was then compared to the experimental obtained values, either from BTO nanocrystals in oil or in air. First, averages over all measured pixels in P-SHG maps show that despite the existence of the shell in the observed nanocrystals in air, the nonlinear anisotropy values obtained are close to that of a tetragonal symmetry as expected from the core crystalline structure  values obtained at the contour of radial-like patterns leads to high I2 values that resemble pure dipolar responses  dependence obtained for the BTO unit cell symmetry can be compared to that of a pure KTP crystal  dependence for KTP was generated using its reported nonlinear coefficients30 and orientations defined according to its crystallographic data  dependence is thus the fingerprint of a bulk crystal symmetry as read-out by SHG, allowing possible unambiguous determination of its 3D orientations. It does not however reveal possible nanocrystal spatial heterogeneities, for which the previous analysis of the P-SHG response map is required.The . A polarized beam splitter  was used to make the excitation lasers linearly polarized after the scan mirrors. This ensured that the dichroic mirror  received a p-polarized laser light during the entire experiment. After the dichroic mirror, the linear polarization angle of the excitation laser was controlled by an achromatic half-wave plate  mounted on a motorized rotational mount .The setup is based on a two-photon scanning microscope which uses a Ti:Sapphire femtosecond laser and optical parametric oscillator (OPO) combination , at excitation wavelength in the range 800–1500 nm (Coherent Inc.). The total power delivered to the sample lied in the range of 1.0–1.2 mW. The laser beams sizes were expanded to fill the back aperture of the objective 40×/1.15 water immersion Nikon Instruments Inc.). Imaging was performed on an inverted microscope  using a pair of galvanometric scan mirrors . The transverse optical resolution is estimated to be 250 nm. The pixel size is 50 nm  before being detected by a Photomultiplier tube . Depending on the excitation wavelength, the SHG signal was separated using an appropriate dichroic beamsplitter of 400/40 nm. The signal detection path consisted of imaging pairs of lenses which image the back aperture of the objective on to the detector window. This configuration keeps the signal steady on the detector even though the excitation spot is continuously scanning the sample. Scanning and data acquisition was performed using an in-house LabVIEW  program. The data is acquired by a data acquisition board . Data analysis is performed using Matlab .For drift correction, the images are first denoised (by a Gaussian filter) and the centroid of each nanoparticle image is calculated with pixel size accuracy (multiple nanoparticles within the same field of view can be tracked). Sometimes, the centroid cannot be automatically detected  and the center of the peak is manually chosen. The obtained drift information is used to register all nanoparticle images in each polarization stack. Since the measurement is repeated (8 times), the drift-corrected nanoparticle stacks can be averaged to increase signal-to-noise which benefits the Fourier-analysis in P-SHG.3 (BTO) nanocrystals used in this study are commercially available in the form of a powder from Techpowder S.A. and Nanostructured & Amorphous Materials, Inc. All particles have an average diameter of either about 100 nm or 300 nm. The size of the nanocrystal stated in this work is deduced from Dynamic Light Scattering experiments, which provide an average with a standard deviation depending on the sample. To give an indication, one batch of used BTO nanoparticles had a size distribution of 137 nm in average and a full width half maximum (FWHM) of 69 nm. The methodology used to prepare the BTO samples is straightforward. A stock suspension of nanoparticles in ethanol (1.2 mg to 1 mL) is sonicated (30 min). For a sample, 1 μL of the stock was diluted by 1:100 and a drop of 10 μL is deposited on a clean cover slip, which is then placed in vacuum spin coater (at 100 RPM for 9 s and 2000 RPM for 30 s) to dry the suspension and immobilize the nanoparticles.The BaTiO−5 mbar) in a FEI Magellan 400 system .The BTO nanoparticles are deposited on a ITO covered cover slip to avoid charging during scanning electron microscope (SEM) measurements. High resolution SEM micrographs are obtained in vacuum . A diluted KTP-water solution is sonicated and a droplet placed onto a cleaned cover slip, previously cleaned and set in a UV-Ozone bath to make it hydrophobic.Potassium titanyl phosphate (KTP) is an efficient nonlinear optical material, usually grown to bulk crystals of macroscopic dimensions, but for which nanocrystals can be obtained with stable, high SHG radiation efficiency. In this work we use KTP nanocrystals fabricated by a co-precipitation methodSupplementary information


In additional experiments we also analyzed the timing of abnormal uptake of Zn2+ into the cytoplasm (TZnCy) relative to TCaCy and TSP. We focused on those NRVMs which survived anoxia, as evidenced by at least 50% recovery of ΔΨm and the absence of detectable SP. In these cells, we found a consistent sequence of critical events in the order, from first to last, of TCaCy, TCaMi, TMPT, TSP. After detecting TCaCy and TCaMi, abrupt switches between 1.1 mM and nominally zero [Ca2+] in the perfusate quickly propagated to the cytoplasmic and mitochondrial [Ca2+]. Depletion of the sarcoplasmic reticulum with ryanodine (5 μM)/thapsigargin (1 μM) accelerated all events without changing their order. In the presence of ZnCl2 (10–30 μM) in the perfusate we found a consistent timing sequence TCaCy < TZn ≤ TSP. In some cells ZnCl2 interfered with Ca2+ uptake, causing “steps” or “gaps” in the [Ca2+]Cy curve, a phenomenon never observed in the absence of ZnCl2. Together, these findings suggest an evolving permeabilization of NRVM’s sarcolemma during reoxygenation, in which the expansion of the pore size determines the timing of critical events, including TMPT.While an ischemic insult poses a lethal danger to myocardial cells, a significant proportion of cardiac myocytes remain viable throughout the ischemic episode and die, paradoxically, only after the blood flow is reinstated. Despite decades of research, the actual chronology of critical events leading to cardiomyocyte death during the reperfusion phase remains poorly understood. Arguably, identification of the pivotal event in this setting is necessary to design effective strategies aimed at salvaging the myocardium after an ischemic attack. Here we used neonatal rat ventricular myocytes (NRVMs) subjected to 20–30 min of simulated ischemia followed by 1 hour of “reperfusion”. Using different combinations of spectrally-compatible fluorescent indicators, we analyzed the relative timing of the following events: (1) abnormal increase in cytoplasmic [Ca In different prior studies one can find almost all possible permutations between these factors in terms of their temporal sequence and cause-effect relationship, depending on the stated hypothesis  was available. In that study, SP monitored by cellular uptake of calcein occurred “minutes” after MPT. The authors could not explain the source of Ca2+ waves preceding MPT, but speculated that those could be due to a “canonical” mechanism of spontaneous Ca2+ release from the sarcoplasmic reticulum.Our study mentioned above  lacked in et al.  monitore2+ overload, mitochondrial Ca2+ overload, MPT and SP by simultaneous monitoring of multiple (3 or 4) fluorescent reporters in individual myocytes during I/R. We used NRVM monolayers transfected with a gene encoding for a mitochondrial Ca2+ indicator LAR-GECO1.2 Cy) and mitochondrial ([Ca2+]Mi) Ca2+ concentrations, SR Ca2+ stores were depleted by a combination of ryanodine (5 μM) and thapsigargin (1 μM) applied 15 min before the onset of simulated I/R and maintained throughout the whole experiment (Rya-Thap group).Experiments in the 2+ influx across the sarcolemma during simulated I/R was investigated in three experimental series. The first included the application of 3-min pulses of baseline solution with nominally zero [Ca2+] before I/R, and after a perceptible onset of [Ca2+]Cy overload during reperfusion (Zero-Ca group). In the second series we applied Ni2+ (5 mM) to block Na+-Ca2+ exchange (NCX) Cy, [Ca2+]Mi, ΔΨm, and the onset of SP (as the cellular loss of Fluo-4).To track Cy) was monitored with a zinc-specific dye, Newport Green DCF . Rhod-2 AM  was employed to detect [Ca2+]Cy and SP instead of Fluo-4 AM, to avoid overlap with the emission spectrum of Newport Green DCF. Both dyes were loaded by incubation in Optimem for 35 mins at room temperature, and MitoView633 was used identically to the other groups. The Zinc group was not infected with the LAR-GECO1.2 indicator, as it would overlap with the emission spectrum of Rhod-2. Thus, in the Zinc group, four parameters were also tracked, but [Ca2+]Mi was replaced with [Zn2+]Cy. In some experiments, the SP indicator TO-PRO3  was used as an additional method to detect SP. In spite of its overlapping spectra with MitoView633, the two indicators are spatially distinct  and in practice TO-PRO3 uptake almost always occurred after MitoView633 loss. TO-PRO3 is normally cell impermeable and similar to the commonly used SP indicator propidium-iodide, in that it binds to nucleic acids upon entering permeable cells which amplifies the fluorescent intensity over 100-fold.In the 2+ transients were captured as a continuous time-series every 100 ms for 80 frames, equaling a recording total of 8 secs. The field of view (FOV) was set to a 128 x 128 pixel resolution and a 4.5x magnification with the 20x objective, along with an imaging speed of 1400 to achieve the stated continuous time resolution. For tracking all parameters throughout the full experimental period, time-series were utilized again with a frame rate of either 30 secs or 1 min depending on the experiment. These frame rates were chosen as the best balance between better time resolution and avoiding phototoxicity and bleaching due to excessive laser exposure induced by higher frame rates, such as every 15 sec. The spatial resolution of 512 x 512 pixels at a magnification of 1.3x yielded a 450 x 450 μm FOV. During a time-series, if there appeared to be a slight drift in imaging plane then the series was stopped and the plane was quickly refocused to minimize the time between the acquired series.NRVMs were placed in the perfusion bath and imaged on a Leica SP8 confocal microscope. An oil immersion 20x lens was employed for the collection of all images. CaAll long time-series images were obtained in three channels sequentially. In the first channel, either Fluo-4 or Newport Green was excited with a 488 nm wavelength laser and the emitted signal was filtered through the Leica SP8 prism. Light between 491 and 551 nm was collected with a HyD detector. The second channel, also collected with a HyD detector, imaged either LAR-GECO1.2 or Rhod-2 and was excited with a 561 nm laser and band passed between 566 and 630 nm. In the third channel MitoView633, and sometimes additionally TO-PRO3, were excited at 633 nm and long pass filtered to a PMT detector at 643 nm. Using the Leica SP8 prism technology and imaging each channel sequentially, as opposed to simultaneously, avoided possible crosstalk between signals and ensured quality intensity curves upon analysis.2+ transients at baseline); (2) having clearly demarcated cell boundaries to allow for accurate cell segmentation; (3) having overt signs of “ischemic” effects including at least 50% loss in the ΔΨm and cell shrinking, and (3) surviving within 6 minutes of “reperfusion”, as demonstrated by at least 50% recovery in the ΔΨm during MitoView633 restaining combined with the lack of SP . These criteria were imposed due to the fact that these characteristics are observed in the whole heart model Cy (TCaCy) and a critical increase in [Ca2+]Mi (TCaMi) as the time when the respective signals (Fluo-4 or LAR-GECO1.2 fluorescence) increased above the 50% level between the minimum and maximum. We defined the time of MPT (TMPT) as the decrease of MitoView633 fluorescence below the 50% level. Finally, we defined the time of SP (TSP) as the time when Fluo-4 fluorescence decreased below the 50% level. We interpreted the loss of the Fluo-4 signal during “reperfusion” as evidence of dye leakage from cells through sarcolemmal pores. This interpretation was confirmed in separate experiments, in which the loss of Fluo-4 signal during “reperfusion” correlated well with the uptake of the normally cell-impermeable dye, TO-PRO3 , we set the threshold of detection at 10% level of the normalized signal, as opposed to 50% level used in other series. The low threshold was chosen to facilitate early detection of Zn2+ and TO-PRO3 uptake, which developed in time rather slowly, and by visual inspection was deemed to be robust enough not to produce false-positive detections.For the chondria  althoughchondria . Howeveruo-4 see . Hence, www.jmp.com). Statistical tests included Student t-tests and one-way ANOVA with Newman-Keuls post-hoc test for pairwise comparisons , as indicated in specific figure legends. Differences with p < 0.05 were considered statistically significant.All values within groups are presented as the mean ± standard error of means. Quantification plots portray a variety of data including 1) the detected values according to the analysis criteria, 2) box and whiskers plots which portray median values, the upper and lower quartiles, and the highest and lowest observations excluding outliers, and 3) mean diamond plots which incorporate the mean values and 95% confidence intervals. These plots were generated with JMP trial software  is expected when mitochondria hyperpolarize and increase the pH gradient across the inner mitochondrial membrane. However, during “reperfusion” we observed a large increase in LAR-GECO1.2 signal just before the loss of ΔΨm, and this increase was sustained after the loss of ΔΨm when the mitochondrial matrix is expected to become more acidic. Hence, it seems that under the tested conditions, changes in LAR-GECO1.2 fluorescence reflected a true [Ca2+]Mi increase, even if attenuated due to expected acidification of depolarized mitochondria.LAR-GECO1.2 appears to be highly co-localized with MitoView633, as seen in the yellow areas of Panel C, and thus is expressed exclusively in mitochondria without other offhand targets within the cell. The limited green areas in Panel C that are devoid of MitoView633 staining represent temporarily depolarized mitochondria, and reflect mitochondrial depolarization-repolarization cycles (flicker) sometimes observed at baseline. In sham experiments, the cell-averaged level of LAR-GECO1.2 fluorescence remained relatively constant over a full 2-hour test period . A known2+ handling. Coverslips were incubated with Fluo-4 AM to detect [Ca2+]Cy, and subsequently placed in the imaging perfusion chamber. Cells were paced at 1 Hz and continuously imaged every 100 ms. The resulting Ca2+ transients can be seen in 2+ transient is shown in 2+ transients for the entirety of a 2-hour experimental duration, and had not undergone any detectable SP . The application of CsA (0.2–0.4 μM) (CsA group), an MPT inhibitor which decreases the MPT pore sensitivity to Ca2+, did not change the timing or order of the critical events  and thapsigargin (1 μM), keeping ryanodine receptor channels in a continually open state while also inhibiting Ca2+ uptake into the SR by blocking SERCA2a (Rya-Thap group). Panel A in Rya-Thap cell during reperfusion, analyzed identically to cells in the Control group. Most notably, the major sequence of events remained the same as observed in Control, with [Ca2+]Cy overload occurring first, followed by [Ca2+]Mi overload, then MPT and SP. When quantified and compared to Control (Panel B), the sequence of critical events indeed remained the same as seen in Control, with TCaMi, TMPT, and TSP occurring significantly later than the respective previous event . Interestingly, in spite of the retention of the event sequence, all critical events in the Rya-Thap group occurred significantly earlier than their Control counterpart . The cause and/or meaning of this observation will be deliberated in the Discussion section.Previously, a Call death . To test2+]Cy and [Ca2+]Mi increase during reperfusion, we hypothesized that the source of these increases is an influx of Ca2+ ions across the sarcolemma. To prove this point, we applied 2–3 min pulses of nominally zero Ca2+ solution (0 Ca2+) after the initial phase of the [Ca2+]Cy overload during “reperfusion” became evident during real-time monitoring in pre-selected regions of interest. 