KL impacts the aggressiveness of liposarcomas, in which IGF-1R signaling is frequently upregulated. Indeed, we observed that a higher KL expression in liposarcomas is associated with a better outcome for patients. Moreover, KL is downregulated in dedifferentiated liposarcomas (DDLPS) compared to well-differentiated tumors and adipose tissue. Because DDLPS are high-grade tumors associated with poor prognosis, we examined the potential of KL as a tool for overcoming therapy resistance. First, we confirmed the attenuation of IGF-1-induced calcium (Ca2+)-response and Extracellular signal-Regulated Kinase 1/2 (ERK1/2) phosphorylation in KL-overexpressing human DDLPS cells. KL overexpression also reduced cell proliferation, clonogenicity, and increased apoptosis induced by gemcitabine, thapsigargin, and ABT-737, all of which are counteracted by IGF-1R-dependent signaling and activate Ca2+-dependent endoplasmic reticulum (ER) stress. Then, we monitored cell death and cytosolic Ca2+-responses and demonstrated that KL increases the reticular Ca2+-leakage by maintaining TRPC6 at the ER and opening the translocon. Only the latter is necessary for sensitizing DDLPS cells to reticular stressors. This was associated with ERK1/2 inhibition and could be mimicked with IGF-1R or MEK inhibitors. These observations provide a new therapeutic strategy in the management of DDLPS.By inhibiting Insulin-Like Growth Factor-1-Receptor (IGF-1R) signaling, Klotho (KL) acts like an aging- and tumor-suppressor. We investigated whether  The heterogeneous family of soft-tissue sarcomas (STS) encompasses rare malignant tumors that arise from mesenchymal tissue. The histological and molecular analyses of these tumors allow for the identification of numerous histotypes, including liposarcomas (LPS) that display an adipocytic differentiation and are divided into four histological subtypes: well-differentiated (WDLPS), dedifferentiated (DDLPS), myxoid round cell (MRCLPS), and pleomorphic (PLPS) liposarcomas ,2. WDLPSFor now, complete surgical resection is the mainstay of therapy for LPS patients. Due to the resistance of these tumors to standard chemotherapies , patientIGF-1 and IGF-2 mRNA ER nor enhances the reticular Ca2+ pools releasable by ionomycin, but significantly enhances the amplitude of the [Ca2+]ER decrease induced by TG  confirmed a greater accumulation of Ca2+ in mitochondria of IB115-KL cells (2+] in KL-overexpressing cells compared to the control cell line (2+-stores depletion) and a probable mitochondrial Ca2+ overload.This implies that, in resting conditions, an increased CaKL cells . Moreoveell line . Therefo2+-content of control cell line after 24 and 48 h of treatment  by BTP2 did not protect IB115-KL cells from gemcitabine-induced cell death  channel. Indeed, in neural cancer cells, TRPC6 can be translocated to the reticulum and this localization results in an increase in passive Ca2+-efflux without any modification of the reticular Ca2+ content  increase was observed in IB115-empty vector cells bathed in the Ca2+-containing medium, but not in Ca2+-free medium, meaning that the previous cytosolic Ca2+ increase was due to a Ca2+ influx through TRPC channels at the plasma membrane  transient Ca2+ peak, which was only partially affected by the absence of Ca2+ in the extracellular medium  that activates Cachannels . After tmembrane A. In IB1r medium B. This s release C, sugges2+-free medium and stimulated with Hyp9, a specific activator of TRPC6. Similar to OAG stimulation, a cytosolic Ca2+-response was recorded in IB115-KL but not the B115-empty vector cells (2+-response to OAG (2+-leak. Pretreatment with a specific inhibitor of TRPC3 (Pyr3) combined to larixyl acetate suppressed OAG-response  reduced the TG-induced CaKL cells H. ActualKL cells H, while KL cells I. Howeveed by TG ,45,46. T2+-release and so, probably promote ER stress, these last results altogether suggest that TRPC6 sequestration at the ER is probably not the only and major mechanism by which Klotho sensitizes to TG- and gemcitabine-induced cell death.Despite the fact that intracellular TRPC6 channels take part in TG-induced Ca2+-leak is the translocon (TLC) cyt followed by Ca2+-oscillations in IB115-KL cells  . Indeed,pression , Ca2+-reuromycin  and to Turomycin , and alsuromycin  or gemciuromycin . To concenotypes .2+-leakage.Then, we investigated which IGF-1 signaling pathway is implicated in the resistance to ER stressors and is inhibited by Klotho and addressed its impact on reticular Ca2+-response  and MEK (PD98059) inhibitors . The Wor2+-leakage. First, the pretreatment of control cells with BMS-754807 .Finally, we addressed the consequence of IGF-1R and MEK inhibition on reticular CaS-754807 D or PD98S-754807 E restoreresponse F without-content . TherefoIn this work, we evaluated the relevance of Klotho as a biomarker and as a tool for overcoming drug resistance in high-grade LPS.KL expression is associated with better survival of LPS patients. Moreover, its expression is detected at higher levels in WDLPS, which has a good prognosis. KL expression is critical for adipose tissue homeostasis but is altered in DDLPS, probably by epigenetic regulations as in several other cancers cyt). One field was acquired from each coverslip and the data were pooled from six independent coverslips on three different days. Data were processed using OriginPro 7.5 software . On graphs, data were summarized as the mean ± standard deviation (SD).For calcium imaging, 100,000 cells were seeded onto glass coverslips (25 mm dia.) and incubated in a complete culture medium for 48 h. Single-cell cytosolic calcium imaging was performed, using Fluo2 LR-AM calcium dye as previously described . FluoresKL expression values (estimated with robust multi-array average (RMA)) were obtained with the web tool GEO2R. Patients were divided into groups according to the KL expression value . Survival rates were estimated using the Kaplan–Meier method and compared using the log-rank test using XL-STAT v2018.1  software.The cohort of 140 primary liposarcomas is from Gobble et al.  (GSE3092t-test and the results were represented as the mean ± standard deviation (SD). Otherwise, a Mann–Whitney test was used and the median with interquartile range (IQR) was plotted. For western blot experiments, paired tests were used. Significant differences are represented as * if p-value p < 0.05, ** if p < 0.01 and *** if p < 0.001.Each experiment was repeated at least three times. To examine the statistical significance of the results, analyses were performed with the Prism6 v6.01  software. The normal distribution of datasets was examined with a Shapiro–Wilk normality test. If the data passed the normality test, the statistical significance between the two conditions was assessed with an unpaired 2+ leak and is induced by at least the attenuation of the IGF-1/ERK signaling pathway. Further experiments should be performed in order to identify the molecular players modulating TLC opening and regulated by ERK and Klotho. This would provide new therapeutic targets for overcoming drug resistance of DDLPS.In this study, we evidence that Klotho is downregulated in DDLPS tumors and that its re-expression in DDLPS cell lines enhances the apoptotic response to ER stressors. Its mechanism of action mainly relies on the opening of the TLC, which mediates reticular Ca


There is an increased association between chronic urticaria (CU) and thyroid autoantibodies. We presented a case of a patient suffering from CU and newly diagnosed Hashimoto’s thyroiditis in which levothyroxine therapy completely resolved the symptoms of urticaria. A 58-year-old female with a past medical history of recurrent urticaria presented with complaints of fatigue and weight gain. Laboratory investigations showed a high thyroid stimulating hormone (TSH) level and a low free thyroxine (T4) level. She had elevated levels of antithyroid peroxidase (anti-TPO) and anti-thyroglobulin antibodies (anti-TG). She was diagnosed as a case of Hashimoto's thyroiditis and started on levothyroxine therapy. She noticed that her uncontrolled recurrent urticaria started to get better and after a few months of therapy, she stopped taking her topical ointments and antihistamines. The reason for the association between positive serological tests for thyroid autoimmunity and CU is unclear. The resolution of chronic urticaria with levothyroxine in our patient with Hashimoto's thyroiditis suggests a common underlying mechanism between the two pathologies. Chronic urticaria (CU) is defined as recurrent episodes of urticaria, at least twice a week, occurring for six weeks . HashimoA 58-year-old female with a past medical history of hypertension, diabetes mellitus type 2, hyperlipidemia, vitamin D deficiency, obesity, allergic rhinitis, and uncontrolled recurrent urticaria presented to the clinic with fatigue and weight gain. Review of her medical records showed that her vitals were in the normal range, with blood pressure ranging from 132/70 mmHg, pulse rate 72/min, and weight 210 lbs with a body mass index (BMI) of 38.1 kg/m2. She denied smoking and alcohol intake. She was taking metformin 500 mg twice daily, rosuvastatin 50 mg, hydrochlorothiazide 25 mg, antihistamines, and nystatin-triamcinolone topical 100,000 units/G-0.1% ointment.Laboratory investigations showed raised thyroid stimulating hormone (TSH) levels as 14 mlU/ml and low levels of free thyroxine (FT4) as 0.4 ng/dl. hemoglobin A1c (HbA1c) was 6.1, eosinophil count was raised 6.5%  and eosinophils (absolute) 0.53x103 (N: 0.0-0.4x103), high antithyroid peroxidase antibodies (anti-TPO) 250 IU/ml (0.0-35 IU/mL), and antithyroglobulin antibodies (anti-TG) 437 IU/ml (N: <40 IU/Ml).Based on investigations, she was diagnosed as a case of Hashimoto’s thyroiditis. She was started on 50 mcg levothyroxine therapy, which was raised to 125 mcg to achieve euthyroid levels. She noticed that her uncontrolled recurrent urticaria started to get better, and after six months of levothyroxine therapy, her TSH was 1.77 mlU/ml and T4 level was 1.2 ng/dl, and the recurrent urticaria completely resolved. She quit taking her topical ointments and antihistamines that she had been using for urticaria. She is on regular follow-up every six months for the last two years and is symptom-free since then.Chronic urticaria (CU) has many possible etiologies. Establishing the cause of urticaria and its complete resolution is not always possible . Anti-FcAt the molecular level, TSH has many of the attributes of a cytokine, and it can regulate the immune responses mainly by direct T cell, B cell, and dendritic cell activation. The receptors of thyroid releasing hormone (TSH) and human prolactin expressed on the cells of the immune system. The mononuclear cells, monocytes, and splenocytes release a considerable concentration of serum TSH when treated with TSH releasing hormone. The cytokine receptors, particularly interleukin (IL) IL-1, 2, and 6, and tumor necrosis factor alpha are expressed on the hypothalamic-pituitary loop. When activated, they lead to the inhibition of TSH releasing hormone-induced thyroid stimulating hormone release. This effect gets amplified in Hashimoto’s thyroiditis. It leads to the continual release of various ILs and cytokines  by immune cells that could cause an inflammatory state of target organs such as skin . ThyroidThe resolution of urticaria after levothyroxine treatment, irrespective of the initial thyroid function status, has been reported by some authors. Aversano et al. studied CU and Hashimoto’s thyroiditis and found 80% of patients had an improvement of urticaria after 12 months of starting levothyroxine therapy . Kiyici In Hashimoto’s thyroiditis, thyroid antibodies are more likely present with overt hypothyroidism. Patients with antibody-negative Hashimoto’s thyroiditis had a milder form of hypothyroidism at the time of diagnosis. This could represent an earlier stage or a less aggressive form of Hashimoto’s thyroiditis . ThyroidThe resolution of chronic urticaria with levothyroxine in our patient with Hashimoto’s thyroiditis suggests a common underlying mechanism between the two pathologies. Definitive studies are needed before a strong recommendation for levothyroxine as a treatment for CU in patients can be made.We reported a case of a patient in which levothyroxine therapy completely resolved the symptoms of chronic urticaria. Solid pieces of evidence, based on well-conducted, controlled studies, are desirable to determine if there is a therapeutic role for levothyroxine treatment in ameliorating the manifestations of chronic urticaria.


OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non‐sensory cells of the greater epithelial ridge cause, via ATP‐induced activation of P2X3 receptors, the increase and synchronization of the Ca2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience‐independent Ca2+ signals from sensory and non‐sensory cells.Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine‐tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca The differentiation, maturation and maintenance of these neuronal connections require precise timing and coordination between genetic programmes and physiological activity  activity before the onset of function , OHCs show spontaneous Caet al, 2+ activity in immature OHCs and its modulation by non‐sensory cells in the greater (GER) and lesser (LER) epithelial ridges  for Ca2+ measured from active OHCs  extension of Ca2+ waves in the GER  had no significant effect on the correlation, all 19 of the larger waves analysed were able to synchronize the activity of several OHCs . We also found that both the increased OHC Ca2+ activity and the degree of correlation (rsavg) were independent from the amplitude (ΔF/F0) of the Ca2+ waves  from an average resting membrane potential of −70.8 ± 1.6 mV . In order to test whether Deiters’ cells mediate signal transfer from the GER to the OHCs, we analysed Ca2+ signals after removing a few Deiters’ cells beneath the area of interest  or in CaV1.3−/− mice , providing further evidence that ATP‐induced Ca2+ signals from these non‐sensory cells directly modulate OHC activity. Altogether these data indicate that ATP, acting through P2Y receptors in Deiters’ cells and P2X receptors in OHCs, can coordinate the activity of nearby OHCs.To determine the molecular mechanism linking activity in the Deiters’ cells with OHC synchronization, we pharmacologically probed the basolateral membrane of OHCs deprived of their surrounding Deiters’ cells Fig A. Deiternses Fig B. The OHt 100 μM . Under wtion Fig E and I amice Fig  indicateOHCs Fig F and I. OHCs Fig . When th2+ waves was also present in the intact cochlear preparation , large Ca2+ waves originating in the GER were still able to reach the LER, but failed to increase, and as such synchronize, the Ca2+ signals in OHCs .We then sought to investigate whether the purinergic signalling from Deiters’ cells to OHCs during Ca2+ waves originating in the GER are able to travel to the LER, where the OHCs reside, and induce the release of ATP from the non‐sensory Deiters’ cells. ATP activates P2X3 receptors in the basolateral membrane of OHCs, leading to OHC depolarization and an increased open probability of voltage‐gated CaV1.3 Ca2+ channels. This depolarization will increase the action potential frequency of the OHCs within the area of the Ca2+ wave, thereby increasing the probability of synchronized firing among adjacent OHCs.The above data show that Caet al, 2+ activity, we used Cx30−/− mice  was significantly reduced compared to that of wild‐type mice . Furthermore, the frequency of the larger Ca2+ events . The few remaining large Ca2+ waves in Cx30−/− mice were still able to synchronize the bursting activity of adjacent OHCs . As seen in wild‐type mice (rsavg was independent of the amplitude (ΔF/F0) of the Ca2+ signal measured as a pixel average over the entire spread of the Ca2+ wave  and Cx30 . The total K+ current in mature OHCs was similar between the two genotypes . The total outward K+ current (IK) and the isolated IK,n recorded from OHCs of Cx30−/− mice (P10–P12) were similar in size to that of wild‐type cells , which was obtained by subtracting the control currents from the currents in the presence of 100 μM ACh  and Cx30−/− . We further confirmed that the ACh‐induced currents in Cx30−/− OHCs were carried by the nAChRs and SK2 channels since they were blocked by strychnine , immature IHCs from Cx30−/− mice have been shown to be normal  and after (P10) their onset of functional maturation at ~ P8 , which are required for hair cell exocytosis. OHCs in P11 Cav1.3−/− mice are present and healthy  but not P4 (one‐way ANOVA P = 0.4497) compared to wild‐type cells  tunnel‐crossing outer spiral fibres per 100 μm along the length of the organ of Corti. Compared to wild‐type, Cx30−/− mice had fewer peripherin‐labelled outer spiral fibres  compared to wild‐type mice (P < 0.01). This result is in agreement with the significantly lower number of ribbon synapses in OHCs from Cx30−/− mice  compared to wild‐type mice but not significantly different from Cx30−/− mice  and IHCs  synchronizes Ca2+ activity between nearby OHCs via release of ATP from Deiters’ cells. This ATP acts directly via P2X receptors on the OHCs. The same activity in the GER synchronizes APs in IHCs  Act 1986 and were approved by the University of Sheffield Ethical Review Committee. Some experiments were performed in the USA, and the animal work was licensed by the Baylor University IACUC  as established by U.S. Public Health Service.Cx30−/− , amino acids and vitamins were added from concentrates . The pH was adjusted to 7.5 . The dissected organ of Corti was transferred to a microscope chamber, immobilized using a nylon mesh fixed to a stainless steel ring and viewed using an upright microscope . Hair cells were observed with Nomarski differential interference contrast optics (×63 water immersion objective) or Dodt gradient contrast (DGC) optics (×60 water immersion objective) and either ×10 or ×15 eyepieces.Apical‐ and basal‐coil OHCs from wild‐type mice or transgenic mice of either sex were studied in acutely dissected organs of Corti from postnatal day 0 (P0) to P13, where the day of birth is P0. Transgenic mice include 2, 1 EGTA‐KOH, 5 Na2ATP, 5 Hepes‐KOH and 10 Na‐phosphocreatine . In the experiments designed to investigate the effect of extracellular ATP, Na2ATP was omitted from the above solution. For cell‐attached recordings, the pipette contained (in mM): 140 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 NaH2PO4, 5.6 D‐glucose and 10 Hepes‐NaOH . Data acquisition was controlled by pCLAMP software (RRID:SCR_011323) using Digidata 1320A, 1440A or 1550 boards . Recordings were low‐pass filtered at 2.5 kHz (8‐pole Bessel) and sampled at 5 kHz and stored on computer for offline analysis . Membrane potentials in whole‐cell recordings were corrected for the residual series resistance Rs after compensation  and the liquid junction potential (LJP) of −4 mV measured between electrode and bath solution. The extracellular application of a Ca2+‐free solution or solutions containing 40 mM KCl, ATP  or acetylcholine  was performed with a multibarrelled pipette positioned close to the patched cells.Membrane currents and voltage responses were investigated either at room temperature (20–24°C) or near body temperature (33–37°C), using Optopatch  or Axopatch 200B  amplifiers. Patch pipettes, with resistances of 2–3 MΩ, were pulled from soda glass capillaries, and the shank of the electrode was coated with surf wax  to reduce the electrode capacitative transient. For whole‐cell recordings, the pipette intracellular solution contained (in mM): 131 KCl, 3 MgClet al, For calcium dye loading, acutely dissected preparations were incubated for 40 min at 37°C in DMEM/F12, supplemented