Treatment with a small molecule inhibitor of neddylation, MLN4924, results in the activation of Ataxia Telangiectasia Mutated (ATM). ATM phosphorylates Mdm2, converting Mdm2 to a ubiquitinating enzyme which leads to the destabilization of MdmX. These data show how distinct signaling pathways engage neddylating or ubiquitinating activities and impact the Mdm2-MdmX axis.Mouse double minute 2 (Mdm2) and MdmX dimerize in response to low levels of genotoxic stress to function in a ubiquitinating complex, which signals for destabilization of p53. Under growth conditions, Mdm2 functions as a neddylating ligase, but the importance and extent of MdmX involvement in this process are largely unknown. Here we show that when Mdm2 functions as a neddylating enzyme, MdmX is stabilized. Furthermore, we demonstrate that under growth conditions, MdmX enhances the neddylation activity of Mdm2 on p53 and is a substrate for neddylation itself. Importantly, MdmX knockdown in MCF-7 breast cancer cells resulted in diminished neddylated p53, suggesting that MdmX is important for Mdm2-mediated neddylation. Supporting this finding, the lack of MdmX in transient assays or in  Post-translational modifications such as ubiquitin, sumo, nedd8, and ISG15, are essential for a myriad of fundamental processes in the cell. These modifications can induce conformational changes to recruit binding partners, prevent or enhance interactions, or result in changes in cellular localization. E1, E2 and E3 enzymes are responsible for conjugating these covalent modifications to proteins. Mouse double minute 2 (Mdm2) is one of over a thousand E3 ubiquitin ligases. Mdm2 is an oncoprotein that is elevated in 10% of all human cancers and 40-80% of high-grade human tumors . After confirmation of Mdm2 knockout  and is unable to interact with Mdm2 . Figure The Mdm2-mediated decrease of MdmX could be due solely to the inhibition of neddylation by MLN4924. Alternatively, we postulated that MLN4924 could be converting Mdm2 to be a ubiquitinating enzyme as MLN4924 was previously reported to activate ATM . ATM hasAs p53 is a substrate for ATM, we wanted to confirm that ATM was activated by MLN4924. Upon MLN4924 treatment, pS15 of p53 (an established ATM phosphorylation site) was detected, thus indicating ATM activation Figure . This phPreviously, we have shown that increased p53 levels do not necessarily result in increased activity. To assess p53 activity, we investigated the effect of MLN4924 treatment on p21 and found that p21 was also increased with MLN4924 treatment Figure . This reLastly, we conducted fluorescence-activated cell sorting to determine if there were any aberrations in the cell cycle. Treatment of actively growing MCF7 cells with MLN4924 for 18 h resulted in a higher proportion of cells in the G1 phase and less cells in the G2 phase of the cell cycle Figure . DisruptIt is well established that Mdm2 and MdmX can form heterodimers via their RING finger domains , 13–15. Furthermore, since MdmX levels of all mutants were increased under conditions of growth, signaling through those pathways dependent on the wild-type residues are unlikely to be involved. Although the C464S mutant was tested Figure , it has There has been considerable interest in the development of therapeutics that modulate the regulation of kinase pathways and limited development on those that impact other post-translational modifications. One such small molecule, MLN4924, is a specific inhibitor of the NEDD8-activating enzyme in Phase 2 clinical trials. However, its effect on the regulation of p53 has not been fully explored. There have been reports that MLN4924 can elicit DNA stress type responses –38 in adThe majority of studies on the Mdm2-MdmX interaction have been conducted under conditions of DNA damage. We have shown that c-Abl phosphorylation of Mdm2 results in Mdm2-MdmX complex formation and ubiquitination of MdmX . Other sAnother component that could be contributing to MdmX degradation under DNA damaging conditions is the activation of NEDP1. This isopeptidase is induced by chemotherapy and can de-neddylate Mdm2 . It is cThe seminal finding of this study is the contribution of MdmX to the neddylation of p53. Initial work suggested the involvement of MdmX as part of a neddylating complex and reported that while co-expression of MdmX did not significantly affect Mdm2-mediated neddylation of p53, it was sufficient to rescue Mdm2 mutants that had impaired neddylation activity in transient transfections . HoweverTaken together, our results show that during growth conditions when Src is active, Mdm2 and MdmX are in a complex together to neddylate p53 and MdmX. Inhibition of this activity with MLN4924 results in ATM activation, MdmX degradation, and increased p21 Figure . This stThe shMdmX, MdmX, and ΔC MdmX constructs were obtained . Lentivi2 in DMEM high glucose and 8-10% fetal bovine serum. Transient transfections were performed using 1:1 DNA to PEI (H1299). Equal amounts of DNA were transfected into cells except for neddylation assays (see figure legend). Unless indicated, cells were treated with 0.3 μm MLN4924 (Life Sensors), 2 mM Caffeine and/or 10 μM ATMi (KU-55933 (Millipore) for 18 h.Viral transductions were conducted as described . H1299, 2PO4, 6M Urea) on ice for 2 hours and sonicated before centrifugation. After quantitation, proteins were analyzed by western blotting. All antibodies are listed in Cell pellets were lysed in Urea Lysis Buffer  and sonicated. Antibodies were preabsorbed, washed and then incubated with lysate at 4°C for 2 h. All antibodies are listed in Cells were lysed in 25 mM Tris-HCl, 150 mM NaCl, 1% IGEPAL, protease cocktail, 1 mM Na2, 3 mM MnCl2, 3 μm Na3VO4, 20 mM ATP) using 0.1 μg of Src (Millipore). Kinase reactions were incubated with glutathione-sepharose beads that were pre-washed in 0.1% DTT for 30 minutes. This incubation was conducted for 10 minutes in a 0.1% DTT buffer. Complexes that formed were spun down and washed four times in a high salt buffer . The bound proteins were eluted in SDS-Loading Buffer and western blot analysis was performed.Kinase reactions were performed at 30°C for 60 minutes in kinase buffer  prewashed with lysis buffer and incubated for 2 h at room temperature. The beads were successively washed in each of the following: 6 M guanidinium–HCl, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris–HCl pH 8.0 plus 10 mM ß-mercaptoethanol; 8 M urea, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris–HCl pH 8.0, 10 mM ß-mercaptoethanol; 8 M urea, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris–HCl pH 6.3, 10 mM ß-mercaptoethanol plus 0.2% Triton X-100; 8 M urea, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris–HCl pH 6.3, 10 mM ß-mercaptoethanol plus 0.1% Triton X-100. After the last wash the beads were eluted with 200 mM imidazole in 5% SDS, 0.15 M Tris–HCl pH 6.7, 30% glycerol, 0.72 M ß-mercaptoethanol. Eluates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting.H1299 cells were transfected with His-nedd8 and other plasmids using PEI (1 DNA:1 PEI ratio). Twenty-four hours after transfection, cells were lysed in 500 μl of 6 M guanidinium–HCl, 0.1 M NaMCF7 cells growing on coverslips were serum starved for 72 hours and then treated for EGF for 8 hours. Cells were washed in PBS and then fixed for one hour at room temperature in 4% paraformaldehyde. After 3 washes in PBS, cells were permeabilized in 0.1% Triton-X 100 for 20 minutes. and washed 3X in PBS. After blocking in 2% BSA, coverslips were incubated with MdmX and/or Mdm2 antibodies  overnight at 4° C and washed 3X in PBS. Coverslips were then incubated with AlexaFluor 488 and 637 linked to anti-mouse and anti-rabbit secondary antibodies (respectively) for 1 hour, washed 3X in PBS, mounted on slides with Prolong Diamond Antifade Mountant with DAPI and visualized with a Leica SP8 MP confocal microscope at room temperature. Images were obtained using HC PL APO 40x/1.3 oil CS2 objective and the Leica Application Suite Advanced Fluorescence Software provided by the Indiana Center for Biological Microscopy core.5) were plated onto 6-well plates in triplicates and serum starved for 24 h. Cells were released from starvation with DMEM complete and treated with DMSO or MLN (0.6 μM) for 18 h, cells were washed with 1X PBS, harvested with trypsin, and spun at 300g x 5 minutes. FACS analysis was performed on the Muse (Millipore) with the Muse Cell Cycle Kit, according to manufacturers instructions . Significance was calculated using Two-tailed Student’s T-Test.MCF-7 cells (3×10


Widely recognized as a fundamental player in systemic Ca2+ homeostasis, the CaR is ubiquitously expressed in the human body where it activates multiple signalling pathways. In this review, old and new notions regarding the mechanisms by which extracellular Ca2+ microdomains are created and the tools available to measure them are analyzed. After a survey of the main signalling pathways triggered by the CaR, a special attention is reserved for the emerging concepts regarding CaR function in the heart, CaR trafficking and pharmacology. Finally, an overview on other Ca2+ sensors is provided.The current interest of the scientific community for research in the field of calcium sensing in general and on the calcium-sensing Receptor (CaR) in particular is demonstrated by the still increasing number of papers published on this topic. The extracellular calcium-sensing receptor is the best-known G-protein-coupled receptor (GPCR) able to sense external Ca These extracellular Ca2+ changes are sensed by the CaR (and other Ca2+ sensors) and transduced by means of different signalling pathways that modulate organ-specific physiological processes. Over the last few years, new concepts have emerged regarding the CaR’s role in cancer and cardiac physiology and pathology. Also, exciting news have come into view in the fields of CaR signalling, trafficking, and pharmacology. In addition, it is now clear that a number of different Ca2+ sensors share important tasks with the CaR. Therefore, the aim of this review is to summarize for the reader of International Journal of Molecular Science these new and exciting notions.The present review will examine the potential conditions under which extracellular Ca2+ concentration (~1.4 mM) is usually precisely regulated by a number of cooperative organs, including parathyroid glands, bones, kidney, and intestine  changes  in the restricted domain of both the intact amphibian gastric mucosa  in the sub-epithelial spaces of the intestine [2+] in the gut lumen or perturbations in whole-animal Ca2+ homeostasis were shown to cause significant changes in the external [Ca2+] underlying the epithelium of these tissues.Finally, Mupanomunda and colleagues used in situ microdialysis techniques in anesthetized Wistar rats to monitor extracellular  changes in HEK-293 cells, showing that Ca2+ extruded to the extracellular space by PMCA, as a consequence of intracellular Ca2+ events, activates a positive feedback through the CaR  changes, PTH secretion, and intracellular Ca2+ signals was finally demonstrated by Nemeth and Scarpa ext .2+ concentration is reduced, CaR-induced parathyroid hormone (PTH) secretion is elicited by the parathyroid glands. Consequences of PTH release are: (a) reduced excretion of Ca2+ by the kidney; (b) increased intestinal absorption of Ca2+, which is mediated by PTH-induced synthesis of the active vitamin D metabolite 1,25-dihydroxyvitamin D; and (c) net release of skeletal Ca2+ under sustained PTH secretion. All these tasks cooperate to increase serum Ca2+ back to physiological levels. On the other hand, when serum Ca2+ levels are increased over the physiological range, the receptor is activated inhibiting PTH synthesis and secretion  are the voltage-gated Human ether-à-go-go-related gene (HERG) encoded K+ channels, which have been shown to be implicated in a variety of physiopathological mechanisms, such as neuronal spike frequency adaptation and tumor cell growth [2+ under physiological conditions shift the voltage dependence of channel activation to more positive membrane potentials, allowing for fine-tuned cell repolarization [Another class of ion channels modulated by dynamic changes of extracellular [Cal growth . Johnsonrization . Under trization .2+ in hippocampal and pyramidal neurons and aid an inward cation current [2+-sensing nonselective cation channels, IcsNSC), which are almost silent at the 2.0 mM extracellular Ca2+ level, become robustly activated by small decreases in extracellular Ca2+ [Finally, the Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) was shown to sense a reduction of extracellular Ca current ,319,320.lar Ca2+ .The history of research on the CaR’s role in physiology and pathological states is a perfect example of the relevance of basic research for ameliorating human health. It is in fact clear that none of the clinically relevant discoveries in the pharmacological field today could have ever taken place without a deep knowledge of the specific mechanisms underlying the functionality of the tissues and organs on whom CaR mutations have their more striking effects.It is also clear that the novel methodological tools available to researchers in the field of GPCRs and signal transduction will be of paramount importance to further improve the knowledge of basic signalling modes, a step that is indispensable for the design of smart drugs able to rescue GPCR defects in human diseases.