2+ pulses applied after the steep rise in [Ca2+]Cy. As is apparent, both [Ca2+]Cy and [Ca2+]Mi drop immediately upon the application of 0 Ca2+, and return directly back upon the reapplication of normal [Ca2+]. Note the almost symmetric response of [Ca2+]Cy to decreases and increases of [Ca2+]o, suggesting equal permeability for Ca2+ in both transmembrane directions.Since the depletion of SR did not affect the dynamics of [Ca2+ observed during “reperfusion” could be mediated Na+-Ca2+ exchanger (NCX) [2+]Cy overload we applied 5 mM Ni2+ to the perfusate in the Nickel group. At this concentration, Ni2+ blocks NCX by 80% Cy dynamics in such a way that in the majority of cells the criterion of TCaCy as the time when [Ca2+]Cy curve crossed the 50% level of the ascending phase of the dynamic range during reperfusion  . To inveX by 80% . Ni2+ afsion see  was not Control group. However, the post-reperfusion recovery of fluor-4 signal reflecting [Ca2+]Cy was very poor, the minimum level barely scratching the 50% of the dynamic range. Also, the recovery of LAR-GECO1.2 signal, reflecting the combination of changes in [Ca2+]Mi and pH was extremely slow as compared to Control cases . In analyzed Control cells maximal post-reperfusion level of Fluo-4 signal reached 154±6% of the maximal baseline level, whereas in analyzed Nickel cells it was 100±14% of the maximal baseline level (p < 0.0001 by unpaired two-tail t-test).To provide a quantitative support for the notion that Ni2+ described above nominally supported the role of NCX as the conduit of post-reperfusion [Ca2+]Cy overload, we had our doubts  concentrations. Also, under normal conditions, the exposure of cardiomyocytes to extracellular Zn2+ at 10 to 100 μM concentrations does not cause a detectable increase in [Zn2+]Cy, and does not adversely affect cardiomyocytes apart from a slight inhibition of ICa,L Cy and [Zn2+]Cy, respectively. TO-PRO3 fluorescence was collected in the same channel as MitoView633, since the signals could be easily separated spatially and temporally  in the range between minimum and maximum measured during “reperfusion”. In each cell, the onset of [Ca2+]Cy rise was detected the earliest, followed by the onset of Zn2+ uptake, and finally uptake of TO-PRO3 (colored open circles in m loss (solid red line), whereas the final and sustained increase (dashed red line) indicates uptake of TO-PRO3 signifying “canonical” SP. Overall, the timing of the three permeability events varied between cells, but it was always the case that Zn2+ uptake started after Ca2+ uptake and before, or simultaneously with, TO-PRO3 uptake .Zinc group, there exists a phenomenon in some, but not all cells, that is not present in Control experiments. This phenomenon is a ‘step’ or a ‘gap’ in the rising phase of the [Ca2+]Cy curve during “reperfusion”. This ‘step’ can be detected by visual inspection and quantified as the time difference between the 20 and 80% levels of the rising phase of [Ca2+]Cy. Zinc group with varying ‘step’ durations, including a cell without a step (similar to those observed in Control cases), cells with a median and long step, and one with a “gap” (two phases of [Ca2+]Cy increase separated by a period of [Ca2+]Cy decrease). The thin black lines indicate the 20 and 80% levels of [Ca2+]Cy rise and the colored open circles are the detected time points . The time difference between the points demonstrates how the step size may vary between different myocytes.When scrutinized more closely, it became apparent that in the 2+ effects, the time interval between T20Ca and T80Ca was not statistically different between the Control and Zinc groups. However, when the Zinc group was divided into two subgroups with and without a “step” in [Ca2+]Cy rise, statistical analysis showed that the subset of Zinc cells with a “step” had a significantly longer interval between T20Ca and T80Ca as compared to Control cells and Zinc cells without the “step” . Third, we provide evidence of specific interactions between Ca2+, Zn2+, and TO-PRO3 uptake which might suggest that their permeation during reperfusion occurs through the same common path.The important findings of this study are threefold. First, we established a consistent chronology of critical events during reperfusion, in which cytoplasmic Ca2+], reactive oxygen species (ROS), and inorganic phosphate , but cannot prevent MPT pore opening when a sufficiently high [Ca2+] in the vicinity of the pore is achieved Cy. However, any vicious circle has to start from some event outside the feedback loop(s). There has to be an external trigger either for MPT, or [Ca2+]Cy elevation, or both. To that effect, a canonical view has been developed which emphasized events occurring at the moment of restoration of oxygen and ionic imbalances at the onset of reperfusion. It is generally believed that in this setting a net Ca2+ influx takes place due to a coupled action of the sodium-proton exchanger and NCX in response to abrupt restoration of extracellular pH upon reperfusion . There g to MPT . Anotherg to MPT  or succig to MPT  by a posg to MPT  electrong to MPT . Traditig to MPT . Howeverto hours , 9, 32. 2+]Cy elevations consistent with an activation of NCX in the first minutes of “reperfusion” . We will now discuss the possible source of the [Ca2+]Cy increase in reperfusion, and whether findings of our current study can be reconciled with earlier observations, including our own.The findings of the current study are in line with prior observations in whole hearts , 9, 32 tion” see , those dgenation . In our genation . In our 2+ shifts between various cellular compartments during reperfusion. It is universally accepted that a net influx of Ca2+ into cells upon reperfusion is a critical component of I/R injury. However, the sources, the sinks, and the conduits for abnormal Ca2+ transport during reperfusion are not well established. The canonical view implicated reverse-mode NCX as a major source of Ca2+ influx into the cell during reperfusion Cy overload is the prerequisite for hypercontracture, whereas [Ca2+]Mi is the determinant of MPT. Upon SR Ca2+ release, RyR channels situated in close apposition to mitochondria may create a much higher [Ca2+] near the outer opening of the mitochondrial Ca2+ uniporter channel than the bulk [Ca2+]Cy, creating a SR-mitochondrial “Ca2+ cross-talk” which was shown to significantly contribute to the regulation of [Ca2+]Mi Mi . This cr[Ca2+]Mi . It was ntracture or a reg2+, the most reliable blocker of NCX, overall blunted [Ca2+]Cy dynamics and limited the maximum level of [Ca2+]Cy observed during “reperfusion”. This can be considered as the evidence that NCX is a significant source of Ca influx triggering the death sequence in our model. However, there are several arguments against the possibility that reverse-mode NCX operation is the actual mechanism for the massive post-reperfusion increase in [Ca2+]Cy observed in our model. First, this event occurred at variable delays after the restoration of normal extracellular milieu. The activation of reverse-mode NCX during reperfusion is linked to the abrupt restoration of the extracellular pH, which triggers influx of Na+ through sodium-proton exchanger, which in turn activates reverse-mode NCX extruding Na+ and bringing Ca2+ in. This sequence of events is expected to occur within the first minute of “reperfusion”. It is not clear why it would occur 15–20 minutes after reintroduction of oxygen and normalization of pH. Second, Zn2+ is not transported by NCX and does not influence transport of Ca2+ by NCX, and yet in our experiments Zn2+ influx was correlated with Ca2+ influx, and also interfered with Ca2+ influx. Alternative explanation of Ni2+ effect may be related to the competitive hinderance by Ni2+ of a putative pore which presumably also passes Ca2+, Zn2+ and TO-PRO3. At the high concentration necessary to block NCX (5 mM) and having the hydrated radius  smaller.11 Å  sm2+ loading in the absence of a major Ca2+-absorbing entity.Considering the role of SR, depletion of SR with ryanodine/thapsigargin in our experiments did not affect the sequence of critical events. In fact, depletion of SR accelerated the timing of all critical events see . Specula2+ from mitochondria to the cytoplasm at any time of reperfusion studied. In all cases, [Ca2+]Mi elevation followed [Ca2+]Cy elevation at some delay, and after MPT (as indicated by a precipitous loss of ΔΨm) [Ca2+]Mi stayed constant or further increased, with the exception of occasional fluctuations . Howeve2+]Cy. Since treatment attempting to prevent MPT has not been effective [2+]Cy elevation may be an effective strategy to salvage cardiomyocytes in the wake of I/R insult, provided we understand the mechanism of this elevation. We believe our data suggests that the critical Ca2+ influx occurs through a non-selective sarcolemmal pore expanding with time. A revealing observation here is the relationship between uptake of Ca2+, Zn2+, and TO-PRO3. The onset of detectable uptake of these species correlated with their estimated size when dissolved in water. The hydrated radii of Ca2+ and Zn2+ are 4.1 and 4.3 Å, respectively. Stokes radius of TO-PRO3 was not published but the estimated stokes radius of YO-PRO1, a close analog of TO-PRO3, is ~5 Å Cy rise . These channels can be formed by proteins identified as connexins or pannexins.Connexin channels are known to be present at the membrane of many cell types, including cardiac myocytes, and forming structures known as hemichannels . These hTrypanosoma cruzi infection, which mediate Chagas disease Cy increase due to Ca2+ influx through a possibly expanding non-selective transmembrane pore. Once a sufficient [Ca2+]Cy increase occurred, the subsequent [Ca2+]Mi increase followed by MPT are logical and probably unavoidable consequences. The nature of the putative pore remains unknown, but the fact that the abnormal Ca2+ uptake route can be hindered by extracellular Zn2+, and that Ca2+, Zn2+, and TO-PRO3 may pass through the same route, should provide initial guidance for steps towards establishing the molecular identity of the putative pore/channel. In