with Fluo‐4 AM . The incubation medium contained also pluronic F‐127  and sulfinpyrazone (250 μM) to prevent dye sequestration and secretion  based on a mode‐locked laser system operating at 800 nm, 80‐MHz pulse repetition rate and < 100‐fs pulse width . Images were formed by a 60× objective, 1.1 NA  using a GaAsP PMT (Hamamatsu) coupled with a 525/40 bandpass filter . Images were analysed offline using custom‐built software routines written in Python  and ImageJ (NIH) . ΔF = F − F0, where F is fluorescence at time t and F0 is the fluorescence at the onset of the recording.Ca3 antagonist A‐317491 (Sigma) was performed using a Picospritzer or bath application. The pipettes used for local perfusion (diameter 2–4 μm) were pulled from borosilicate glass using a two‐step vertical puller . Pressure was kept at a minimum (< 3 psi) to avoid triggering mechanically induced calcium signals. Responses in each experimental condition were normalized to control experiments, carried out on the same day under the same imaging and dye‐loading conditions.The extracellular application of solutions containing ATP, ryanodine, the P2X antagonist suramin and PPADS (Tocris), the P2Y agonist UTP , the phospholipase C inhibitor U73122  and the P2X2+ variations and the exponential decay in fluorescence due to photo‐bleaching were removed by subtracting a polynomial fit of order 5 to each trace. Detrended traces were normalized to the maximum value in the recording. (iii) The noise floor level was estimated by calculating the power spectral density of the signal using Welch's method and averaging over the large frequencies (greater than 66% of the Nyquist frequency). (iv) A spike inference algorithm [spikes  if S was above (below) a predetermined threshold. (v) Cells that were classified as active (or inactive) and had a maximum signal below (or above) 4 standard deviations were manually sorted. (vi) The entire dataset was independently reviewed by two experimenters. Cells that had discording classification based on the above criteria  were removed from the analysis. For the experiments in which we calculated the Ca2+ spike frequency from Ca2+ imaging data  region. OHC fluorescence traces were computed as pixel averages from square ROIs (side = 3.7 μm) centred on each OHC. OHCs were classified as either active or inactive using the following algorithm: (i) imaging traces were smoothed using a moving average temporal filter of length 3. (ii) Slow Caing data , we firsa . Recordings showing large drifts of the preparation were discarded from the analysis to avoid potential artefacts in the computation of correlation. Calcium waves were manually identified using thresholding, and a ROI was drawn around the maximum extension of each multicellular calcium event. Only events that initiated within the field of view of the microscope were considered for this analysis. GER fluorescence traces were computed as ROI pixel averages, and as such they give an indication of the average cytosolic calcium increase in non‐sensory cells participating in the propagation of the Ca2+ wave. To measure the degree of correlation between OHCs during Ca2+ activity in the GER, we first computed the pairwise Spearman's rank correlation coefficient (rs) between every pair of OHCs in the field of view  to check whether OHC correlation coefficients during spontaneous Ca2+ activity in the GER were significantly (P < 0.001) greater than those computed over a time window of 13.2 s (400 frames) during which no Ca2+ waves were observed in the GER.To test for the increase in coordinated OHC activity, we used the Mann–Whitney 2+ waves in non‐sensory cells, we measured the integral of the Ca2+ trace in the same 400 frames (see above) in the absence of Ca2+ waves (baseline) and during Ca2+ waves. Traces were smoothed using the Savitzky–Golay filter  and normalized to the baseline F0 before computing the integral.To quantify the change in OHC activity during the Ca2+ waves were triggered by applying high‐intensity laser pulses using a second mode‐locked laser system operating at 716 nm . The laser was merged into the excitation light path using a long‐pass dichroic mirror  and focused on the preparation through the imaging objective . Two galvanometric mirrors were used to steer the laser beam across the photo‐damage area (6.6 × 8.4 μm), which typically comprised one or two non‐sensory cells of the GER. The number of repetitions, and thus the total amount of energy delivered, was set to the minimum able to trigger a Ca2+ wave .Photo‐damage‐induced CaCx30−/− and CaV1.3−/− mice (n ≥ 3 for each set of experiment) were fixed with 4% paraformaldehyde in phosphate‐buffered saline  for 5–20 min at room temperature. Cochleae were microdissected, rinsed three times for 10 min in PBS and incubated for 1 h at room temperature in PBS supplemented with 5% normal goat or horse serum and 0.3% Triton X‐100. The samples were then incubated overnight at 37˚C with the primary antibody in PBS supplemented with 1% of the specific serum. Primary antibodies were as follows: mouse anti‐myosin7a , rabbit anti‐myosin7a , rabbit anti‐peripherin , mouse anti‐CtBP2  and rabbit anti‐prestin . All primary antibodies were labelled with species appropriate Alexa Fluor secondary antibodies for 1 h at 37°C. Samples were then mounted in VECTASHIELD. The z‐stack images were captured either with a LSM 800 with Airyscan (Carl Zeiss) system with GaAsP detectors or with a Nikon A1 confocal microscope. Image stacks were processed with Fiji Image Analysis software.Dissected inner ears from wild‐type and t‐test or, for multiple comparisons, analysis of variance (one‐way or 2‐way ANOVA followed by Bonferroni's test) and Mann–Whitney U‐test  were applied. P < 0.05 was selected as the criterion for statistical significance. Mean values are quoted in text and figures as means ± SEM (electrophysiology and imaging) and ±SD (fibre counting). Only mean values with a similar variance between groups were compared. Animals of either sex were randomly assigned to the different experimental groups. No statistical methods were used to define sample size, which was defined based on previous published similar work from our laboratory. Animals were taken from multiple cages and breeding pairs. The electrophysiological and morphological (but not imaging) experiments were performed blind to animal genotyping.Statistical comparisons of means were made by Student's two‐tailed FC, AH, J‐YJ, SLJ, FS, JE, JO, DDS and WM collected and analysed the data. All authors helped with the interpretation of the results. FC, AH, MCH, CJK, DDS and WM wrote the paper. FM helped with the design of the 2‐photon imaging system. WM conceived and coordinated the study. SLJ is a Royal Society University Research Fellow.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Movie EV1Click here for additional data file.Movie EV2Click here for additional data file.Movie EV3Click here for additional data file.Movie EV4Click here for additional data file.Movie EV5Click here for additional data file.Movie EV6Click here for additional data file.Movie EV7Click here for additional data file.Movie EV8Click here for additional data file.Review Process FileClick here for additional data file.