These changes can be correlated with modifications in caveolin-3 and L-Type Ca2+ channel distributions across the sarcolemma. Channel regulation is also compromised since cAMP-dependent PKA activity is enhanced, increasing the probability of L-Type Ca2+ channel opening events. Immunofluorescence reveals that cholesterol depletion abrogates sarcomeric organization, changing spacing and alignment of α-actinin bands due to increase in proteolytic activity of calpain. We propose a mechanism in which cholesterol depletion triggers a signaling cascade, culminating with contraction impairment and myofibril disruption in cardiomyocytes.Cholesterol regulates numerous cellular processes. Depleting its synthesis in skeletal myofibers induces vacuolization and contraction impairment. However, little is known about how cholesterol reduction affects cardiomyocyte behavior. Here, we deplete cholesterol by incubating neonatal cardiomyocytes with methyl-beta-cyclodextrin. Traction force microscopy shows that lowering cholesterol increases the rate of cell contraction and generates defects in cell relaxation. Cholesterol depletion also increases membrane tension, Ca Cholesterol plays a fundamental role in regulating plasma membrane fluidity, integrity, and in compartmentalizing intracellular signaling events672+ dynamics in cardiomyocytes being responsible for gathering and regulating the activity of important ion channels, such as L-Type Ca2+ channel (LTCC)16172+ cycling in those cells.Flask-shaped and cholesterol enriched domains, known as caveolae, are fundamental for cardiomyocyte functioning10122+ entry is an important step for Ca2+ cycling within the cardiomyocyte and it is mediated by LTCC. Regulation of LTCC opening and closing events is fundamental to guarantee a synchronous Ca2+ signalingv1.2, the pore-forming subunit of LTCC, keeping it open and promoting extracellular Ca2+ entry to the sarcoplasm2+ plays a pivotal role in neonatal cardiomyocyte contraction since those cells lack t-tubules2+-induced-Ca2+-release mechanism for contraction in adult cardiomyocytesExtracellular Ca2+ but also myofibrillar organization and stability are critical for muscle cell contraction. Myocytes have an extremely organized and stable actomyosin network that is responsible for uniform and long-range contraction2+ binds to troponin-C changing tropomyosin conformation and releasing the site where myosin binds to actin allowing the filaments to slide past each other2+ and release of this ion to either the sarcoplasmic reticulum or to the extracellular milieu is important for proper relaxation of the myofibers since Ca2+ mishandling can cause cardiac issues2+ can trigger activation of proteases like calpainsNot only Cain vivo as a way of facilitating the reverse cholesterol transport in which accumulated cholesterol is removed from the vessel walls and transported to the liver for degradation decreasing the incidence of atherosclerosis in hyperlipidemic rabbitsTo investigate the importance of cholesterol in regulating cardiomyocyte physiology we incubated the cells with a cyclodextrin called methyl-beta cyclodextrin (MβCD) to acutely deplete plasma membrane cholesterol in isolated primary neonatal rat cardiomyocytes. MβCD is a cyclic oligosaccharide that has a hydrophobic core (with a high affinity for cholesterol molecules) and a hydrophilic surface that facilitates its solubilization in aqueous media2+ spikes and intracellular Ca2+ concentration. We correlated these results with the fact that caveolin-3 and Cav1.2 change distribution across the sarcolemma upon cholesterol depletion which, in turn, might impact the regulation of the LTCC. In order to test this hypothesis we measured cAMP-dependent PKA activation and we saw an increase in the enzymatic activity consequent to cholesterol depletion. This result corroborates the increase in intracellular Ca2+ after MβCD treatment since PKA activity is directly involved in Cav1.2 opening events. Immunofluorescence revealed that cholesterol depletion abrogated the organization of myofibrils, changing the spacing and alignment of α-actinin bands and this change was correlated with increase in proteolytic activity of calpain probably due to enhancement in cytoplasmic Ca2+ concentration.Traction Force Microscopy (TFM) experiments revealed that cells with lower cholesterol levels had higher and variable contraction rates and relaxation defects. MβCD treatment increased the cardiomyocytes cortical mechanical properties values, the frequency of CaThis work suggests for the first time a complete mechanism by which cholesterol depletion, due to MβCD incubation, impairs contraction in neonatal cardiomyocytes.For more details in the methods section visit the All animals were maintained at The University of Chicago Animal Resources Center. Animal husbandry and all experimental procedures were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). Experimental protocols were approved by the University of Chicago IACUC (protocol number 72347).Neonatal rat hearts (p0-p3) were submitted to enzymatic digestion steps that were adapted from a previously stablished protocolTo deplete cholesterol the cardiomyocytes were incubated with either 5.0 or 7.5 mmol/L MβCD, for 45 minutes, at 37 °C.v1.2. Next, the cells were rinsed and incubated with secondary fluorescent antibodies. Images of cells were taken on Ti-E Nikon inverted confocal microscope using either 60 × 1.49 NA ApoTIRF or 40 × 1.30 Plan Fluor oil-immersion objectives.After fixation, cardiomyocytes were incubated with the following primary antibodies: mouse monoclonal anti-sarcomeric α-actinin, rabbit polyclonal anti-caveolin-3, mouse monoclonal CaCardiomyocytes were plated on polyacrylamide gels with a shear modulus of 8.64 KPa embedded with fluorescent beads as previously described34353435373839An infrared optical tweezers (OT) setup was used to extract tethers from the surface of the cardiomyocytes. The OT setup and force calibration were performed as previously described414412+ probe (Thermo Fisher Scientific) was acquired for control and treated cardiomyocytes and cytoplasmic Ca2+ sparks were analyzed using ImageJ. Conversion of fluorescence values into Ca2+ concentration was based on the following equationEpifluorescence signal from the Fluo-4 AM Ca2+ dye used, R is the fluorescence ratio 0 is the minimum fluorescence recorder for a specific cell analyzed), [Ca2+]rest is approximately 140 nMK is the dissociation constant of the CaTo measure cAMP-mediated PKA or calpain activities, cell extracts were submitted to either enzymatic assay using PKA activity kit (Enzo Life Sciences) or using calpain Activity Fluorometric Assay Kit (BioVision Inc) according to manufacturer’s instructions.0 and R were propagated by taking the derivatives of the 0 and R. Statistical differences were labeled with asterisks. Fourier analysis of contraction and Ca2+ sparks as well as quantification of α-actinin bands were done using MATLAB scripts described in the Student’s t-test was used to compare between control and treated groups. Data was represented by mean values ± standard error unless otherwise stated. The experimental errors for FTo deplete plasma membrane cholesterol we used a cyclodextrin called MβCD. To directly measure how cholesterol depletion affects contractility of neonatal cardiomyocytes, we used time-resolved Traction Force Microscopy (TFM). Cell contractility is defined as the ability of the cell to generate contractile forces via actomyosin interactions within myofibrils. For the case of cells attached to a flat substrate, we can measure their contractile behavior using TFM. Traction stress maps were obtained for 40 seconds to capture the spontaneous beating of cardiomyocytes plated on fibronectin coated gels. The maximal and minimal traction stresses obtained during this time lapse are shown in m) and then abruptly decreases to a constant value F0. Fm corresponds to the moment that precedes the tether formation whereas F0 corresponds to the force applied to form and elongate the tether. We measured F0 for all tested conditions .In order to uncouple contributions from the plasma membrane and from the cytoskeleton to cortical mechanical properties we decided to induce plasma membrane vesicles (PMVs)  μm . In contCaveolin-3, localized in cholesterol invaginated domains known as caveolae, has been previously shown to be important for adult cardiomyocytes to adapt to sudden changes in membrane tension462+ is another fundamental piece of the contractile machinery in muscle cells, we sought to investigate whether cholesterol depletion was also changing sarcoplasmic Ca2+ cycling in cardiomyocytes. For that, control and cholesterol depleted cardiomyocytes were incubated with Fluo-4 AM and imaged in order to track changes in intracellular calcium spike dynamics. In 2+ fluorescence for control and MβCD treated cardiomyocytes in the contracted and relaxed states. By comparing contracted states, we see that cytoplasmic Ca2+ fluorescence intensity increases when cholesterol is depleted. For the relaxed states we also noticed that cholesterol depleted cells cannot buffer the cytoplasmic Ca2+ efficiently during relaxation. Quantification of intracellular Ca2+ fluorescence . It is known that PKA phosphorylates Cav1.2 in order to control the opening of the LTCC pore2+ entry. In conjunction with our previous results, this evidence suggests that cholesterol depletion activates cAMP-dependent PKA activity leading to enhancement in Ca2+ entry, which in turn deregulates contraction dynamics in neonatal cardiomyocytes. In addition, the clear disruption of myofibers in cells treated with MβCD raised the question whether some Ca2+ -dependent cytoplasmic protease might have been activated in the process. Since we saw an increase in cytoplasmic Ca2+ concentration we decided to test activity of calpain, one of the main proteases in muscle tissue that is activated by intracellular Ca2+ 2+-dependent protease might be degrading the myofibers due to mishandling of cytoplasmic Ca2+ caused by cholesterol depletion.Since extracellular Calabeling  or 7.5 mlabeling  MβCD theIt is widely known that cholesterol plays a pivotal role in controlling permeability, diffusion and receptor clustering at the plasma membrane level234678The contractility of cardiomyocytes is controlled by two different actin cytoskeletal architectures: sarcomeric myofibrils and the sub-sarcolemmal actin. There is substantial evidence that the sub-sarcolemmal actin contractility is regulated in a similar manner as in non-muscle cells312+ handling, adrenergic signaling cascade, sarcomeric integrity and caveolae domains are thought to be important to all of them  shows a decrease in cell contractility. In addition to that, the increase in trough strain energy and decrease in overall contractility suggests impairment in cell shortening and increase in isometric force generation in low cholesterol scenarios. Interestingly, isometric cardiac contraction is correlated with myofibrillar disarray in left ventricles obtained from patients with idiopathic hypertrophic subaortic stenosis of them .Caveolae are flask-shaped and cholesterol-enriched plasma membrane invaginations that regulate many cellular processes including lipid homeostasis and adaptation to membrane tension4760102+ plays a fundamental role in contraction44484950512+ entry to calcium-induced-calcium-release that is essential for adult ventricular cardiomyocytes contraction652+ to contract  is activated and accelerates the production of cAMP, which in turn activates PKA. PKA phosphorylates the pore-forming subunit of LTCC, Cav1.2, promoting opening of the channel and Ca2+ entry. It had been shown via immunoprecipitation that caveolin-3, Cav1.2, β2-AR (but not β1-AR), AC and PKA are closely associated in neonatal murine