future studies it would be important to reproduce the chronology of critical cellular events observed here in a whole heart model of I/R. If the sequence is confirmed, then the putative sarcolemmal pore mediating the abnormal Ca2+ influx during reperfusion has to be identified and explored as a target for therapies aimed at salvaging myocardium in the wake of an I/R episode.In this study, we established a methodological framework which enabled us to reveal a consistent chronology of critical cellular events during simulated I/R in NRVMs. Despite a highly complex and multi-facetted mechanistic concept of myocardial I/R injury, in our NRVM model cell death upon reperfusion seemed to follow a quite specific path which begins with a pathological [CaS1 Fig(PDF)Click here for additional data file.S2 FigNote a relatively constant level of LAR-GECO1.2 and MitoView633 fluorescence, and a continuous presence of Ca transients throughout 110 minutes of continuous recordings in NRVMs perfused with normal oxygenated solution (recordings started 10 min after placing the NRVM monolayer in the perfusion chamber). There was a trend for a slow increase in LAR-GECO1.2 and MitoView633 signal perhaps reflecting slow mitochondrial Ca loading and energizing in beating cells, and a slow decrease in Fluo-4 signal perhaps reflecting an expected slow leak of the dye from the cell. However, there were no loss of MitoView633, or a step-wise increase in LAR-GECO1.2, or a catastrophic increase followed by full dissipation of Fluo-4 signal, as observed in NRVMs subjected to simulated I/R see .(PDF)Click here for additional data file.S3 FigCells were perfused with normal HEPES solution in which pH was varied between 6 and 8. Data presented as mean ± standard deviation .(PDF)Click here for additional data file.S4 FigA, fluorescence intensity curves during “reperfusion” from a representative cell in the CsA group. All labeling and notations are the same as in B, when quantified, the sequence of critical events in the CsA group (grey bars) is the same as in Control group (black bars). *, p < 0.05 as compared to the timing of previous event in the same group (paired t-test); §, p < 0.05 as compared to the timing of the same event in different group (unpaired t-test). In CsA group, TCaMi  is significantly earlier than in Control group by t-test, but this cannot be explained by the effects of CsA with respect to the MPT pore, and the scientific meaning of this observation remains unclear.(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Figm. Also, the uptake of Ca2+, Zn2+, and TO-PRO3 occur within a minute of each other. We interpret it as the case when the expansion of a putative sarcolemmal pore occurred very quickly, leading to an immediate catastrophe.The time course of fluorescence from 4 different indicators during simulated I/R in a single NRVM, as labeled in the Figure. This cell was selected as an infrequent case where all the critical events were very tightly coupled (2-min time window indicated by light blue), reminiscent of our findings previously published (Ref. 7 in the manuscript). Note that the fluorescence of MitoView633 and TO-PRO3 was recorded in the same channel. However, when segmented separately for the nucleus (grey) and the cytoplasm (red), it was evident that the signal from nucleus, dominated by nucleic acid stain TO-PRO3, started to rise perhaps a minute before the sharp decrease in the cytoplasmic signal, dominated by MitoView633 and reflecting ΔΨ(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.S5 Dataset(XLSX)Click here for additional data file.


We determine that NPC1 inhibition or disease mutations potentiate store-operated Ca2+ entry (SOCE) due to a presenilin 1 (PSEN1)–dependent reduction in ER Ca2+ levels alongside elevated expression of the molecular SOCE components ORAI1 and STIM1. Associated with this dysfunctional Ca2+ signaling is destabilization of neuronal dendritic spines. Knockdown of PSEN1 or inhibition of the SREBP pathway restores Ca2+ homeostasis, corrects differential protein expression, reduces cholesterol accumulation, and rescues spine density. These findings highlight lysosomes as a crucial signaling platform responsible for tuning ER Ca2+ signaling, SOCE, and synaptic architecture in health and disease.Niemann-Pick type C1 (NPC1) protein is essential for the transport of externally derived cholesterol from lysosomes to other organelles. Deficiency of NPC1 underlies the progressive NPC1 neurodegenerative disorder. Currently, there are no curative therapies for this fatal disease. Given the Ca  Following the release of ER Ca2+, two major mechanisms serve to refill depleted stores: (1) sarco-ER calcium ATPases (SERCA) and (2) store-operated Ca2+ entry (SOCE). Classically, the molecular identity of the proteins choreographing SOCE are STIM proteins in the ER membrane and ORAI channels in the PM  in an external 0 mM Ca2+ solution, followed by a 2 mM Ca2+-containing solution. This experimental protocol allows us to analyze the Ca2+ released from the ER (as the area under the curve [AUC] between 150 s and 450 s) and SOCE (AUC >450 s). We found that NPC1I1061T fibroblasts have significantly altered Ca2+ dynamics . To fully underscore that these alterations in Ca2+ signaling were due to NPC1 dysfunction and not to adaptation of this patient cell, we measured Ca2+ dynamics under four additional conditions of NPC1 dysfunction: two additional NPC1 disease patient cell lines (NPC1I1061T/I1061T and NPC1I1061T/P1007A), NPC1−/− cells, and mouse embryonic fibroblasts (MEFs) treated overnight with an inhibitor of NPC1 . Collectively, these data show lysosomal cholesterol is associated with altered Ca2+ dynamics in NPC1 disease and support the hypothesis that NPC1-mediated cholesterol efflux tunes Ca2+ homeostasis.Next, we tested the hypothesis that Cadynamics . Quantitreatment . The conntensity  and Furantensity . Calibra2+ influx into NPC1I1061T cells, we transfected cells with a genetically encoded Ca2+ sensor targeted to the PM  higher intensity of NPC1I1061T fibroblasts, suggesting greater Ca2+ influx across the PM. To test if STIM–ORAI interactions represent the molecular identity of this differential Ca2+ flux, we treated cells with an inhibitor of ORAI1 channels, AnCoA4  conductance from NPC1I1061T fibroblasts  and suggests enhanced resting ORAI1 conductance in NPC1 disease cells.To determine the molecular identity of the enhanced Ca, AnCoA4 . Measure2+ conductance in NPC1I1061T disease we performed Western blot analysis and superresolution nanoscopy. Western blot analysis revealed significant increases in total protein levels of ORAI1 (33% ± 11%) and STIM1 (20% ± 4%) from NPC1I1061T fibroblasts relative to healthy patient cells , which also appeared to be larger than their healthy cell counterparts . These data suggest that STIM1 and ORAI1 are not only preassociated in resting NPC1I1061T fibroblasts but also may form puncta more quickly following depletion of intracellular Ca2+ stores.Given the enhanced Ca+ influx  in NPC1I compare . Quantit compare . Next, wI1061T fibroblasts, we next tested if this occurs as a consequence of reduced ER luminal Ca2+ levels  between CFP and YFP fluorophores to increase the FRET ratio , while NPC1I1061T cells at a significantly lower ER Ca2+ concentration of ∼170 µM . To confirm this observation, we measured the total Ca2+ released from intracellular stores following application of the Ca2+ ionophore ionomycin (in the absence of extracellular Ca2+). Experiments from Fluo-4–loaded cells treated with ionomycin (2.5 µM) revealed a significant reduction in the pool of Ca2+ mobilized to the cytoplasm in NPC1I1061T fibroblasts  Ca2+ buffering proteins, (2) Ca2+ reuptake pumps, and (3) rate of intrinsic Ca2+ leak. We hypothesized that loss of NPC1 function may alter the protein levels underlying one or more of these parameters. To test this hypothesis, we performed Western blots for several proteins that may alter ER Ca2+ load. We began with SERCA, the ER’s primary pathway of refilling and maintenance of luminal Ca2+. Western analysis of SERCA isoforms 2A/2B revealed a significant increase of 29% ± 9% in NPC1I1061T fibroblasts  in the absence of extracellular Ca2+. Decreases in FRET ratio following inhibition of the SERCA pump represent Ca2+ leaking from the ER , overexpression of the catalytically inactive PSEN1D257A (accumulates holoprotein form) decreases ER Ca2+ levels and increases the rate of Ca2+ leak from the ER , and PSEN−/− cells have increased TG-mediated Ca2+ release . These data align with PSEN1’s putative role as an ER Ca2+ leak channel. As a final piece of evidence that PSEN1 mediates the reduction in ER Ca2+ found in NPC1 disease, treating PSEN−/− cells with UA did not alter Ca2+ dynamics . These findings suggest that changes in the expression of PSEN1 influence ER Ca2+ concentrations and contribute to reduced ER Ca2+ levels in NPC1 disease.An increase in PSEN1 expression and/or activity is predicted to result in a leakier ER, which may account for the observed decrease in resting ER Caand SOCE . 