FMS-like tyrosine kinase 3- internal tandem duplication (FLT3-ITD) remains as one of the most frequently mutated genes in acute myeloid leukemia (AML), especially in those with normal cytogenetics. The FLT3-ITD and FLT3-TKD (tyrosine kinase domain) mutations are biomarkers for high risk AML and are associated with drug resistance and high risk of relapse. Multiple FLT3 inhibitors are in clinical development, including lestaurtinib, tandutinib, quizartinib, midostaurin, gilteritinib, and crenolanib. Midostaurin and gilteritinib have been approved by FDA for Flt3 mutated AML. Gilteritinib  is a small molecule dual inhibitor of FLT3/AXL. The ADMIRAL study showed that longer overall survival and higher response rate are associated with gilteritinib in comparison with salvage chemotherapy for relapse /refractory (R/R) AML. These data from the ADMIRAL study may lead to the therapy paradigm shift and establish gilteritinib as the new standard therapy for R/R FLT3-mutated AML. Currently, multiple clinical trials are ongoing to evaluate the combination of gilteritinib with other agents and regimens. This study summarized clinical trials of gilteritinib for AML. Recurrent and novel genetic mutations are increasingly discovered through FISH, PCR and next-generation sequencing studies of leukemia specimens –5. TheseFLT3 gene encodes a receptor type tyrosine kinase which plays a key role in the proliferation, differentiation, and survival of hematopoietic stem cells. FLT3-ITD leads to constitutive activation of the FLT3 tyrosine kinase, resulting in uncontrolled cell proliferation and high WBC counts in AML patients , 13.The FLT3-ITD and FLT3-TKD (tyrosine kinase domain) mutations are biomarkers for high risk AML and are associated with drug resistance and high risk of relapse , 15, parGilteritinib is a small molecule dual inhibitor of FLT3/AXL. In a phase I/II study in relapsed /refractory (R/R) AML with or without FLT3 mutations, gilteritinib was given as once-daily doses in dose-escalation and dose-expansion cohorts  (Table n = 4). Four of 11 patients with wild-type FLT3 also responded. This study also established the 120 mg once-daily as the recommended dose in the Japanese patients.Gilteritinib was studied in another phase 1 study in Japanese patients with R/R AML . Gilteritinib was given as daily escalating doses in 6 cohorts, with doses ranging from 20, 40, 80, 120, 200, to 300 mg/day. In the published report, 24 subjects were enrolled . Grade 3Gilteritinib was compared with salvage chemotherapy in R/R AML with mutated FLT3 in an open-label, multicenter, randomized phase III study  . Gilterin = 29/138), with 11.6% CR and 9.4% CRh. The median duration of remission (DOR) was 4.6 months (range 0.1–15.8). The median time to response was 3.6 months (range 0.9–9.6). Based on these data, gilteritinib was approved by US FDA for R/R AML patients [In the pre-planned interim analysis of 138 patients enrolled in the gilteritinib group, CR/CRh was 21% . The rates of OS at 1 year were 37.1% for gilteritinib group and 16.7% for the SC group. In the full analysis of the 371 patients randomized, the CR/CRh rate for gilteritinib group (34%) was significantly better than that in the SC group . Cytopenia were the common serious adverse events (SAE) related to gilteritinib, including anemia, febrile neutropenia, and thrombocytopenia. Other clinically significant AEs that were observed in the clinical trials of gilteritinib included prolonged cardiac ventricular repolarization , pancreatitis (5%), posterior reversible encephalopathy syndrome (1%), and differentiation syndrome (3%). Dexamethasone 10 mg IV every 12 h  should be initiated once differentiation syndrome is suspected. Careful hemodynamic monitoring should be done until clinical improvement. Steroids should be administered for a minimum of 3 days and can be tapered once the symptoms resolve.Among the 371 patients, 247 were randomized to gilteritinib and 124 to salvage chemotherapy . The medIn conclusion, gilteritinib as a single oral agent at 120 mg daily led to significantly longer OS and higher ORR than salvage chemotherapy. The overall safety profile also favors gilteritinib. These data from the ADMIRAL study may lead to the therapy paradigm shift and establish gilteritinib as the new standard therapy for R/R FLT3-mutated AML.The role of gilteritinib in combination regimens remains unclear. It is possible that adding gilteritinib to the commonly used chemotherapy agents and regimens may improve clinical efficacies. Currently, multiple clinical trials are ongoing to evaluate the combination of gilteritinib with other agents and regimens Table .n = 10/15). In conclusion, the combination of gilteritinib with AZA was well tolerated. These data from the safety cohort led to the decision to use a dose of 120 mg gilteritinib plus AZA in the randomized portion of the 3-arm study. The preliminary data showed antileukemic responses in these newly diagnosed FLT3mut + elderly unfit AML patients. This trial could provide evidence for a new regimen for elderly AML patients [Epigenetic dysregulation plays a major role in leukemogenesis –39. Hypopatients .n = 24/27). In conclusion, gilteritinib in combination with intensive chemotherapy for induction was well tolerated. Two different gilteritinib schedules in combination with idarubicin or daunorubicin induced high ORR in those patients with FLT3 mutations. Overall survival and long-term outcome are still being monitored.In an ongoing open-label, dose-escalation /expansion phase 1 study, gilteritinib is being studied in combination with front-line 7 + 3 induction chemotherapy in adult patients with newly diagnosed AML (NCT02236013). This study also includes consolidation phase with high-dose cytarabine, and maintenance therapy with single-agent gilteritinib. Dose escalation of gilteritinib was planned at 40, 80, 120, or 200 mg/day. During the initial 2 cycles of a standard 7 + 3 induction regimen . Once the dose-expansion cohort using Schedule 1 was completed, a new cohort of six patients were enrolled, with gilteritinib given on days 8–21 (Schedule 2). For the consolidation phase, cytarabine was planned at 1.5 g/m2 every 12 h on days 1, 3, and 5 together with gilteritinib on days 1–14. For responding subjects with appropriate donors, HSCT was allowed. For the maintenance phase after consolidation or transplantation, gilteritinib was given daily as a single agent for ≤26 cycles. In the update at the 2018 ASH annual meeting, 62 patients were enrolled, with 60 eligible for safety analysis . Among tMidostaurin has been approved for combination with induction chemotherapy for newly diagnosed AML patients with FLT3 mutations , 50, 51.Among the FLT3 inhibitors in clinical trials, crenolanib is in multiple trials for R/R AML as well as for frontline regimens for newly diagnosed AML , 52, 53.Sorafenib, midostaurin as well as gilteritinib are being studied as maintenance therapy after HSCT for AML with FLT3 mutations , 10, 56.Gilteritinib has been approved for R/R AML with FLT3 mutations. The ADMIRAL study showed that longer overall survival and higher response rate are associated with gilteritinib in comparison with salvage chemotherapy for R/R AML. These data from the ADMIRAL study may lead to the therapy paradigm shift and establish gilteritinib as the new standard therapy for R/R FLT3-mutated AML.