cardiomyocytesv1.2 subunit of LTCC changes distribution within the cells being excluded from the perinuclear region. We also demonstrated, by enzymatic assay, that cAMP-dependent PKA activity increases when cholesterol is depleted even in the absence of β2-AR agonists, which might increase the number of Cav1.2 opening events leading to increase of Ca2+ influx. In fact, we saw that cholesterol reduction led to an increase in the Ca2+ probe fluorescence in the cytoplasm of the cardiomyocytes. Moreover, the Ca2+ spikes were more frequent and less synchronized in the MβCD treated cells, which correlates to our TFM results and corroborates our hypothesis that cholesterol sequestration impairs both contraction dynamics and Ca2+ handling by the cardiac cells.Some members of the G-protein Coupled Receptors (GPCRs) family, known as adrenergic receptors , regulate cardiac CaThe last pillar that governs cardiac contraction is sarcomeric integrity. Since we measured impairment in cardiomyocyte contraction dynamics and also changes in membrane tension in those cells we hypothesized that MβCD treatment was somehow perturbing the sarcomeric actin cytoskeleton that plays an important role in cardiomyocyte contraction2+-dependent proteolytic activity in the sarcoplasm. In order to test that, we measured cytoplasmic calpain enzymatic activity. It is known that cytoplasmic Ca2+ triggers calpain activation which in turn can degrade sarcomeric proteins such as troponins, tropomyosins and titins leading to abrogation of the z-bands organization2+ upon cholesterol depletion we hypothesized that it might be activating calpain degradation of myofibers impacting the normal contraction behavior of those cells. In fact, we saw an increase in calpain activity due to cholesterol depletion and that correlates with the disorganization of the z-bands that we measured.In addition, we decided to investigate whether the disruption of the myofibers was caused by Ca2+ concentration, calpain proteolytic activation and disorganization of myofibers.Overall this work points out to a new mechanism in which sarcoplasmic cholesterol depletion by MβCD incubation leads to impairment in cardiomyocyte contractility and calcium handling increasing cortical mechanical properties values, cytoplasmic CaHow to cite this article: Hissa, B. et al. Cholesterol depletion impairs contractile machinery in neonatal rat cardiomyocytes. Sci. Rep.7, 43764; doi: 10.1038/srep43764 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We explain the observed phenomenon with the coherent photogalvanic effect model, which correctly agrees with the retrieved experimental parameters.Efficient second harmonic generation in integrated platforms is usually achieved by resonant structures, intermodal phase-matching or quasi-phase matching by periodically poling ferroelectric waveguides. However, in all these structures, it is impossible to reconfigure the phase-matching condition in an all-optical way. Here, we demonstrate that a Watt-level laser causes a periodic modification of the second-order susceptibility in a silicon nitride waveguide, allowing for quasi-phase-matching between the pump and second harmonic modes for arbitrary wavelengths inside the erbium band. The grating is long-term inscribed, and leads to a second harmonic generation enhancement of more than 30 dB. We estimate a  Phase-matching and quasi-phase-matching in periodically poled ferroelectrics ensure efficient harmonic generation, but only statically. Here, Billat et al. demonstrate all-optically reconfigurable second harmonic generation by inscribing a stable grating in CMOS-compatible nonlinear waveguides. However, integrated waveguides showing significant second-order optical nonlinearity are key to enabling a new range of on-chip applications such as self-referencing of chip-based frequency combs3, or telecom signal up-conversion in order to perform direct detection with integrated silicon photodiodes4. On-chip frequency down-conversion for quantum optics5 is also an option, as second-order processes facilitate the pump rejection in photon-pair generation experiments6.Over the last decade, integrated photonics allowed the demonstration of micrometre-scale and low power optical nonlinear devices. In particular, complementary metal–oxide–semiconductor (CMOS) compatible materials such as silicon and silicon nitride (SiN) are the most promising for nonlinear optical signal processing based on third-order processes9. To enhance second harmonic generation (SHG) in SiN, researchers used resonant structures like microresonators10 and waveguide gratings11, but at the expense of using fixed operational wavelengths. Moreover, momentum conservation for SHG in integrated waveguides is generally achieved through intermodal phase-matching, implying that the effective index of the pump mode is equal to the second harmonic one, i.e. np = nsh. Phase-matching is therefore constrained by waveguide design. To overcome this restriction, quasi-phase-matching (QPM) techniques are routinely employed by periodically poling waveguides made of polar materials such as lithium niobate12, lithium tantalate13 or suitable polymers14. However, their integration on silicon photonic circuits still faces limitations15. Up to now, none of the presented integrated platforms allows the all-optical and permanent configuration of the phase-matching condition in the waveguide, nor its dynamic update.SiN exhibits a very large transparency window, from ultraviolet to mid-infrared, and moderate second-order nonlinearity due to interface symmetry breaking, higher multipole bulk terms and a non-isotropic distribution of the SiN dipoles inside the amorphous matrix16, developed to explain the poling occurring in optical fibres illuminated by kilowatt near-infrared lasers17.Here we demonstrate an optically induced and dynamically reconfigurable SHG enhancement in an integrated photonic platform. We report the growth of the SHG signal over time when pumping a SiN waveguide with a pulsed laser in the communication band (designated as pump). By probing the waveguide with a tunable continuous wave (CW) laser (designated as probe) following the SHG growth, we observe a clear phase-matching peak centred on the pump wavelength. Shifting the pump to another arbitrary wavelength in the 1534–1550 nm range, we notice a similar SHG growth as well as a subsequent phase-matching peak at the new wavelength. Moreover, the waveguide features the same SHG level when probed over a few days, demonstrating a persistent change in the phase-matching condition caused by the pump. Our experimental observations fit well within the framework of the coherent photogalvanic theoryFigure 18 (see Methods for fabrication details), which guarantees a crack-free nitride layer deposition and void-free structures of high aspect ratio. The devices are made of stoichiometric Si3N4 buried in SiO2 and exhibit a very low attenuation (0.2 dB/cm). The waveguides are folded in meanders and have trapezoidal cross sections  within 25–30 min. In this interval, we kept the pump power constant and observed that any second harmonic generated was TM polarized, as the pump. The SHG growth as a function of time can be triggered by different pump wavelengths, even widely separated, in the same waveguide. This behaviour is illustrated in Fig. We first injected the pulsed pump in waveguide (i) with 90 W of coupled peak power into the transverse magnetic (TM) mode, which corresponds to an intensity of about 9 GW/cmOwing to the waveguide birefringence, changing the pump polarization alters the phase mismatch between the pump and the second harmonic, similar to what happens when changing the pump wavelength. To test this behaviour, we alternatively pumped the transverse electric (TE) and TM modes of waveguide (i), keeping the same pump wavelength (1544 nm) and peak power (90 W). At the output of the chip, we measured the SHG component parallel to the pump polarization using a polarizer positioned after the filter assembly. We first pumped the TE mode, and observed the TE second harmonic component growth until saturation. Rotating the polarizer by 90°, we observed that the TM component of the SHG was 30 dB weaker than the TE. Setting the pump to the TM mode, the TM component of the second harmonic grew by three orders of magnitude. After saturation of the TM SHG, the output polarizer was rotated by another 90° transmitting only a negligible TE SHG component. However, switching the pump back to the TE mode systematically re-triggered the SHG growth on the TE polarization, which went back to the initial saturation level in an identical amount of time. Figure We then tested waveguide (ii) by pumping in the TE fundamental mode. In this configuration, green light generation is greatly suppressed, as THG is not phase-matched. As for waveguide (i), SHG growth with a pulsed pump is observed. Figure χ(2) grating derives from an all-optical poling mechanism. This QPM condition is fulfilled every time the pump wavelength is changed, in spite of the waveguide dispersion. The peak 3 dB bandwidth (FWHM) is about 2.5 nm (at the pump wavelength) while their maxima are located 10 dB higher than what can be considered as the floor level for SHG.After SHG saturation, we probed the waveguides with a CW tunable laser, keeping the coupled probe power constant at 350 mW. Under such light exposure, we did not notice any SHG evolution over time. We plot the second harmonic power as a function of wavelength in Fig. We also probed waveguide (ii) by coupling a 130 mW CW laser, after pumping at 1535 nm with 90 W peak power and at 1548 nm with 60 W peak power. We again observed phase-matching peaks around the pump wavelengths see Fig. . In addi17. Researchers identified the coherent photogalvanic effect (CPE) as the main underlying mechanism19. Optical poling has also been reported in polymer fibers21, however in that case the physical origin of the poling has been ascribed to an orientation mechanism of the molecules inside the polymer matrix, with no apparent contribution from a space-charge field20.The effect reported here is qualitatively similar to the all-optical SHG enhancement reported in silica fibres pumped by a kilowatt-level pulsed Nd:YAG laser2-related defects in silica). It takes place under illumination by a pump and its frequency-doubled counterpart, as the result of the interference from different coherent multiphoton absorption processes involving the two waves. The spatially preferential emission of electrons gives rise to a photogalvanic current (see Supplementary Note EDC)22. As schematically illustrated in Fig. EDC by the CPE process. EDC has a periodicity that directly depends on the coherence length between the pump and its second harmonic16. The effective χ(2), which allows QPM of the SHG at the pump wavelength23, thus arises as the product of χ(3) and the built-in field, a process known as electric field induced second harmonic generation (EFISHG)24. The gratings are long-term inscribed. In fact, the ejected electrons, responsible for the built-in EDC, are eventually trapped by deep and localized defect states19.The CPE consists in an asymmetric photoemission of electrons from defect centres with energy levels lying inside the band gap of the material  to the counter-acting effects of CPE involving photons from THG and direct re-ionization also due to third harmonic photons.The Si-Si defects in deposited SiN, extensively studied in the frame of electronic memories−1, which corresponds to about 3∙10−3% W−1 cm−2. Assuming quasi-phase-matched SHG with a sine-modulated χ(2) grating29, we can also estimate the amplitude of the χ(2) in waveguide (i) by fitting the data from Fig. ωsh is the second harmonic frequency, Pp the coupled pump power, L the waveguide length and S the