48-h trand SOCE  and TG-mand SOCE  elevatiocontrols . Further2+ have been shown to adapt NPC1 proteostasis, increase the functional amount of NPC1 in lysosomal membranes, and rescue the aberrant storage of cholesterol  is responsible for the enhanced expression of key Ca2+ handling proteins, including PSEN1, in NPC1 disease , while treatment of NPC1I1061T fibroblasts with PF-429242 significantly increased the ionomycin-mediated elevation in cytoplasmic Ca2+  differential expression of key Ca2+ signaling and SOCE regulating proteins, (2) a reduction in luminal ER Ca2+, (3) an increase in resting STIM1–ORAI1 interactions, and (4) enhanced SOCE, leading to (5) increased cytoplasmic Ca2+ concentrations. Importantly, we also report that inhibition of NPC1 results in a decrease in neuronal spine density. Our experimental evidence suggests that these changes are mediated through the SREBP pathway. This hypothesis is supported by data showing that SREBP pathway inhibition normalizes mRNA and protein expression profiles back to normal ranges and, in parallel, rescues Ca2+ signaling in NPC1I1061T fibroblasts. Collectively, these data suggest that lysosomal NPC1 protein is a key gatekeeper in not only cholesterol metabolism but also Ca2+ homeostasis and synaptic architecture.Mechanisms that link cholesterol metabolism to regulation of cellular Canditions . Here, w2+ handling proteins with important roles in Ca2+ homeostasis and SOCE can have their protein expressions altered via SREBP. Of note, we find that PSEN1 (discussed below), STIM1, and ORAI1 can have their mRNA and protein abundances increased by SREBP in circumstances of reduced NPC1 cholesterol egress (such as NPC disease) to decrease ER Ca2+ concentrations and subsequently increase SOCE.SREBPs are a family of transcription factors often described as master regulators of lipid homeostasis through their ability to acutely sense the concentration of ER cholesterol and maintain its concentration within a precise physiological range . SREBPs 2+ homeostasis in NPC1 disease. Our evidence for the involvement of PSEN1 is robust: (1) PSEN1 is elevated in NPC1I1061T patient cells, (2) PSEN1 siRNA is sufficient to abrogate the changes in ER luminal Ca2+ and SOCE, (3) overexpression of the holoprotein recapitulates NPC1 ER Ca2+ loss, and (4) PSEN1−/− cells do not exhibit UA-dependent changes in Ca2+ signaling. PSEN1 has been proposed to act as low-conductance, passive ER Ca2+ leak channel, with mutations in the PSEN1 protein leading to disruption or abolishment of ER Ca2+ leak activity in familial Alzheimer’s disease (3R) to prolong IP3R opening and Ca2+ leak permeability  that facilitates continuous interactions between STIM1 and ORAI, leading to constitutive Ca2+ entry  were purchased from Coriell Institute. Additional fibroblast cell lines derived from patients, NPC1I1061T, I1061T and NPC1I1061T, P1007A, were generously provided by Dr. Forbes D. Porter . Fibroblasts were cultured in MEM supplemented with 2 mM L-glutamine, 15% non–heat-inactivated FBS, and 0.2% penicillin/streptomycin. Cells were passaged twice weekly and incubated in 5% CO2 at 37°C. PSEN+/+ (WT) and PSEN1/PSEN2 double-knockout (PSEN−/−) MEFs were a generous gift from Drs. David Kang, Angels Almenar, and Lawrence S.B. Goldstein . WT, NPC1−/−, and SCAP−/− CHO cells were kindly provided by Daniel Ory . MEFs, CHO cells, and tsA201 cells were cultured in DMEM supplemented with 10% FBS and 0.2% penicillin/streptomycin. Hippocampal Neurons were isolated from rats at gestation day 18. Neurons were cultured in Neurobasal  supplemented with B27 , Glutamax , 5% FBS, and 0.2% penicillin/streptomycin. On DIV 7, cytosine-D-arabinofuranoside  at 1:1,000 was added to neuronal cultures to inhibit astrocyte growth.Fibroblast cell lines derived from an apparently healthy male (GM05659) and a male patient with the NPC1I1061T knock-in mice .C57BL/6 WT mice and NPC1-in mice  were kinCells were washed with PBS and fixed in 3% PFA and 0.1% glutaraldehyde for 10 min at 21°C. Cells were then incubated with sodium borohydrate (10 mM) for 5 min and subsequently stained with filipin (3 mg/ml in PBS) for 2 h at room temperature or overnight at 4°C. Cells were excited using a 405-nm LED and light collected using a Plan-Apochromat 63×/1.40 oil-immersion lens and a Zeiss 880 Airyscan microscope. Images were acquired in PBS solution at room temperature (21–23°C) using Zen software.2+ Ringer’s solution  with 0.1% pluronic acid to permeabilize cells. Cells were then moved to a Fluo-4–free Ringer’s solution to deesterify for 30 min. Following deesterification, Fluo-4–loaded cells were excited with a 488-nm laser and the resulting fluorescence monitored using an inverted microscope with a Plan-Apochromat 63×/1.40 oil objective, connected to an Andor W1 spinning-disk confocal with a Photometrics Prime 95B camera. Images were acquired every 2–5 s in Ringer’s solution at room temperature (21–23°C) using Micromanager software. Hippocampal neurons were imaged at DIV 21; Ca2+ measurement regions of interest in neurons were restricted to dendritic spines. SOCE was induced by initially perfusing cells in 2 mM Ca2+ solution; cells were then perfused in Ca2+-free solution , followed by 500 nM TG in Ca2+-free solution. After 5 min of TG incubation, cells were exposed to 2 mM Ca2+ solution to measure resulting SOCE. Resting Fluo-4 intensities were measured by normalizing to the minimum fluorescence in an ionomycin-containing Ca2+-free solution. Ca2+ dyes and the D1-ER FRET probe were calibrated using standard empirical calculations as previously described (Cells were incubated for 30 min in 5 µM Fluo-4 in 2 mM Caescribed .The following cDNA plasmids were used in this study (in order of appearance): pGP-CMV-GCaMP6s-CAAX , mCherry-STIM1 , ORAI1-GFP , D1-ER-CFibroblasts were fixed in 4% PFA for 15 min followed by permeabilization with 0.1% Triton X-100 for 10 min, blocked with 4% FBS for 1 h at 21°C, and stained with rabbit polyclonal anti-ORAI1  or rabbit polyclonal anti-STIM1  overnight at 4°C. Fibroblasts were then washed and incubated for 1 h in Donkey Anti-Rabbit Alexa Fluor 647  secondary antibody. Superresolution imaging was performed as previously described  using a g at 4°C. Concentration of protein lysate was quantified using Pierce BCA protein assay kit . Protein samples were resolved in 4–12% Bis-Tris gels under reducing conditions. Proteins were transferred from gels onto 0.2-µm polyvinylidene difluoride membranes  using a Mini-Bolt system . using rabbit monoclonal anti-NPC1 , rabbit polyclonal anti-ORAI1 , rabbit polyclonal anti-STIM1 , rabbit polyclonal anti-STIM2 , mouse monoclonal anti-SERCA2 , mouse monoclonal β-actin  Calreticulin , mouse monoclonal anti-SREBP1 , rat monoclonal anti-PSEN1 N-terminal fragment , and mouse monoclonal anti-PSEN1 C-terminal fragment . Blot bands were detected using fluorescent secondary antibodies goat anti-rabbit 680RD , goat anti-Mouse 800CW , and goat anti-rat 680 . ImageJ was used to calculate the fluorescent density of each band. Abundance of proteins were normalized to β-actin; treatment group protein expression was normalized to protein abundance from the appropriate controls.Protein from cultured cells was harvested using RIPA Buffer  with complete, Mini, EDTA-free protease inhibitor  for 25 min at 4°C. Samples were sonicated, and postnuclear supernatant was isolated by centrifugation for 25 min at 13,200 mRNA was isolated from fibroblasts using RNeasy Plus Mini Kit . cDNA was synthesized from 1 µg of total RNA using AffinityScript cDNA Synthesis Kit . Quantitative real-time PCR was performed using PowerUp SYBR Green Master Mix . C-terminal fragment values were normalized to β-actin. The following primer sequences were used: β-actin forward, 5′-AGAGCTACGAGCTGCCTGAC-3′; β-actin reverse, 5′-AGCACTGTGTTGGCGTACAG-3′; STIM1 forward, 5′-AGTCACAGTGAGAAGGCGAC-3′; STIM1 reverse, 5′-CAATTCGCGAAAACTCTGCTG-3′; ORAI1 forward, 5′-GACTGGATCGGCCAGAGTTAC-3′; ORAI1 reverse, 5′-GTCCGGCTGGAGGCTTTAAG-3′; PSEN1 forward, 5′-GAAGCTCAAAGGAGAGTATCCA-3′; PSEN1 reverse, 5′-CCCTAGATGACTGTCCCTCT-3′; PSEN1 forward, 5′-GAAGCGTATACCTAATCTGGGA-3′; and PSEN1 reverse, 5′-CACAGAAAACAAAGCCTCTTGAG-3′.Cultured hippocampal neurons were initially washed with PBS and fixed at DIV 21 in 4% PFA for 15 min followed by permeabilization with 0.1% Triton X-100 for 10 min. The neurons were then blocked with 4% FBS for 1 h at 21°C and stained with rabbit polyclonal anti-MAP-2  and mouse monoclonal anti-PSD-95  overnight at 4°C. Neurons were then washed and incubated 1 h in goat anti-rabbit Alexa Fluor 647  and Goat anti-mouse Alexa Fluor 555  secondary antibodies and subsequently imaged in PBS using a Plan-Apochromat 63×/1.40 oil DIC M27 objective . Images were acquired at room temperature (21–23°C) using Zen software.I10161T mice between 70 and 90 d old. Golgi staining