The healing stage and the stroma recovery were also analyzed by means of histological sections. Results show that the CXL effects depend on the initial arrangement of the corneal collagen. While the treatment increases the order in corneas with a low level of initial organization, corneas presenting a fairly regular pattern are hardly affected. Histological samples showed active keratocytes in anterior and middle stroma, what means that the recovery is still in progress. The combination of SHG imaging and the ST is able to objectively discriminate the changes suffered by the collagen arrangement after the CXL treatment, whose effectiveness depends on the initial organization of the collagen fibers within the corneal stroma.Corneal cross-linking (CXL) is a surgical procedure able to modify corneal biomechanics and stabilize keratoconus progression. Although it is known that CXL produces changes in corneal collagen distribution, these are still a topic of discussion. Here we quantitatively compare the corneal stroma architecture between two animal models four weeks after Collagen cross-linking (CXL) is a minimally invasive technique based on a photochemical reaction carried out by a photosensitizer  exposed to ultraviolet A light UVA, 370 nm). During this operation, reactive oxygen species are produced, which induces covalent bonds  between 70 nm. DuCXL long-term effects have been analyzed in a number of clinical trials (both nonrandomized and randomized) –12. AlthSecond harmonic generation (SHG) microscopy is a powerful nonlinear imaging technique used to visualize nonstained collagen-based structures, such as the corneal stroma . SHG imaAlthough most of these experiments used a standard CXL protocol, the changes visualized in the SHG images are not completely coincident. Among others, the reasons might be the initial collagen organization of the different animal models , or the The changes detected in CXL corneas have mainly been analyzed in a qualitative way. Only a few explored the changes in the cornea morphology after CXL treatment in a quantitative manner , 27. TanAlthough the FFT has been used to quantify stromal organization in healthy and pathological corneas , 29, 30,A number of animal models with different stromal organizations have been employed to study the CXL effects. Results have been shown to depend on the sample and not to be conclusive. Moreover, to our knowledge, there is a lack of studies comparing morphological changes induced by CXL treatment in different animal models under similar experimental conditions. Two animal models (rabbit and chicken) presenting very different stromal organization  were invThe SHG microscope setup used in this work was based on a custom instrument previously reported . This coIber Braun adult chickens (N=12) and New Zealand adult rabbits (N=13) were used in this experiment. Animals were provided by Ibertec  and Granja San Francisco , respectively. The entire experiment followed the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The use of tissue samples from animals and the protocol herein were approved by the Animal Ethics Committee of the Universities of Valladolid and Murcia.2 of irradiance) for 30 minutes by using a UV-X™ radiation system . This corresponds to a total dose of 3.4 J or a total radiant exposure of 5.4 J/cm2. During the irradiation, the cornea was soaked with riboflavin/dextran solution every 5 minutes.The CXL treatment was performed in the left eye of both animal models (the fellow eye was used as control). The animals were anesthetized with intramuscular ketamine hydrochloride , xylazine , and topical application of 0.5% tetracaine hydrochloride and 1 mg of oxybuprocaine . The corneal epithelium was firstly removed with a scalpel. Drops of riboflavin 0.1% solution in 20% dextran  were instilled onto the cornea every 5 minutes for a duration of 30 minutes. Then, the cornea was irradiated with UVA light  under general anesthesia. Eyes were enucleated just after dead and the corneas excised by using ophthalmic scissors. A 2 mm scleral rim was left. Corneas were then fixed with 4% buffered paraformaldehyde for 24 h and rinsed in buffer phosphate (0.1 M). Manipulation and excision procedures were carried out by a well-experimented technician. All corneas appeared clear during the entire experiment. The CXL-treated corneas were compared to the control ones (fellow eyes).μm) filled with buffer phosphate for SHG imaging. For each corneal location, three sequential SHG images (210x210 μm2) corresponding to the central cornea  were acquired. Every final SHG image was the average of those three individual frames. This simple image processing optimizes the signal-to-noise ratio. Along the Z-direction, images were acquired at different depth locations (20 μm apart) across the entire corneal depth. In this work, the 0 μm corneal depth position was established as the first location within the stroma where the SHG signal appeared . Throughout this work, anterior (or outer), mid, and posterior (or inner) cornea refer to the areas of the stroma located within the corresponding thickness . For instance, for a 240 μm thick cornea, the anterior stroma corresponds to corneal layers located between Bowman's layer and an 80 μm depth plane.These nonstained samples were placed on a glass bottom dish . Further technical details on this tool have been extensively reported in . In partTo quantitatively differentiate the stromal arrangement between control and post-CXL corneas, the statistical analysis was carried out in three datasets: PO histograms, SHG total intensity, and DoI parameter. PO histograms were analyzed and compared using the Kolmogorov-Smirnov nonparametric test of the equality of continuous, avoiding making assumptions about the data distribution. This analysis was performed using the IBM SPSS Statistics software and differences were considered as significant when p-value was smaller than 0.05. The analysis concerning SHG intensity and DoI was carried out applying the t-test analysis (p<0.05) provided by OriginLab® software.