overlap integral between the pump and second harmonic mode.We verified the quadratic relationship between the CW probe power and the second harmonic power at the phase-matching peak in the 1544 nm pump case, as shown in Fig. 10, the QPM grating period is twice the initial coherence length. From the simulated mode profiles, we estimate a peak χ(2) value of about 0.3 pm/V, and we calculate the grating period to be Λ = 2π/|βsh − 2βp| ≈ 43 μm (where β is the mode propagation constant). For waveguide (ii), the SHG is expected to happen on the 8th TE mode. In this case, we retrieve from Fig. χ(2) ≈ 0.15 pm/V, consistent with the estimation in waveguide (i), and a grating period of about 64 μm at 1535 nm. As the second harmonic always has the same polarization as the pump, these values refer to the diagonal χ(2) tensor element. All the mode simulations and calculations are detailed in Supplementary Note χ(2) = 3χ(3)EDC19, we calculate a space-charge field magnitude of 108 V/m, assuming a third-order susceptibility of 10−21 m2/V230. Finally, it has to be noted that because of intermodal phase-matching, the FWHM of the SH depends on the difference between the slopes of the fundamental and the second harmonic effective index curves, at the second harmonic wavelength. According to Supplementary Fig. 30, different optical absorptions at different waveguide’s positions would lead to a deviation of the coherence length between the pump and the second harmonic along the waveguide. This coherence length distribution is equivalent to a chirping of the QPM grating, known to broaden the SHG conversion efficiency.With the help of a mode solver, we compute the effective indices and the mode overlap of the TM modes at the pump and SHG frequencies in waveguide (i). We assume that the SHG occurs on the TM 8th dipolar mode (modes are numbered according to their effective index), which features the best phase-matching and mode overlap with the pump. Indeed, since the natural initial SHG takes place on a higher order mode thanks to intermodal phase-matchinga peak χ value ofa peak χ value of−1 (3∙10−3% W−1 cm−2) is comparable to the one obtained in similar SiN waveguides designed to fulfil the intermodal phase-matching condition at telecom wavelengths31, or in SiN ring resonators10. This shows the effectiveness of the reported all-optical QPM method. The χ(2) value of few tenths of pm V−1 is also in agreement with previous measurements in SiN waveguides at the same wavelengths31. The estimated DC field magnitude, assuming the coherent photogalvanic effect as the underlying physical mechanism, is comparable to the one obtained in ref. 31, where a voltage is applied across the waveguide structure to enhance SHG.We have experimentally demonstrated an all-optical and reconfigurable SHG enhancement by more than 30 dB in a popular SiN waveguide platform, ready to be fabricated on a large scale in multi-project wafer runs. The enhancement results from the persistent inscription of a second-order susceptibility grating in the waveguide. The grating period automatically adapts to a modified coherence length between the pump and the harmonic, allowing for quasi-phase-matched SHG over the whole C-band. The measured conversion efficiency of 0.05% W32 or by leveraging the EFISHG through a p-i-n junction created by ion implantation33. The presented findings however feature an improved and appealing versatility compared to the current strategies for on-chip integration of second-order nonlinearity. In fact, they show a unique method to configure dynamically QPM in passive devices fabricated with CMOS compatible steps.In parallel, silicon waveguides recently achieved remarkable SHG efficiencies, either thanks to a SiN strain layers17. Various options for improving the conversion efficiencies can be explored. As shown here, the efficiency of the process increases with the length of the waveguide and the pump power. In addition, the saturation of the CPE is given by the pump-second harmonic mode overlap, the density of the defect states and the χ(3) value. Simulations indicate that seeding the process with a second harmonic wave injected on the fundamental mode, in order to improve the mode overlap, may increase the SHG efficiency by 40%. Moreover, the low propagation loss (0.2 dB/cm) at the pump wavelength results in a long effective length (about 22 cm), such that longer waveguides can be considered for enhanced conversion efficiency. Employing materials with a larger χ(3), such as Si-rich SiN34, is also an option to increase the SHG by at least an order of magnitude35.Compared to previous works in silica optical fibres, the use of integrated waveguides allows to increase the conversion efficiency by two orders of magnitude, and to decrease by the same factor the SHG growth duration18. The process starts by patterning the waveguides as well as a dense filler pattern into a hard mask of amorphous silicon on a 4 μm thick wet thermal silicon oxide. The structures are then transferred into the preform using a dry etch process based on He and C4F8. Next the waveguide trenches in the preform are filled with low pressure chemical vapour deposition silicon nitride, deposited in one run up to the desired thickness. The dense filler pattern efficiently releases film stress and prevents cracking of the SiN thin film. The excess SiN is removed using chemical mechanical polishing, providing a smooth and planar wafer surface. Finally, the wafer is annealed to drive out residual hydrogen in the films  and cladded with low temperature oxide (LTO), before being separated into individual chips.The waveguide devices were fabricated using the photonic Damascene processThe data that support the findings of this study are available from the authors on reasonable request, see author contributions for specific data sets.Supplementary Information


Human mesenchymal stem cells are promising tools for regenerative medicine due to their ability to differentiate into many cellular types such as osteocytes, chondrocytes and adipocytes amongst many other cell types. These cells present spontaneous calcium oscillations implicating calcium channels and pumps of the plasma membrane and the endoplasmic reticulum. These oscillations regulate many basic functions in the cell such as proliferation and differentiation. Therefore, the possibility to mimic or regulate these oscillations might be useful to regulate mesenchymal stem cells biological functions.2+ spikes caused by the penetration of Ca2+ from the extracellular medium, through the transiently electropermeabilized plasma membrane, in human adipose mesenchymal stem cells from several donors. Attached cells were preloaded with Fluo-4 AM and exposed to the electric pulse(s) under the fluorescence microscope. Viability was also checked.One or several electric pulses of 100 μs were used to induce Ca2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. None of the pulses used here, even those with the highest amplitude, caused a loss of cell viability.According to the pulse(s) electric field amplitude, it is possible to generate a supplementary calcium spike with properties close to those of calcium spontaneous oscillations, or, on the contrary, to inhibit the spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca2+ spikes, is reported here. Indeed, the direct link between the microsecond electric pulse(s) delivery and the occurrence/cancellation of cytosolic Ca2+ spikes allowed us to mimic and regulate the Ca2+ oscillations in these cells. Since microsecond electric pulse delivery constitutes a simple technology available in many laboratories, this new tool might be useful to further investigate the role of Ca2+ in human mesenchymal stem cells biological processes such as proliferation and differentiation.An easy way to control Ca Mesenchymal stem cells (MSCs) are multipotent stromal cells  originatHuman-adipose MSCs (haMSCs), derived from adipose tissue are amongst the most easily accessible MSCs, with high quantities, and without aggressive extraction procedures. They are more available than other MSCs as, for example, the human bMSCs (hbMSCs). In addition, they have a phenotype, surface markers , and gen2+ oscillations, implicating Ca2+ channels and pumps of the plasma membrane (PM) and the endoplasmic reticulum (ER) [2+ release from the ER and further amplification from plasma membrane store-operated Ca2+ channels (SOCCs). Afterwards, the excess of Ca2+ is removed from the cytosol by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), the plasma membrane Ca2+ ATPase (PMCA), and the Na+/Ca2+ exchanger (NCX) [2+ is one of the most important second messenger in the cell, and it regulates many important cellular processes such as ATP synthesis, apoptosis, cellular motility, growth, proliferation and gene expression. Hence, Ca2+ oscillations contain embedded information that has to be decoded by the cell, and Ca2+ signalling pathways play a key role in controlling cell behaviour and differentiation processes of MSCs.These cells present spontaneous Calum (ER) . These olum (ER)  followeder (NCX) . Ca2+ is2+ oscillations frequency is different between undifferentiated MSCs and MSCs on route to differentiation and it differs between the various outcomes of the differentiation process (the level of differentiation and the differentiated cell type). While the MSCs exhibit regularly repeated Ca2+ oscillations, MSCs undergoing osteodifferentiation display a decrease in the frequency of their spontaneous Ca2+ oscillations while primary myoblasts present still another pattern of oscillations [2+ oscillation frequency and shape, but the exact correlation between Ca2+ oscillations and MSCs differentiation is still unclear.It was shown that the Callations . This sh2+ is an ion that only crosses the plasma and ER membranes through channel proteins. Applying PEFs to cells in a medium containing Ca2+ (amongst other compounds) allows Ca2+ entry from the cell outside, and, if the electric field amplitude and pulse duration are high enough, Ca2+ is released from the cell inner stores [2 for 250 μs) [Presently, pulsed electric fields (PEFs) are widely used in research as a non-invasive physical means to permeabilize cellular membranes. Using one or several pulses of ultrashort duration causes changes in the cell membrane structure that permits access to the cell cytosol to molecules that cannot cross the plasma membrane under normal conditions . Normallr stores . The eler stores  or bipha 250 μs) . These f2+ oscillations of the cells. Attached haMSCs presenting normal spontaneous Ca2+ oscillations were exposed to one single pulse with a duration of 100 μs and different electric field amplitudes. At low and moderate electric field amplitudes, the Ca2+ spike induced by such PEFs can be very similar to a spontaneous oscillation. At high electric field amplitudes, the electrically induced Ca2+ spikes inhibit the spontaneous oscillations occurrence for some time after which oscillations reappear. This inhibition allowed us to impose on the cells experimentally induced Ca2+ spikes at a desired frequency. Hence, we show here that it is possible to control haMSCs spontaneous Ca2+ oscillations by microsecond pulsed electric fields (μsPEFs) with a complete preservation of the cell viability. The delivery of μsPEFs constitutes an easy way to control Ca2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca2+ spikes. Indeed, unlike chemical factors, which cause continuous effects, the direct link between the microsecond electric pulse(s) delivery and the ulterior occurrence/cancellation of the cytosolic Ca2+ spikes allowed us to mimic and regulate the Ca2+ oscillations in these cells. Since microsecond electric pulses delivery constitutes a simple technology available in many laboratories, the electrical control of