was performed using an FD Rapid GolgiStain Kit . Slices were imaged with transmitted light using a 63×/1.40 oil objective connected to an Andor W1 spinning-disk confocal coupled to a Photometrics Prime 95B camera; only Purkinje neurons were assessed for imaging. ImageJ was used to develop representative binary skeletons for quantitative analysis of dendritic spine branching.Cerebellums were isolated from paired age- and sex-matched WT and NPC1UA (Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 1 µM. AnCoA4 was dissolved in DMSO and used at a final concentration of 50 µM. PF-429242 (Tocris) was dissolved in DMSO and used at a final concentration of 500 nM. TG (Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 500 nM. Ionomycin (Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 2.5 to 5 µM. ML-SA1 (Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 10 µM.t tests were conducted to determine significance; P values <0.05 were considered as statistically significant. P values and number of cells are detailed in each figure or figure legend.Microsoft Excel, GraphPad Prism, and IGOR PRO were used to analyze all data. ImageJ was used to process and analyze images. Parametric Student’s 2+, and SOCE across multiple models of NPC1 disease. Fig. S2 shows enhanced ICRAC current and STIM1/ORAI1 activation in NPC1 disease fibroblasts. Fig. S3 demonstrates the involvement of PSEN1 as a regulator of ER Ca2+ levels in healthy and NPC1 disease cells. Fig. S4 shows the involvement of the SREBP pathway in mediating transcriptional and functional changes in Ca2+ signaling in NPC1 disease. Fig. S5 shows the effect of SREBP inhibitor PF-429242 in appropriate control cells.Fig. S1 shows impaired cholesterol distribution, ER Ca


Leukemia is one of the most common cancers in patients of reproductive age. It is well known that chemotherapy, used as anti-cancer therapy, adversely affects male fertility. Moreover, the negative effect of leukemia on sperm quality, even before chemotherapy treatment, has been reported. However, the mechanisms behind this disease’s effect on sperm quality remains unknown. In this study, we examine the direct effect of leukemia and chemotherapy alone and in combination on sperm parameters and male fertility. For this, we developed an acute myeloid leukemia (AML) mouse model (mice were treated with AML cells C1498 and developed leukemia); these mice then received cytarabine chemotherapy. Our findings reveal a significant reduction in sperm concentration and motility and a significant increase in abnormal morphology and spontaneous acrosome reaction of the sperm following AML and chemotherapy treatment, alone and in combination. We also found a reduction in male fertility and the number of delivered offspring. Our results support previous findings that AML impairs sperm parameters and show for the first time that AML increases spontaneous acrosome reaction and decreases male fertility capacity and number of offspring. Leukemia is one of the most common cancers of patients of reproductive age. Treatment of these patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) has known side effects on the reproductive system that may lead to permanent male sterility and, in a large proportion of patients, to oligospermia or azoospermia . This efRecently, it was reported that sperm parameters from leukemia patients were impaired even before anti-cancer treatments ,13,14,15Systemic and local effects of a cancer can impact fertility, although the exact mechanism has not yet been clarified . LeukemiIn the present study, we used a mouse model, in which mice were treated with acute myeloid leukemia (AML) cells and developed leukemia, to examine the direct effect of AML and chemotherapy (cytarabine)—alone and in combination—on sperm parameters and male fertility.Our results show that injection of AML cells (C1498) led to gradual death of the treated mice beginning two weeks after injection . There was 60% survival after three weeks and 0% survival (100% death) after four weeks . InjectiOur results showed a constant concentration of sperm in the control group mice within four weeks of the experiment. However, injection of AML cells (C1498) significantly decreased the sperm concentration after 1–3 weeks compared to the control . FurtherOur results show constant sperm motility in the control group mice within the four weeks of the experiment . HoweverSperm with normal morphology were considered those with a normal head, neck, and tail A–I. HoweIn order to understand whether the fertility declines of the leukemic mice were related only to poor quality of sperm parameters  or also due to the inability of the sperm to undergo the necessary processes of capacitation and acrosome reaction in order to fertilize the egg, we performed an acrosome reaction test A and evaAfter we demonstrated that leukemia has an effect on sperm parameters, we examined whether there was an effect of the above treatment on male mouse fertility. Our results showed that three weeks after injection of C1498, male fertility was significantly reduced (50%) compared to the control A. HoweveIn the present study, we showed that the AML-developed animal model led to the death of the mice within four weeks and that treatment of these mice with cytarabine chemotherapy extended their life to eight weeks (doubled their life). Using this model, we demonstrated that AML directly affected the different sperm parameters . These results are in harmony with previous studies in cancer and AML patients ,25,26,27In conclusion, our results support previous findings that AML disease impairs sperm parameters, and we show, for the first time, its role in increasing spontaneous acrosome reaction and its possible involvement in decreasing male fertility capacity. We also suggest possible different roles for AML and cytarabine in the generation of sperm, in spermiogenesis in the testis, and in sperm motility and acrosome reaction in the epididymis.Thus, AML alone can affect male infertility, and the addition of chemotherapy will increase this pathology. Understanding the mechanisms of the action of AML and chemotherapy may lead to the development of future therapeutic strategies for male infertility in cancer patients. This study was performed in accordance with the Guiding Principles for the Care and Use of Research Animals Promulgated by the Society for the Study of Reproduction. It was confirmed by the Ben-Gurion University Ethics Committee for Animal Use in Research (IL-70-11-2016). Six-week-old C57/black mice were purchased from Envigo Laboratories, Jerusalem, Israel. 2, at 37 °C. Then, 105 cells/100 µL were injected intravenously per mouse.Murine C1498 (TIB-49) AML cells were purchased from American Type Culture Collection . They were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (0.1 mg/mL), and 10 mM HEPES (pH = 7.4) in a humidified atmosphere of 95% air and 5% COCytarabine (powder) was purchased from Sigma  and dissolved in sterile PBS. Mouse weight was evaluated before injection, with 100 µL of Cyt (3 mg/kg) injected intraperitoneal into each mouse. The injections were performed 24 h after the injection of C1498 cells, 3 times every 12 h  were examined for concentration and motility using a Makler counting chamber. For sperm concentration determination, 10 µL of each sample was transferred to a chamber, and cells were counted at a total microscope magnification of ×400. For sperm motility evaluation, only motile cells were counted without differentiation of motility type.Semen smears were made and stained by use of Quik Stain . Evaluation of sperm morphology was performed using an upright microscope under a magnification of ×1000 (using immersion oil). This was performed according to the WHO criteria .2O at 5-min intervals, and mounted with DAPI  to stain cell nuclei. For each experiment, at least 200 cells per slide (on duplicate slides) were evaluated . Cells with green staining over the acrosomal cap were considered acrosome intact; those with equatorial green staining or no staining were considered acrosome reacted.The percentage of acrosome-reacted sperm was determined microscopically on air-dried sperm smears using fluorescein conjugated peanut agglutinin (FITC-PNA) . An aliquot of sperm cells was smeared on a glass slide and allowed to air dry. The sperm were then permeabilized by methanol for 15 min at room temperature, washed three times at 5-min intervals with PBS, and air dried. Thereafter, the slides were incubated with FITC (50 µg/mL in PBS) for 30 min, washed twice with HMale mice were injected with PBS (control), C1498 cells, cytarabine alone, or a combination of both cytarabine and C1498. Two weeks after treatment, a single male from each treatment was mated with 2 females (8 weeks old). After two weeks, the females were each separated in different cages. The number of pregnant females and the number of offspring for each female was examined after 4–5 weeks of separation.