Ca2+ oscillations could be in the future a promising tool to further investigate the role of Ca2+ in MSCs physiology and to better understand the correlation between the Ca2+ oscillations and MSCs biological processes such as proliferation and differentiation.The novelty of our study is the use of PEFs of a high field amplitude that are capable of permeabilizing the cell PM or both the PM and the ER. Our objectives were to test the effect of these pulses on the Ca2. Cells were passed twice a week (every passage corresponds to one doubling time of the population). The multipotency capabilities of the cells were assessed by submitting them to differentiation conditions as previously reported in Liew et al. [HaMSCs were isolated from surgical waste of individuals undergoing elective lipoaspiration. Samples were obtained after written informed consent from all the donors, in accordance with French and European legislations. The lipoaspirates were surgical waste and as such the French legislation (Art.L. 1245-2 du Code de la Santé Publique) establishes that the authorisation from an ethics committee is not required. Every experiment was done on cells before passage 8 from at least two donors . All haMSCs reacted similarly to the electric pulses, whoever the donor. The cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax and supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin and were cultured at 37 °C in a humidified incubator with 5% COw et al. . Cells f3 cells/cm2. After 1 day, the cells were incubated for 30 minutes with 5 μM of Fluo-4 AM (Fischer Scientific), a fluorescent Ca2+ marker, in a humidified 5% CO2 atmosphere at 37 °C in complete DMEM. The incubation buffer also contained 375 nM of the nuclear fluorescent dye Hoechst 33342. After incubation, the attached cells were rinsed three times with PBS  and 500 μl of complete DMEM were added to the cells.One day prior to the experiments, cells were seeded in 24-well plates at a density of 20 × 10One single micropulse of 100 μs was delivered in all the experiments. A Cliniporator™  was used for the generation of the microsecond pulsed electric fields (μsPEFs). In order to treat the cells under the microscope, the pulse generator was connected to two parallel stainless steel rods of 1.2 mm diameter used as electrodes. The distance between them was 5 mm, and they were shaped to enter a 24-plate well and to reach the bottom of the dish. The whole system was set under a Zeiss Axiovert S100 epifluorescence inverted microscope.2 containing two slots was designed to fit into a Petri dish of the same dimensions. Two plate electrodes of 2 mm thickness and 2 cm length were slipped into the slots until they touched the bottom of the Petri dish. This system does not permit observation of the cells under a microscope, it was used because it allows covering a larger surface (in order to have enough cells) than the system described above.In order to test the cell viability, another model of electrodes was designed. In this system, a thick cover of 10 cmImages of the cells were taken every 10 seconds for 10 to 20 minutes with a Zeiss AxioCam Hrc camera controlled by the Axio Vision 4.6 software . The electric pulse was delivered after at least 2 minutes of recording, and 2 seconds before the next image. The excitation and emission wavelengths used for Fluo-4 were 496 nm and 515 nm respectively. The nuclear dye Hoechst 33342  was used to recognise the nuclei and to track the cells using the Cell Profiler (version 2.0) software . The software allowed the automatic measurement of the fluorescence intensity signal of each cell on every image. Curves were plotted using a Matlab program . All the observations were done at room temperature. The minimum opening time of the shutter for the fluorescent light was about 500 ms. To decrease the light energy applied on the cells, a 90% density Filter NE110B  was used.3 cells/cm2 in 10-cm2 Petri dishes containing a 12 × 32 mm cover slide. After 24 hours, medium was removed, cells were washed with PBS, and 1 ml of fresh medium was added. The electrodes were placed on the cover slide using the Petri dish cover containing two slots and a 100-μs pulse was delivered to the attached cells. After the pulse, the Petri dish was put in the incubator for 20 minutes. Then, the non-pulsed cells on the cover slides were scratched under a sterile hood. The area covered by the pulsed cells was recognised due to the loss of cells beneath the electrodes after the removal of the latter. The cover slide with the pulsed cells only was transferred to a Petri dish with fresh medium and placed for 2 hours in the incubator. Then medium was removed and the cells were trypsinised, centrifuged, and resuspended in 300 μl of a fresh medium. This volume was distributed in two wells of an opaque-walled 96-multiwell plate and put in the incubator for 24 hours. After 24 hours, cells were visualised on an epifluorescence microscope to compare their shape and level of confluence, and 150 μl of Cell-Titer Glo Reagent  were added to each well according to manufacturer’s protocol. The reagent caused cell lysis and the generation of a luminescent signal proportional to the amount of the ATP present in the medium. A GloMax luminometer (Promega) was used to read the luminescence. The ATP amount and therefore the luminescence intensity reflected the number of cells present in the culture [One day prior to the experiments, cells were seeded at a density of 20 × 10 culture .All the experiments were done at least three times. Data are presented as means and standard deviations. The Spearman correlation was used to analyse the data of Figs. 2+ oscillations as seen in Fig. 2+ oscillations. When one electric pulse of 100 μs was applied on haMSCs, different situations could be observed. As shown in Fig. 2+ spike in all the cells whatever the stage of their Ca2+ oscillation at the time the pulse was applied. After the pulse, the spontaneous Ca2+ oscillations returned to their original cell-dependent frequency and continued normally. When the electric pulse applied was 900 V/cm  and the spontaneous Ca2+ oscillations did not yet resume.Undifferentiated haMSCs present asynchronous Ca2+ spikes in response to a 100-μs duration pulse of different electric field amplitudes are represented in Fig. 2+ spike was observed. At 150 V/cm, the Ca2+ spikes had a lower amplitude than the spontaneous oscillations and presented a relatively small slope. At moderate electric field amplitudes (200 to 450 V/cm), each electric pulse induced a Ca2+ spike. The Ca2+ spike displayed a higher slope than at 150 V/cm, then reached its maximal amplitude and came back to the normal Ca2+ amplitude as before the pulse. The electro-induced Ca2+ spikes had nearly the same shape and amplitude as the spontaneous oscillations at 200 and 300 V/cm. At 450 V/cm, their amplitudes were higher than the spontaneous ones, but they came back to the original baseline. When high electric field amplitudes were applied (600 to 900 V/cm), the electrically induced Ca2+ spike presented larger slopes (with different degree of sharpness depending on the electric field strength) and displayed a higher amplitude than the spontaneous oscillations; after the Ca2+ spike, the Ca2+ amplitude remained relatively high compared to the initial amplitude, and it took a longer time  to come back to normal depending also on the amplitude of the applied electric field.The shapes of the electrically induced Ca2+ spike in response to the electric pulse at 150 V/cm. This percentage increased proportionally to the electric field amplitude, mainly between 150 and 300 V/cm. One hundred percent of the cells responded to the pulse at 450 V/cm. According to Spearman correlation analysis, there was a perfect correlation between the electric field amplitude and the percentage of cells displaying a Ca2+ spike (r = 0.96).According to Fig. 2+ spike had different effects on Ca2+ oscillations. At a relatively low electric field amplitude (300 V/cm), the pulse-induced Ca2+ spike displayed a very similar shape to the spontaneous Ca2+ oscillations .According to the electric field amplitude, the pulse-induced Caons Fig. , and it tes Fig. . The app2+ oscillations inhibited after one electric pulse at 450 V/cm and nearly all the cells at 750 V/cm. Twenty-four hours after the pulse delivery, the cells remained viable under all the conditions tested, even for the highest electric field amplitude . For example  are multipotent stromal cells that have the ability to self-renew and to differentiate into a wide variety of cells of the mesodermal, ectodermal, and endodermal lineages . This is2+ oscillations play an important role in the transduction of many physical stimuli in the cells. For example, tissue strain and shear stress on mouse tibia are transduced by repetitive Ca2+ spikes in the osteocytes. The spikes frequency and amplitude depend on the mechanical magnitude [2+-related mechanisms. The exposition of MSCs to pulsed EMFs for 10 minutes every day results in the enhancement of osteogenesis early stages [Caagnitude . Electroy stages .2+ oscillations are a universal mode of Ca2+ signalling in both excitable [2+ oscillations are typically initiated by a receptor-triggered production of IP3 (after the binding of an agonist to the receptor) [2+ release from the ER [2+ oscillation frequency is different between undifferentiated MSCs and MSCs on route to differentiation and it differs between the various differentiated cell types [2+ oscillations are suppressed after the addition of neuroinductive factors, but reappear in less than 7 days after neurodifferentiation [2+ spikes per 30 minutes of observation, and that this number decreases to 3.66 ± 2.42 after 28 days of incubation with osteoinductive factors [Caxcitable , 25, andxcitable . In non-eceptor)  and the m the ER , which sm the ER . It was ll types . Accordintiation . Sun and factors .2+ oscillations of haMSCs because many of the physical stimuli are transduced by cytosolic Ca2+ concentration rapid changes (oscillations) in the cells. The ultrashort pulses (one thousandth to one ten thousandth shorter than the pulses of 100 μs used in our study) called nanopulses or nanosecond pulsed electric fields (nsPEFs) have been used already to induce Ca2+ bursts in the cell, which resulted from the electropermeabilization of the ER, the plasma membrane or both [In our study, we decided to analyse the effect of a type of short electric pulses termed microsecond pulsed electric fields (μsPEFs) on the spontaneous Ca or both , 30. How2+ concentration detected by the periodic increase of their fluorescence. The videos of the haMSCs showed that not all the cells presented Ca2+ oscillations at a specific time. Some of the cells never displayed oscillations during the recording time (20 minutes). This observation can be related to the fact that MSCs display Ca2+ oscillations only during the phases G1 and S of the cell cycle, and not all the cells in the visualisation area of an experiment were in those phases of the cell cycle. Ca2+ oscillations increase the levels of cell cycle regulators such as cyclins A and E and probably control cell cycle progression and cell proliferation, via the regulation of cyclin levels (amongst other mechanisms) [2+ oscillations displayed them at different rhythms and frequencies: the oscillations were asynchronous, and at a given time, each cell was in a different phase of a Ca2+ oscillation and the oscillations frequencies displayed wide variations between cells. The average duration of an oscillation was around 2 minutes.The haMSCs loaded with the fluorescent marker Fluo-4 asynchronously displayed periodic changes in their cytosolic Cahanisms) . In addi2+ spike was observed synchronously in a given percentage of the cells (as a function of the electric field amplitude). Moreover, the electric field amplitude should be enough to cause the transmembrane voltage to reach a permeabilization threshold. This threshold depends on the size of the molecule to be internalised. This is why, at 100 V/cm, which is a very low electric field amplitude, no Ca2+ spike was observed because there was no permeabilization and hence no Ca2+ entry. 120 V/cm was the lowest electric field amplitude at which some cells presented Ca2+ spikes. This threshold is very low compared to other ones previously reported, because the Ca2+ has a small size compared to other classical markers such as yo-pro-1 iodide, propidium iodide or bleomycin that are those frequently used . At 450 V/cm, 100% of the cells responded to the electric pulse. The origin of the Ca2+ causing the Ca2+ spike was not always the same: it could be the result of Ca2+ entry from the external medium only, or the combination of Ca2+ entry from cells outside and Ca2+ release from the ER. Our group demonstrated recently that the μsPEFs could permeabilize not only the cell membrane  but also the internal organelles membranes . The origin of the Ca2+ spike depends on the electric field amplitude. If the latter is below 500 V/cm, the pulse permeabilizes only the plasma membrane , and the Ca2+ spike is primarily the result of Ca2+ entry through the plasma membrane followed by an amplification due to Ca2+ entry from the voltage-operated Ca2+ channels (VOCCs), activated by the membrane depolarisation [2+ release from the ER . Hence, the Ca2+ spike at high electric field amplitudes, resulted primarily of a massive Ca2+ entry through the plasma membrane (due to the generation of larger pores at the higher field amplitudes) also followed by the external Ca2+ entry through the VOCCs, the Ca2+ release from the ER and the subsequent stimulation of the store-operated Ca2+ channels (SOCCs) [2+ spike induced by electric fields below 500 V/cm had a very similar shape to the spontaneous Ca2+ oscillations with a gradual increase and same amplitude, whereas the Ca2+ spike induced by electric fields above 500 V/cm had higher amplitude than the spontaneous oscillations, and presented a sharper rise (due to the abrupt penetration of Ca2+ through the permeabilized membranes). At 600 V/cm, three different outcomes were observed, mainly due to four factors: first the distribution of the electric field which results in field amplitude small differences in the area between the two electrodes; second, the cells are heterogeneous in size and according to Schwan equation [2+ oscillations inhibited.Electric pulses of 100 μs duration are already applied in many biotechnological and medical applications, notably in anticancer electrochemotherapy , tumour risation . However (SOCCs) . This exequation , the eleequation ; fourth,2+ spikes obtained looked like the Ca2+ spikes induced by even shorter electric pulses . In our2+ oscillations for some time allowed us to study the state of the cell during this period, to determine whether the cell was in a “refractory state” or if it was still able to respond to an electric pulse. The application of several pulses after a 900 V/cm pulse inhibition showed that the cells could still react to the pulse and present Ca2+ spikes with different amplitudes depending on the applied electric field amplitude.The inhibition of the CaSeven days after exposure, MSCs showed similar stem cell surface antigens expression compared with non-treated MSCs.Four days after exposure to the eight electric pulses, MSCs were cultured in osteogenic and adipogenic differentiation media and successfully differentiated into osteoblasts and adipocytes.Eleven days after exposure to the electric pulses, MSCs cultures yielded similar cell growth rates compared with non-treated MSCs cultures.Last but not least, the application of one 100-μs electric pulse did not affect the cell viability 24 hours after treatment Fig. , even wh2+ spikes can have wide effects on the Ca2+ oscillations frequency, amplitude and duration. Whereas the nanosecond pulses allowed only to add a Ca2+ spike to the oscillations [2+ spike without affecting the oscillations, or to inhibit the Ca2+ oscillations and impose on the cell Ca2+ oscillations at a desired frequency and amplitude. It is worth noting that the microsecond technology is cheaper than the nanosecond one, it is more available in the laboratories, and simple generators can deliver it. These considerations add further value to the observations reported here. Taking into account that each type of differentiated cell from MSCs has its own Ca2+ oscillation characteristics, and that these oscillations may play an important role in the decision of MSCs fate, the μsPEFs constitute a novel useful tool to control the oscillations and the differentiation of MSCs.The electrically induced Callations , the mic2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. The delivery of short PEFs of 100 μs, a robust technology easy to apply, could be therefore a promising way to investigate the role of the Ca2+ in MSCs physiology and biological processes such as proliferation and differentiation.hMSCs show great promise for regenerative medicine. As a function of the pulse(s) electric field amplitude, we report here that it is possible to generate in hMSCs a supplementary calcium spike with properties close to those of their spontaneous calcium oscillations, or, on the contrary, to inhibit their spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca


Since cytotoxic Ca2+ overload has such a central role in cell death, the PMCA represents an essential “linchpin” for the delicate balance between cell survival and cell death. In general, impaired PMCA activity and reduced PMCA expression leads to cytotoxic Ca2+ overload and Ca2+ dependent cell death, both apoptosis and necrosis, whereas maintenance of PMCA activity or PMCA overexpression is generally accepted as being cytoprotective. However, the PMCA has a paradoxical role in cell death depending on the cell type and cellular context. The PMCA can be differentially regulated by Ca2+-dependent proteolysis, can be maintained by a localised glycolytic ATP supply, even in the face of global ATP depletion, and can be profoundly affected by the specific phospholipid environment that it sits within the membrane. The major focus of this review is to highlight some of the controversies surrounding the paradoxical role of the PMCA in cell death and survival, challenging the conventional view of ATP-dependent regulation of the PMCA and how this might influence cell fate.The plasma membrane Ca PMCA is encoded by four separate genes (PMCA1-4) and numerous splice variants that give rise to specific tissue distribution, cellular localisation and functional diversity 2+ concentration ([Ca2+]i) below 300 nM (∼100 nM), due to its high affinity for Ca2+  2+ efflux pathway in non-excitable cells 2+]i2+ signalling has steadily increased. PMCA exhibits memory of past [Ca2+]i increases, suggesting an important role in regulating the frequency of Ca2+ oscillations The plasma membrane Ca2+ signalling, the importance of the house-keeping role of the PMCA should not be under-estimated, especially when one considers how important maintaining low resting [Ca2+]i is for cell survival and the prevention of Ca2+-dependent cell death. In this regard the PMCA can be regarded as the “last gatekeeper” for the maintenance of low resting [Ca2+]i; an essential “linchpin” for the delicate balance between cell survival and cell death 2+ overload associated apoptosis, but the PMCA is an ATP-driven pump and since ATP depletion induces necrosis, a decline in PMCA activity will accompany and exacerbate necrosis 2+-dependent apoptosis.Despite the emerging role of the PMCA in dynamic Ca22+]i, the autoinhibitory CaM-binding motif interacts with the catalytic site (first and second cytosolic loops) thereby inhibiting the PMCA  is also a major activator of PMCA and is thought to account for ∼50% of the activity of PMCA at rest In order to understand the role of the PMCA in cell death and survival it is necessary to highlight some of the key structural features and regulatory mechanisms. Structurally, PMCA consists of ten transmembrane domains, two cytosolic loops with both N- and C-terminal cytosolic tails  [1], [2]the PMCA A. When [the PMCA B [5]. ThThe N-terminal tail exhibits the greatest diversity between the different isoforms 33.12+]i and the prevention of cytotoxic Ca2+ overload, in order to understand the role of PMCA in cell death and survival it is important to explore the mechanisms of Ca2+-dependent cell death. Ca2+ has a critical yet paradoxical role in regulating cell death 2+ and mitochondrial Ca2+ are key contributors  2+ and thus facilitate Ca2+ uptake into the mitochondrial matrix only when cytosolic Ca2+ is very high. However, the molecular identity of MCU, and its various pore-forming subunits ) and accessory proteins (MICU1 and MICU2), has revealed that this process is a complex molecular machine 2+-dependency than originally thought 2+ dependent activation of key metabolic enzymes 2+ overload, which can occur when the PMCA is inhibited, MCU can lead to excessive mitochondrial Ca2+ overload and the production of reactive oxygen species (ROS) 2+ can activate the mitochondrial permeability transition pore (mPTP) 2+-mediated apoptosis +/K+-ATPase.Ca2+ can also amplify cell death by activating the proteolytic enzymes, calpains 2+ transport proteins 2+ signalling and cell death.Ca2+ signalling and cytosolic Ca2+ overload clearly promotes both apoptosis and necrosis. Intuitively therefore, impaired PMCA activity or reduced PMCA expression would be expected to promote Ca2+-mediated cell death, whereas maintenance of PMCA activity or PMCA overexpression is generally regarded to be cytoprotective. Although such remodelling of PMCA function and expression may have evolved to attenuate Ca2+-mediated cell death, paradoxically, this may also result in apoptosis resistance which is a major hallmark of cancer.Enhanced Ca3.22+ overload. However, ATP depletion during extensive metabolic stress has been suggested to be the determining factor whether a cell undergoes apoptosis or necrosis 2+ overload. This is largely because ATP is critical for many of the ATP-dependent apoptotic processes, but is not required for necrosis Although the physiological role of the PMCA has been debated for several years, the importance of the PMCA during cellular stress and under pathological conditions is undeniable. The nature of cell death (i.e. necrosis vs apoptosis) will largely depend on the extent of metabolic stress and cytosolic CaInterestingly, the anti-apoptotic factor Bcl-2/xL has been recently shown to inhibit PMCA activity in pancreatic acinar cells 2+ homeostasis can, in some cases, be cytoprotective 2+ signalling has been shown to promote the exocytotic loss of lysosomes, resulting in protection against TNF-induced cell death Paradoxically, the impairment of PMCA function and the subsequent dysregulation of cytosolic Ca2+-dependent necrosis (PMCA1) or caspase-dependent apoptosis (PMCA4) In MDA-MB-231 breast cancer cells the expression of different PMCA isoforms differentially regulate cell fate by protecting against either Ca2+ transport and the majority of the Ca2+ composition within milk produced by normal mammary epithelial cells. During weaning, PMCA2 is down-regulated when the secretory cells die from apoptosis 2+ thereby increasing the sensitivity of Ca2+-dependent apoptosis In breast epithelial cells, the PMCA has an important role in the normal physiology and in tumorigenesis. During lactation PMCA2 is up-regulated at the apical membrane where it contributes to apical Ca2+]i and mitochondrial Ca2+ overload, which intuitively one might expect would protect against Ca2+-dependent apoptosis. However, this also leads to ER Ca2+ depletion, leading to ER-stress (activation of inositol-requiring enzyme 1α/X-box binding protein 1 pathway (IRE1α-XBP1) and inhibition of Activating Transcription Factor 6 (ATF6)) and the consequent activation of caspase-mediated apoptosis Insulin-secreting β-cells (BRIN-BD11) also exhibit a functional paradox. Overexpression of PMCA reduces resting i. Indeed, acute metabolic stress induced by pancreatitis-inducing agents markedly inhibited PMCA in pancreatic acinar cells 2+ overload and necrotic cell death 2+-dependent apoptosis but not necrosis.In most healthy cells under physiological conditions, when the bulk cytosolic ATP concentration is saturating for the PMCA (i.e.