Aim: We investigated the underlying mechanisms in atrial fibrillation (AF) associated with R33Q mutation and Ca2+-triggered activity.Methods and Results: We examined AF susceptibility with intraesophageal burst pacing in the sarcoplasmic reticulum (SR) Ca2+ leak model calsequestrin 2 R33Q (Casq2R33Q/R33Q) mice. Atrial trigger appeared in R33Q mice but not WT mice . AF was induced by 25 Hz pacing in R33Q mice . The mice were given 1.5 mg/kg isoproterenol (Iso), and the incidences of AF increased . Electrophysiology experiments and the recording of intracellular Ca2+ indicated significant increases in the Ca2+ sparks , intracellular free Ca2+ , Ca2+ wave , transient inward current (ITi) , and oscillation in membrane potentials  in the R33Q group, but there was no significant difference in the L-type calcium current. These effects were enhanced by Iso, and the inhibition of calmodulin-dependent protein kinase II (CaMKII) by 1 μM KN93 reversed the effects of Iso on Ca2+ sparks , intracellular Ca2+ , Ca2+ wave , ITi , and oscillation in membrane potential . The reduction of ryanodine receptor 2 (RyR2) stable subunits  rather than RYR2 and the increase in CaMKII, phosphor-CaMKII, phosphor-RyR2 (Ser 2814), SERCA, and NCX1.1 was reflected in the R33Q group.Conclusion: This study demonstrates that the increase in spontaneous calcium elevations corresponding to ITi that may trigger the oscillation in membrane potentials in the R33Q group, thereby increasing the risk of AF. The occurrence of spontaneous calcium elevations in R33Q atrial myocytes is due to the dysfunction of RyR2 stable subunits, CaMKII hyperactivity, and CaMKII-mediated RyR phosphorylation. An effective therapeutic strategy to intervene in Ca2+-induced AF associated with the R33Q mutation may be through CaMKII inhibition. In a canine atrial tachyarrhythmia model, it was reported that intracellular Caafter AF . The inc content .2+ is mainly released from the sarcoplasmic reticulum (SR) by the ryanodine receptor 2 (RyR2) channel, but the number of RyR2 channels in patients with chronic AF is significantly reduced or unchanged  or delayed afterdepolarizations (DADs). It is well known that afterdepolarizations are responsible for atrial-triggered activity and are associated with various Cacurrents . The L-tcurrents , whereascurrents . More reR33Q/R33Q mice have mutated Casq2 genes that provide an SR Ca2+ leak model for this study. Firstly, we organized an in vivo study to check the AF susceptibility in this animal model with an SR Ca2+ leak. Secondly, we planned to demonstrate that Ca2+ changes were associated with the changes in Ca2+-sensitive currents and the oscillation in membrane potentials. Finally, we investigated the effects of interventions including the Iso and calmodulin-dependent protein kinase II (CaMKII) on Ca2+ levels, Ca2+ sensitive currents, and oscillation in membrane potentials.Casq2R33Q/R33Q mouse model was provided by Dr. Nicoletta Rizzi , and the genotype characteristics were determined. Mouse housing and care were provided by PLA General Hospital Laboratories . All our experimental procedures were approved by the Ethics Committee of PLA General Hospital and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health .The Casq2in vivo study. Mice were ECG-monitored for 6 h in our observation unit, and a premature atrial trigger was defined as >3 consecutive supraventricular beats. Up to 3 s, 25 Hz intraesophageal burst pacing was applied to assess the susceptibility to AF. AF was defined as an irregular atrial tachycardia (>800 bpm) lasting for at least 5 s immediately after a burst pacing cycle. After the mice were given 1.5 mg/kg Iso by intraperitoneal injection, the induction of AF was recorded again at 25 Hz atrial burst pacing.Surface II-lead ECG was used to obtain ECG recordings in the 2+-free Tyrode’s solution immediately after removal. The Ca2+-free Tyrode solution with pH of 7.40 ± 0.05 (nmol/L) was composed of NaCl 113, KCl 4.7, KH2PO4 0.6, Na2HPO4 0.6, MgSO4 1.2, NaHCO3 12, KHCO3 12, HEPES 10, taurine 30, glucose 5, and BDM 10. First, isolated hearts were perfused for 4–5 min with Tyrode buffer solution. Then, isolated hearts were perfused for 11–14 min with Tyrode’s solution containing 1 mg/ml collagen II and 0.25 mg/ml trypsin. The left atria were removed and cut into pieces in enzyme solution to single atrial cells. Then, isolated atrial cells were stored in low Ca2+ (0.5 mM) Tyrode’s solution. Atrial myocytes were moved into a 1.8 mM Ca2+-containing Tyrode’s solution for measuring intracellular Ca2+ and membrane currents/potentials.The Langendorff system was used to prepare single atrial myocytes. The hearts of mice were perfused in Ca2+ imaging was used to detect intracellular Ca2+ changes by recording Ca2+ sparks and Ca2+ waves as well as the times to Ca2+ peak and Ca2+ decay. Fluo-4 acetoxymethyl (AM) ester dye (Thermo Fisher Scientific) is a non-ratiometric Ca2+ indicator that is commonly used with 488 nm excitation. Fluo-4-loaded atrial myocytes were exposed in different solutions including a control solution, solution with 1 μM Iso (Sigma), or solution with both 1 μM KN92 (Sigma) and 1 μM KN93 (Sigma) to assess the different effects of these solutions on Ca2+ dynamics. After de-esterification, a laser-scanning confocal microscope  was used to record spontaneous Ca2+ release events (SCaEs) and the constant rate of Ca2+ cycling under different conditions.Confocal Ca2+ level was quantified with Fura-2 (a ratiometric Ca2+ indicator) /(Rmax–R). A different fluorescence intensity was observed for Fura-2 at 340 and 380 nm wavelength (F340 and F380), and R = F340/F380, where Rmin is the value of R in the Ca2+-free solution, Rmax is the value of R when the indicator is saturated with Ca2+, and sf,2/sb,2 is simply the ratio of the measured fluorescence intensity in Ca2+-free and Ca2+-bound solutions; Kd = 224 nM. To measure spontaneous Ca2+ waves, the atrial cells were paced at 1 Hz for 10 s, and then were monitored for 10 s. After the 1 Hz pacing stimulation stopped, the external solution was rapidly switched to a free Na+ and Ca2+ solution to inhibit Ca2+ transportation by NCX; this was followed by 10 s of monitoring to record the Ca2+ spark frequency and another 10 s of monitoring to record the intracellular baseline Ca2+  . A [Ca2+ine Ca2+ .Electrophysiology experiments were used to study the relationship between ICa-L or ITi and the action potential. Currents (ICa-L and ITi) and action potentials were recorded using the whole-cell patch-clamp technique with an Axon-700B amplifier (Axon Instruments). The action potentials and the action potential amplitudes were measured under the current clamp mode with 2.5 ms/1 nA depolarizing pulses. The whole-cell currents including ICa-L and ITi were obtained under the voltage clamp mode. ICa-L was recorded with a 10 mV voltage step from a holding potential of -40 mV to +70 mV. ITi was recorded with an increment of 10 mV voltage potential from -100 mV to +10 mV. These recordings were used to create the