> 1 mM), such close functional coupling between glycolytic enzymes and the PMCA is likely to be of minimal functional significance. In other words, the PMCA does not care whether ATP comes from a mitochondrial or glycolytic source. However, in the face of impaired mitochondrial metabolism, for example under conditions of cellular stress, a glycolytic source of ATP might be critical for maintaining PMCA activity and thus restoring low resting cytosolic [Ca4.1in vitro cell-free assays of ATPase activity, whereby PS/PI was either absent or present in an artificial membrane, making it difficult to extrapolate these findings to intact cells. It is therefore unclear what the critical concentration of PS is to maintain “normal” ATP-sensitivity of the PMCA or whether this relationship is influenced by dynamic changes in Ca2+, Mg2+, CaM or other membrane lipids. However, functional studies in intact endothelial cells have shown that the loss of phosphatidylserine from the inner leaflet of the plasma membrane, following cholesterol depletion with β-methyl-cyclodextrin, inhibited PMCA activity 2+-dependent apoptosis before ATP declines sufficiently to trigger necrosis.Another important caveat when considering the ATP-sensitivity of the PMCA is that acidic phosphoplipids, such as phosphatidylinositol (PI), phosphatidylcholine (PC) and phosphatidylserine (PS) regulate the ATP sensitivity of the PMCA and mimic regulation by CaM 4.22O2) can directly oxidise PMCA and also oxidise calmodulin, which is the main activator of PMCA 2O2 has been reported to reduce the functional expression of PMCA at the plasma membrane of cultured hippocampal neurons within 1–2 h 2+ efflux during metabolic stress even in the presence of continued high ATP levels.Severe mitochondrial stress, whatever the mechanism, often leads to the generation of reactive oxygen species (ROS) 4.32O2 and Ca2+The release of cytochrome C from the mitochondria and the subsequent activation of caspases and calpain 52+-dependent cell death is central.In summary the PMCA represents an important regulator of cell death, both apoptosis and necrosis. Although impaired PMCA activity is generally regarded as being cytotoxic and the maintenance of PMCA activity is regarded as being cytoprotective, this rather simplistic view is by no means clear-cut. The PMCA can be differentially regulated by a vast repertoire of additional signalling pathways, proteolytic cleavage and the ATP-dependency and ATP supply is even more complex than originally thought. This raises the question as to whether the PMCA deserves greater recognition in the Pantheon of calcium signalling machinery than it currently has. Moreover, the regulation of the PMCA may offer a relatively untapped and rich tapestry of novel therapeutic targets for a variety of diseases in which Ca


Ca2+ dysregulation may plausibly be initiated in IBM by immune-mediated membrane damage and/or abnormally accumulating proteins, but no studies to date have investigated Ca2+ regulation in IBM patients. We first investigated protein expression via immunoblot in muscle biopsies from IBM, dermatomyositis, and non-myositis control patients, identifying several differentially expressed Ca2+-regulatory proteins in IBM. Next, we investigated the Ca2+-signaling transcriptome by RNA-seq, finding 54 of 183 (29.5%) genes from an unbiased list differentially expressed in IBM vs. controls. Using an established statistical approach to relate genes with causal transcription networks, Ca2+ abundance was considered a significant upstream regulator of observed whole-transcriptome changes. Post-hoc analyses of Ca2+-regulatory mRNA and protein data indicated a lower protein to transcript ratio in IBM vs. controls, which we hypothesized may relate to increased Ca2+-dependent proteolysis and decreased protein translation. Supporting this hypothesis, we observed robust (4-fold) elevation in the autolytic activation of a Ca2+-activated protease, calpain-1, as well as increased signaling for translational attenuation (eIF2α phosphorylation) downstream of the unfolded protein response. Finally, in IBM samples we observed mRNA and protein under-expression of calpain-3, the skeletal muscle-specific calpain, which broadly supports proper Ca2+ homeostasis. Together, these data provide novel insight into mechanisms by which intracellular Ca2+ regulation is perturbed in IBM and offer evidence of pathological downstream effects.Sporadic inclusion body myositis (IBM) is the most common primary myopathy in the elderly, but its pathoetiology is still unclear. Perturbed myocellular calcium (CaThe online version of this article (doi:10.1186/s40478-017-0427-7) contains supplementary material, which is available to authorized users. Phenoty T cells . The mol T cells , 14. How T cells . Current2+) concentrations (~50nM) are tightly maintained by an array of Ca2+ sensors, channels, transporters, and buffers [2+ elevations can cause various downstream myodegenerative phenomena, including two strongly associated with IBM pathogenesis [2+ dysregulation contributes to abnormal proteostasis by promoting mitochondrial reactive oxygen species (ROS) production and perturbing protein folding in the endoplasmic reticulum (ER) lumen [2+-dependent proteases downstream of Ca2+ dysregulation may cause TDP-43 cytosolic mislocalization (a specific finding in IBM vs. other inflammatory myopathies) [2+ dyshomeostasis is closely linked with mitochondrial dysfunction. Excessive Ca2+ uptake by the mitochondria can stimulate ROS production, initiate formation of the permeability transition pore, and diminish the inner mitochondrial membrane potential critical for oxidative phosphorylation [In healthy skeletal muscle fibers, low resting intracellular calcium  lumen , 31. Addpathies) . Anotherpathies) . As withrylation , 13, 31.2+ dysregulation, abnormal proteostasis, and mitochondrial dysfunction makes Ca2+ regulation a phenomenon of potential pathological significance in IBM. Although Ca2+ regulation has not yet been investigated in human cases of IBM, data exist suggesting that some proteins associated with IBM can mediate an insult to Ca2+ homeostasis [2+ regulation may plausibly stem from membrane pores and osmotic stress, as clonal cytotoxic T-cells (expressing membranolytic enzymes) have been reported in IBM and may have a particularly aggressive phenotype [2+ regulation in IBM, we hypothesized that human cases of IBM would display downstream gene and protein-level evidence of alterations consistent with Ca2+ dysregulation. In this study, we compared muscle biopsies from IBM patients with those of DM (an inflammatory myopathy without prominent degenerative characteristics) patients and non-myositis controls, investigating a panel of Ca2+-regulatory proteins as well as the Ca2+ signaling transcriptomic pathway. To explain observed alterations, we investigated proteolytic activation of ubiquitously expressed calpain-1, expression of the skeletal muscle specific calpain-3, and ER stress-induced translational attenuation.The direct link between Caeostasis , 34, 46.henotype , 20, 44.All procedures were completed under Institutional Review Board (IRB) approved protocols of the University of Maryland and Johns Hopkins University School of Medicine. Patients from the Johns Hopkins Myositis Center consented to use of their snap-frozen, rectus femoris biopsy samples for IRB-approved research purposes. Patients in the IBM group met ENMC 2011 criteria for clinically defined IBM whereas patients in the DM group met Bohan and Peter criteria , 21, 47 Samples were prepared as previously described . Brieflye) value of 7 or higher. Paired-end libraries were prepared using 50 ng of input RNA with the NeoPrep™ system according to the TruSeq™ Stranded mRNA Library Prep protocol (Illumina) and subsequently analyzed using the Illumina HiSeq 2500 machine. Reads were demultiplexed using Casava 1.8.2 and the quality of the resulting fastq files was tested using FastQC 0.11.2. There was no need to mask or trim the reads, as all nucleotide positions had a median Phred score over 30. Reads were aligned to the reference genome (hg19) using Tophat 2.0.134. The fragments per kilobase of exon per million fragments mapped (FPKM) values of each gene in each group were compared using Cuffdiff (Cufflinks 2.2.1) [2+-regulatory proteins were divided by FPKM values for each protein. Group means for IBM and controls were compared using Student’s t test.Samples for RNA sequencing were prepared using a standard TRIzol protocol. Briefly, biopsies were homogenized in TRIzol using 1.4 mm ceramic bead low-binding tubes. After phenol-chloroform extraction, RNA was purified and treated with DNase using the RNeasy Mini Kit (Qiagen). Concentration and quality of the resulting RNA was assessed using standard NanoDrop and TapeStation protocols, respectively. Samples were included in analysis with a RNA quality (RINs 2.2.1)  and the s 2.2.1) . Pathways 2.2.1) . Post-hoCalpain-1 autolysis was assessed by quantifying full-length (80 kDa) calpain-1 band densitometry, in arbitrary units, as well as cleaved (78 and 76 kDa) isoforms using a method previously described . BrieflyTo induce downstream effectors of the UPR, an ER stress sensor, Ire1α, facilitates alternative splicing of X-Box Binding Protein 1 (XBP1) mRNA . We quanAll statistical tests and parameters were established, and all experimental data collection and analysis were performed, while the responsible investigators were blind to sample identity. All protein expression values, as quantified by densitometry, were expressed as fold change vs. controls with error bars representing standard error of the mean (SEM). Student's t test, using pre-specified parameters, was used as the primary moderator of significant differences between experimental groups for immunoblots. Where appropriate, false discovery rate  was used to adjust the significance of analyses for multiple comparisons .2+-regulatory proteins that have been implicated in skeletal myopathy  proteins, SERCA1 and SERCA2a, are critical intracellular Ca2+ buffering proteins in fast and slow skeletal muscle, respectively. The SERCA proteins function to actively transport Ca2+ from cytosol to the SR lumen and are subject to cytosolic Ca2+ concentration-dependent proteolysis by calpains [p < 0.01) and 57% in DM (p < 0.01). SERCA2 was reduced 51% in IBM compared to controls (p < 0.05) and showed a non-significant trend toward reduction compared to DM . Calsequestrin (CSQ) has an important Ca2+-binding function in the SR, serving as a buffer to reduce effective Ca2+ concentration in the SR lumen and augment SERCA function [p < 0.05) and DM (p = 0.05). The mitochondrial Ca2+ uniporter (MCU) is an inner mitochondrial membrane complex that buffers the cytoplasmic concentration of Ca2+ by facilitating entry of Ca2+ into the mitochondrial matrix. We detected a 75% increase in MCU expression in IBM vs. both controls (p < 0.01) and DM (p < 0.01). The skeletal muscle RyR1 is the primary Ca2+ release channel of the SR, has altered Ca2+ gating after exposure to ROS, and is dynamically regulated by calpain cleavage [p < 0.05), but no difference between IBM and DM (p = 0.12). The DHPR is an L-type sarcolemmal Ca2+ channel that allows Ca2+ influx from the extracellular space and regulates RyR1-dependent Ca2+ release during excitation-contraction coupling. DHPR expression was decreased in both IBM (p < 0.05) and DM (p < 0.05) vs. controls, but did not significantly differ between IBM and DM (p = 0.17). We did not detect any differences between groups in expression of leucine zipper and EF-hand containing transmembrane protein (LETM1), a mitochondrial Ca2+/H+ antiporter, or stromal interaction molecule 1 (STIM1), an SR protein that acts a sensor of Ca2+ levels within the SR lumen . Together, these observed alterations are consistent with elevated basal Ca2+ levels in IBM myofibers, which we predicted would also result in transcriptomic alterations.We used immunoblotting to investigate the expression of a pre-specified panel of Cathy Fig. . The sarcalpains . Comparefunction , 42. CSQcleavage , 45. We 2+ signaling pathway (an unbiased gene list), were investigated from whole-transcriptome data. From these 183 genes, 54 (29.5%) were differentially expressed  < 0.05; Fig. 2+ buffer. PVALB mRNA was increased 2.7-fold in IBM samples (q < 0.001). Dysregulation of the canonical Ca2+ signaling pathway, as assessed using Ingenuity Pathway Analysis, was significant (q < 0.01). Using an established statistical approach to relate genes with causal regulatory networks [2+ abundance was a significant upstream regulator of the observed whole-transcriptome changes . Full Ca2+ signaling gene list data, with read quantity, fold change, and q values, are available in Additional file 2+-regulatory proteins assessed in this study, IBM had significantly less (p < 0.05) protein per transcript than control biopsies . However, in IBM samples, we detected prominent 78 and 76 kDa bands, reflecting proteolytically active isoforms  and DM (p < 0.05)  and 50% vs. DM (p < 0.05). DM and controls did not significantly differ in calpain-3 protein expression (p = 0.22).Since our data implicated cytosolic Caproteins , 49. Calmunoblot , 50. Totrms Fig. . Chemilu05) Fig. . Unlike ic triad , 39, 51.ic triad , and tragulation . At the 2+-regulatory proteins. Protein accumulation and Ca2+ dysregulation activate ER stress signaling and the UPR to restore normal proteostasis. Initiated by alternative splicing of XBP1, the UPR up-regulates effector molecules (e.g. chaperone proteins) but broadly halts protein translation through phosphorylation of eIF2α. Compared with controls, XBP1 mRNA was upregulated and preferentially spliced to its UPR effector form in IBM  and DM (p < 0.01)  Fig. . These d2+ is a ubiquitous second messenger in skeletal muscle, regulating processes as varied as secretion, contraction, and apoptosis. In healthy myofibers, intracellular Ca2+ concentration is maintained in a tight homeostasis by various Ca2+-regulatory proteins which function as ion sensors, channels, transporters, and buffers. Diminished Ca2+ regulation, typically resulting in pathologically high levels of cytosolic Ca2+, plays a pathogenic role in several neurodegenerative and neuromuscular diseases and has many upstream causes [2+ dysregulation is linked with RyR1 alterations in Alzheimer’s disease [2+ dysregulation include mitochondrial dysfunction and abnormal proteostasis, two likely contributors to myodegeneration in IBM [2+ regulation in IBM muscle, associate these changes with Ca2+ overabundance, and identify potential mechanisms contributing to Ca2+ dysregulation. This novel study of Ca2+ regulation in myositis patients strongly supports the hypothesis that Ca2+ dysregulation is present in and pathologically relevant to IBM.Cam causes , 56. For disease , mutant  disease , and sar disease . Patholon in IBM . In this2+ concentrations are likely elevated. Compared with controls, both DM and IBM had reduced levels of RyR1 and DHPR protein, which may reflect a non-specific change associated with muscle damage or inflammation. Of note, reduced protein levels of SERCA1, the fast-twitch SERCA isoform, was previously reported in a proteomic study of IBM that describes broad expression reductions in fast twitch proteins [2+-regulatory proteins have not been reported in the three other IBM proteomic studies to date, although these studies each report alterations in only 29 proteins or fewer [Compared with control and DM muscle biopsies, IBM samples had decreased SERCA1, SERCA2, and CSQ protein and increased MCU protein, supporting mechanisms by which both cytosolic and mitochondrial Caproteins . Interesproteins . We obseor fewer , 29, 30,2+ pathway. Primarily, these studies use microarrays to screen for genes with high comparative expression changes vs. controls and other inflammatory myopathies. Our approach differed, pre-specifying an unbiased list of genes  from which to extract transcript expression information and perform bioinformatics analysis. This approach allowed for detection of subtle but still highly significant expression changes at the gene and pathway level. Our analyses revealed the Ca2+ signaling canonical pathway to be significantly altered in IBM, with 54 of 183 genes (29.5%) differentially expressed vs. control muscle after correction for multiple comparisons. Several gene expression alterations imply myocellular compensation for the loss of Ca2+ homeostasis, such as upregulation of PVALB (a Ca2+ buffer) and suppression of phospholamban , which would increase cytosolic Ca2+ buffering and SR Ca2+ uptake, respectively. Using the upstream regulator analysis function within IPA, which predicts activation status of upstream molecules using a large dataset of causal experiment data, whole-transcriptome changes observed in IBM were determined to be highly consistent with myocellular Ca2+ abundance [Prior studies have investigated the IBM transcriptome, although no study to our knowledge has reported on alterations in the Cabundance .2+-activated proteases. Robust (~4-fold) elevations in autolytic activation of the Ca2+-activated protease calpain-1 in IBM provides strong evidence of abnormally high cytosolic Ca2+ concentration, and may explain specific protein expression decreases  that were out of proportion with their respective mRNA decreases. It is worth noting again that neither calpain-1 transcript nor overall protein level were increased in IBM, indicating that calpain-1 abnormalities in IBM are primarily post-translational. In neurons, moderate calpain-1 activation (as we observe in IBM samples) causes cleavage of TDP-43 into aggregation-prone fragments, promoting the TDP-43 cytoplasmic mislocalization (TDP-43 proteinopathy) observed in amyotrophic lateral sclerosis [2+-activated protease investigated in this study, calpain-3, was reduced in IBM samples vs. controls and DM. This finding is consistent with a hypothesis proposed in a previous proteomic study, in which calpain-3 substrates were among the few over-expressed proteins in IBM samples [2+ dysregulation may be initiated or exacerbated and may have broader implications for myofiber adaptation to damage [Perhaps our most interesting finding is that IBM displays alterations in the activation status and expression of Caclerosis . As TDP-clerosis , 33 and clerosis , calpain samples . Moreove samples . Thus, do damage , 39.2+-regulatory proteins. Of additional interest, activation of the UPR can induce local inflammation, which may contribute to repression of the gene encoding calpain-3 (CAPN3), further linking these phenomena [Abnormal proteostasis and downstream activation of the UPR have long been hypothesized to contribute to IBM pathology . Supporthenomena .2+ dysregulation and other pathogenic mechanisms in IBM is complex, but possibly significant for disease progression. To illustrate this relationship, we propose a theoretical three-phase mechanism of Ca2+ dysregulation in IBM that integrates our data with the literature  sarcolemma damage and subsequent osmotic stress stemming from expanded and potentially-aggressive cytotoxic T-lymphocytes (CTLs) [2+ channel function and/or mitochondrial Ca2+ buffering due to cytotoxic protein oligomers or fibrils [2+ homeostasis, Ca2+ dysregulation progresses in the propagation phase. Mitochondria, susceptible to injury in IBM due to conditions of inflammation, mtDNA abnormalities, and impaired proteostasis, are damaged by excessive Ca2+ influx [2+ efflux from the SR [2+ elevations potentiate the activation of calpains , which cleave Ca2+-regulatory proteins like SERCA1 [2+ and redox imbalances in the ER perturb protein folding, inducing UPR signaling [2+-regulatory proteins, and suppression of translation cause progressive increases in cytosolic Ca2+. In the myofiber pathology phase, mitochondrial damage has diminished the capacity for oxidative phosphorylation through reduction of the inner mitochondrial membrane potential, prolonged ER stress has altered myofiber viability through induction of pro-apoptotic factors (e.g. CHOP), and calpain-1 activation has directly contributed to weakness by degrading proteins involved in excitation-contraction coupling (e.g. RyR1).The relationship between Caure Fig. . While ts (CTLs) , 20, 44; fibrils , 34, 46; fibrils , 51. Wit+ influx , 32, 48.m the SR  and ER se SERCA1 , 50. Ca2ignaling , 32. Tog2+ dysregulation in IBM by interrogating the association between indicators of Ca2+ dysregulation  and markers of potential upstream causes (e.g. protein aggregation) and/or attempting to functionally recapitulate aspects of this phenomenon in model systems. A limitation of this study was the use of a restricted number of biopsies, from different patients, in immunoblot and RNA-seq studies. This limits the strength of protein to transcript comparisons, although our finding of reduced protein to transcript amongst Ca2+-regulatory proteins was well supported by subsequent experiments . Finally, while we did not find substantial Ca2+-associated abnormalities in our myositis control (DM) patients, future studies may look to address whether the pathologies of other inflammatory myopathy subsets (e.g. patients with necrotizing myopathies or specific autoantibodies) include Ca2+ dysregulation.This study provides new insights into a potential contributor to IBM pathogenesis, but further research is needed. Future studies may investigate the initiation of Ca2+ dysregulation in the myocellular pathology of sporadic IBM. While it is still unclear which theoretical insult(s) are upstream of Ca2+ dysregulation in IBM, our data suggest that this phenomenon is propagated by reduced expression of calpain-3, abnormal proteolysis secondary to calpain-1 activation, and decreased protein translation downstream of the UPR. While Ca2+ dysregulation is unlikely to be a primary pathogenic mechanism in IBM, it may contribute to muscle atrophy and weakness through its pleiotropic effects on protease dynamics, gene expression, myocellular proteostasis, and mitochondrial function. As such, future investigations may investigate if targeted treatment aimed to restore Ca2+ homeostasis and/or limit the downstream effects of prolonged Ca2+ dysregulation may be a viable therapeutic strategy in IBM.This investigation provides data, from whole-transcriptome analysis to specific proteins alterations, that implicate CaAdditional file 1:2+ signaling pathway, including gene name and locus, mRNA expression (Fragments Per Kilobase of transcript per Million mapped reads), fold change, and comparison false discovery rate . (PDF 289 kb)Electronic Resource 1: RNA-sequencing data for genes in the KEGG CaAdditional file 2:P > 0.10), suggesting a lack of fiber-type specificity in the reduction of SERCA proteins. (DOC 50 kb)Electronic Resource 2: The ratio of SERCA1 to SERCA2 protein is unaltered between groups (all