current–voltage relationship, steady state activation (SSA), or steady-state inactivation (SSI) curves (SSA left shift indicates the activation of ICa-L and SSI right shift indicates the inactivation of ICa-L). The electrophysiology data were analyzed by Clampfit version 10.4 (Axon Instruments) and Origin . Triggered activity was defined as an unstimulated action potential arising from a DAD or an EAD.R33Q/R33Q mice and WT mice. Protein samples were separated on 4–8 and 12–15% gels using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. Western blot was performed under one of the unique antibodies including anti-RyR2 (abcam), anti-phospho-RyR2 (Badrilla), anti-CaMKII (abcam), anti-phospho-CaMKII (abcam), anti-CASQ (ABR), anti-triadin (ABR), anti-junctin (ABR), anti-NCX1.1 (abcam), and anti-SERCA (abcam), and then incubated overnight at 4°C for specialty binding. Chemiluminescent detection was performed with substrate reagents . Densitometric analysis of Western blot data was performed using ImageJ software.Western blot was used to investigate changes in the SR membrane calcium channel receptor RyR2 and its subunits. Total protein was obtained from 12-week-old Casq2post hoc analysis or student’s t-test was used for comparison between groups. Fisher’s exact test was used to compare the incidences of atrial tachyarrhythmia and oscillation in membrane potentials (DADs/EADs) between the groups. A P-value less than 0.05 was considered statistically significant.Statistical analysis was performed using SPSS v.17 software. The data are expressed as the mean ± SD. One-way ANOVA with Bonferroni in vivo at the baseline; this showed that the atrial-triggered activity appears in 5 of 29 (17.24%) Casq2R33Q/R33Q mutation mice in contrast to 0 of 32 (0.00%) WT mice (P < 0.05). The episodes of AF and atrial flutter were labeled together as AF, and AF was induced by 25 Hz burst pacing in 14 of 29 (48.27%) Casq2R33Q/R33Q mutation mice in contrast to 2 of 32 (6.25%) WT mice (P < 0.01). After an intraperitoneal injection of 1.5 mg/kg Iso, the incidences of AF were increased to 19 of 29 (65.51%) Casq2R33Q/R33Q mutation mice compared with 3 of 32 (9.21%) WT mice (P < 0.01)   and spontaneous Ca2+ waves  as compared to WT atrial myocytes. After treatment with 1 μM Iso, Ca2+ waves , Ca2+ sparks , and intracellular Ca2+  were significantly increased only in the R33Q group. Furthermore, the time to Ca2+ peak was shortened by Iso , and the decay time of Ca2+ waves (reflecting the time of Ca2+ restoration by SR) was significantly lowered by Iso  in the R33Q group compared with the WT group , Ca2+ sparks , and intracellular baseline Ca2+  were significantly reduced. KN-92 was used as a control compound to elucidate the inactivity of KN-93. Casq2R33Q/R33Q atrial myocytes with 1 μM KN92 still developed Ca2+ sparks at a higher level  as well as a higher intracellular baseline Ca2+   on the Iso-induced-SCaE in the Casq2R33Q/R33Q and WT groups , and ICa-L was increased in both groups by 1 μM Iso, with a more significant increase in the Casq2R33Q/R33Q group . There was no significant difference in the SSA curve of ICa-L but a slight shift to the right in the SSI curve by Iso in the R33Q mutation group , indicating that ICa-L was increased by Iso without a significant change in the L-type calcium channel function in the R33Q mutation group  and a dramatic increase in ITi after exposure to Iso  in the R33Q group compared with the WT group. However, the effects of Iso were reversed by 1 μM KN93   is the main component of Na–Ca exchange current (INCX). We found that there was a significant increase in the ITi density  increased in the Casq2R33Q/R33Q group as compared to the WT group . This difference was further promoted by 1 μM Iso in the Casq2R33Q/R33Q group compared with the WT group , and with a significant decrease in the proportion of oscillation in membrane potentials by 1 μM KN93 in the Casq2R33Q/R33Q group . There was no significant difference in the resting membrane potential (RMP) between the two groups with or without Iso , indicating that no oscillation in membrane potentials was caused by basal RMP instability , but no significant changes in the levels of RyR2 or SERCA  in the R33Q group. In contrast, there was an increase in the total form of CaMKII and the phosphoryl form of CaMKII or RYR2 . In addition, atrial NCX1.1 protein expression increased in the R33Q group   Figure .R33Q/R33Q mice were more susceptible to AF, mediated by the increase in SCaEs characterized by Ca2+ sparks and Ca2+ waves. SCaEs might be responsible for afterdepolarizations (DAD/EAD) via a change in ITi in atrial cells from Casq2R33Q/R33Q mice. CaMKII inhibition reversed the effects of Iso on SCaE and oscillation in membrane potentials and reduced atrial-triggered activity, and hence may be a promising therapeutic strategy for AF.In this study, we intended to investigate the effect of Casq2 mutation R33Q on AF. Our major findings are that the Casq22+-sensitive current in Casq2R33Q/R33Q intact atrial cells increases the risk of AF.(1) The increase in the SCaE and the Ca2+leak was found in atrial myocytes of chronic AF patients  The molecular consistency of the RYR2 complex changes for SCaEs in the Casq22+ release  are involved in Ca release . A reduc release . Moreove release . It is p release . Franzin2+-handling proteins has been considered as the molecular basis for the onset of AF  Disordered Ca2+ release and the time to Ca2+ peak. Shortening of the decay time of Ca2+ release indicates an increased SERCA2a function  is enhanced in R33Q hearts. in vivo study of Casq2R33Q/R33Q mice, most other results were drawn from in vitro studies. It is unknown whether these changes in Ca2+, ITi, and oscillation in membrane potentials play the same roles in the development of AF in live animals or humans. We were unable to separately investigate EADs and DADs or their different roles in the development of AF due to the constant occurrence of EADs with DAD in our study. We were also unable to study outward currents during phases 3 and 4 of the action potential. Our future direction is to further clarify the individual role of Ca2+-induced EADs and DADs in the development of AF.Although a high susceptibility to AF was demonstrated in this The investigation complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23 revised 1996).J-cZ and H-lW conceived and designed the research, performed the acquisition, analyses, and interpretation of the data, obtained funding, supervised the work, drafted the manuscript, and critically revised the manuscript for important intellectual content. QC, X-tX, TZ, and G-jX performed the acquisition, analyses, and interpretation of the data, and drafted the manuscript. YD, CZ, and LY drafted the manuscript and critically revised the manuscript for important intellectual content. YL and P-lZ conceived and designed the research, obtained funding, supervised the work, and critically revised the manuscript for important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.