μm thick) were refixed in Bouin's solution overnight. After removing the paraffin, these sections were stained with Masson's trichrome. This is a three-colour staining protocol used in histology that includes a sequence of three solutions: Weigert's iron hematoxylin for 10 min, Biebrich scarlet-acid fuchsin for 10-15 min, and light green for 5 min. Histological sections were examined under a bright-field microscope . Photomicrographs were taken using the AxioCam Digital Camera and AxioVision Microscope software provided with the commercial microscope. The analysis of these histological sections will be useful to assess the depth of the affected corneal tissue and the posttreatment healing following the CXL procedure.After the SHG imaging operation, the corneas were dehydrated through a series of graded ethanol and infiltrated with melted wax. Then, the tissues were sectioned using a microtome Minot  and sections (5  in vivo CXL treatment (right column). From a qualitative point of view, it is difficult to comment on the changes caused by the CXL procedure in the arrangement of the collagen fibers. In general, and compared to the intact tissue, the collagen bundles appeared more delineated and less interwoven four weeks after CXL treatment. However, for a better understanding and description, a quantitative analysis must be carried out. In that sense, For both control and post-CXL corneas, the collagen distribution in the anterior and middle stroma presents POs indicated by the maximum values in the histograms. For both locations, the averaged DoI values are associated with a partially aligned collagen distribution  . HoweverThe SHG intensity profiles as a function of depth for control and 4 weeks after CXL corneas are presented in For the chicken model, representative SHG images revealing the stroma in control and post-CXL corneas as a function of depth are depicted in By simple visual examination, the presence of orthogonal interweaving is apparent at the anterior cornea and middle cornea. This seems to be less evident at the posterior location. For the treated eyes, the structure of the stroma shows long collagen fibers running parallel to each other.The corresponding PO distribution histograms of the previous SHG images are presented in As expected from the corresponding SHG image, at the posterior stroma, one of the POs is much less pronounced and almost disappears. As a result of the CXL and posterior wound healing, for the treated cornea, only one PO is maintained. This finding was similar for all the chicken samples involved in the experiment. For both control and post-CXL corneas, DoI values always corresponded to partially aligned distributions. Changes in DoI after the treatment were not found at any corneal location. Moreover, it is interesting to notice that for this animal model the differences between pairs of PO histograms were not significant . This indicates that the CXL treatment for the adult chicken cornea was not as efficient as it was in the rabbits.In For a better understanding of the CXL effects and the changes (if so) produced in the collagen distribution of both animal models, the DoI values provided by the ST (which inform on the collagen arrangement) have been explored more in depth.For all the specimens involved in the experiment, The specimens used for SHG imaging were also analyzed with a bright-field microscope once the corresponding histological sections were prepared (see Methods above).  in vivo treatment. The results were compared with those of nontreated control corneas .In this work, we have investigated the performance of a standard CXL treatment in two animal models (rabbits and adult chickens) and the ability of SHG imaging microscopy to detect changes produced in the corneal stroma four weeks after theAlthough CXL treatment has been reported to be a very efficient clinical procedure in keratoconus human eyes, the effects at microscopic scale and the changes in collagen arrangement are still under discussion. Some authors compared the effects of using riboflavin alone or irradiating UVA without riboflavin instillation on the corneal stroma by analyzing TPEF signals , 43–45.  ex vivo, etc.), direct comparisons cannot be done. This has been overpassed in the present work by using the same experimental conditions in two animal models. Here, the spatial distribution of the stroma fibers after four weeks of CXL has been compared with control specimens at different corneal depth locations.In order to understand the effects of CXL, SHG imaging has been used in different animal models in the past. However, due to the broad experimental conditions of the corneas involved  right after the CXL procedure and during a maximum of 2 hours. Short-time changes in corneas treated with riboflavin (but not followed by UV irradiation) were also explored. Here, the CXL treatment was carried out in in vivo conditions and the animals were sacrificed one month after the procedure. This permits the analysis of both CXL effects and wound healing response by means of SHG imaging.The experiment carried out here is very different from the previously reported by one of the authors of this work . In thatKrüger and colleagues explored bidirectional SHG images of trephined rabbit corneal buttons 3 days, 6 days, and 6 weeks after CXL treatment  . BackwarQuantitative tools such as the FFT , 29, 31 In the conventional CXL treatment here used, the cornea is firstly deepithelialized. Then, during UVA radiation, stromal cells are killed . This was also reported in post-CXL porcine corneas by Gupta et al. . During The histological analysis here presented corroborates that the CXL treatment covered the entire stroma of both animal models , 36. HowIn summary, the ST applied to SHG images of corneas represents a quantitative and accurate approach to objectively describe the degree of organization of the stromal architecture. Our findings show that the overall organization of corneas presenting a well-organized collagen assembly (such as the adult chicken or any other bird) is hardly affected by CXL treatment. On the contrary, there is a significant increase in collagen organization in CXL-treated corneas whose initial distribution is partially organized or nonorganized (such as the rabbit cornea). A paradigmatic example of a nonorganized collagen arrangement is a keratoconic cornea –22. At tAlthough during the last 20 years SHG imaging of the cornea has been a powerful tool to explore the structure of the stroma under very different experimental conditions, those experiments were carried out in ex-vivo conditions. Measurements in living human eyes were always thought to be very challenging. However, we have recently reported in vivo SHG imaging of the human cornea for the very first time . This de


Atopic dermatitis (AD) is a chronic recurrent dermatitis with profound itching, which could be the first manifestation of acute myeloid leukemia (AML).A 53-year-old Chinese man suffered a 6-month history of systemic symmetrical dermatitis, accompanied with profound itching. The patient was diagnosed as “eczema” in several hospitals, and the effects of antihistamine and topical steroid creams were poor. Nocturnal sleep was seriously affected by aggravating pruritus. Laboratorial examination was compatible with AML-M4.AML-M4 with AD as first manifestation.IA regimen (ayninen and cytarabine) were used in induction chemotherapy. However, the patient did not achieve complete remission, and although his rash had improved, he still experienced severely general body itching. On the seventh day of chemotherapy, the patient entered the period of granulocyte deficiency with infection.The patient died due to septic shock after chemotherapy.The case strengthens the awareness of AML with AD as first manifestation and raises oncological vigilance in patients with AD refractory. And there were no reports of AML with atopic dermatitis (AD) as first manifestation. Herein we report a 53-year-old man who was finally diagnosed as AML, with AD as a first manifestation.Studies reported that acute myeloid leukemia (AML) had a wide range of cutaneous manifestations in clinical and morphological findings such as nodular lesions and plaques. These manifestations could be divided into specific and nonspecific.29/L with 35.50% lymphocytes, 41.40% neutrophils, 8.3% monocytes, 14.50% eosinophil, hemoglobin of 144 g/L, and platelet count of 155 × 109/L. According to the Chinese Criteria for AD proposed by Zhang et al, the patient was diagnosed as AD and was treated with antihistamines, Tripterygium wilfordii, and the dermatitis was treated with oral prednisone (20 mg/d). After 1 and a half months, he felt consciously relieved of itching and the rash was relieved. But there was still development of new rashes.A 53-year-old Chinese male visited the hospital due to suffering systemic symmetrical dermatitis for more than 6 months Fig. . The pat9/L with 46.70% lymphocytes, 21.90% neutrophils, 15.40% monocytes, hemoglobin of 134 g/L, and platelet count of 190 × 109/L. A peripheral blood smear showed the normal total leukocyte count with 33% blasts, and Auer corpuscles can be seen in certain cells  was given. The patient had a fever after receiving chemotherapy for 3 days; we reduced the dose of ayninen to continue chemotherapy. After anti-allergic and chemotherapy, the rash was flatter than before, but the skin was too itchy to bear. On the seventh day of chemotherapy, the patient entered a period of granulocyte deficiency and went into septic shock. Finally, the patient was transferred to the ICU and died in June 2018.According to National Comprehensive Cancer Network Guidelines,3 The specific lesions called leukemia cutis (LC) are considered as malignant lesions, which are localized or disseminated infiltrations of the skin by leukemic cells. And it may involve all layers of the skin. LC may often present with nodular lesions and plaques. While the clinical manifestations like erythematous macules, blisters, and ulcers are rare, and they may appear alone or in combination. LC has been described in patients with AML, chronic myelogenous leukemia, myelodysplastic syndromes, and myelodysplastic and lymphoproliferative diseases. At present, many reports about AML–LC have been published.–11 And LC was somewhat higher in AML-M4 and AML-M5 than in other types of leukemia.Leukemia has a wide range of cutaneous manifestations in clinical and morphological findings, which are traditionally divided into nonspecific lesions and specific lesions. There were about 30% of cases of AML with nonspecific lesions. To the best of our knowledge, nonspecific lesions associated with AML have been reported already.,12 Rao et al proposed that nonspecific lesions were with a wide spectrum of etiologies, including infection, drug rash, purpura, graft versus host disease, vasculitis, the Sweet syndrome, and intraepidermal blistering disorder. Several pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-1 elevated significantly in leukemia,,15 we presumed that these elevated cytokines may be associated with nonspecific lesions. The origins of these cytokines are unknown, but these proteins may be secreted by leukemic cells in the bone marrow.Nonspecific lesions showed varied morphology and could be difficult to distinguish from specific cutaneous lesions, both clinically and histopathologically. proposed 3 features as the criteria for AD: eczema for more than 6 months; personal and/or family history of atopic diseases; elevated total serum IgE level and/or positive allergen-specific IgE and/or eosinophilia. One meta-analysis indicated that there was an inverse association between atopy/allergies and childhood ALL while no association was observed for AML or leukemia overall. Compared with other AML patients, the case we reported was finally diagnosed as AML, with AD as a first manifestation and there was no report about this before.AD is a chronic recurrent dermatitis with profound itching. Zhang et al Further research is needed to investigate the pathogenic relevance between AML and the elevated level of eosinophils in AD.Whether there is an inevitable relationship between AML and AD remains unclear. One hypothesis is that AD may be a hypersensitive reaction of the skin immune system to leukemia cells and presents as a significant increase in total serum IgE level and/or eosinophils. One article revealed that the increased numbers of eosinophils at all stages of maturation in AML-M4 could be associated with abnormal eosinophil precursors.The case of AML-M4 with AD as a first manifestation would shed light on both clinical work and research in the future. AD may be the first presentation of AML. Clinically, leukemia with rash as first clinical manifestation is easily misdiagnosed and missed in clinical. Therefore, with a patient of AD, we should take leukemia as consideration and perform a thorough work-up. Furthermore, AD may suggest a poor prognosis for patients with AML. So, prompt and accurate identification of AD accompanied by AML is crucial for timely management.As an engineer, the patient had a long history of exposure to chemical substances and maybe this is the direct cause of the dermatitis. Perhaps this plays a role in the pathogenesis of leukemia. But it needs further confirmation.In brief, we showed a rare case of AML-M4 associated with AD, which strengthens the awareness of AD associated with AML and raises vigilance against oncology in patients with AD.Data curation: Xu-Feng Yin, Hou-Fang Liu.Writing – original draft: Wei-Wei Ma.Writing – review & editing: Wei-Wei Ma, Lorna Martin Kasyanju Carrero, Bing-Rong Zhou.


In a pathophysiological setting pharmacological modulation of mitochondrial Ca2+ uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca2+-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca2+ accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca2+ uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na+/Ca2+ exchanger, and MCU inhibitor Ru360 in rat ventricular myocytes (VMs) from control rats and rats with hypertrophy induced by thoracic aortic banding (TAB). In periodically paced VMs under β-adrenergic stimulation, treatment with kaempferol (10 μmol/L) or CGP-37157 (1 μmol/L) enhanced mitochondrial Ca2+ accumulation monitored by mitochondrial-targeted Ca2+ biosensor mtRCamp1h. Experiments with mitochondrial membrane potential-sensitive dye TMRM revealed this was accompanied by depolarization of the mitochondrial matrix. Using redox-sensitive OMM-HyPer and ERroGFP_iE biosensors, we found treatment with kaempferol or CGP-37157 increased the levels of reactive oxygen species (ROS) in mitochondria and the sarcoplasmic reticulum (SR), respectively. Confocal Ca2+ imaging showed that accelerated Ca2+ accumulation reduced Ca2+ transient amplitude and promoted generation of spontaneous Ca2+ waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca2+ release channel ryanodine receptor (RyR). Western blot analyses showed increased RyR oxidation after treatment with kaempferol or CGP-37157 vs. controls. Furthermore, in freshly isolated TAB VMs, confocal Ca2+ imaging demonstrated that enhancement of mitochondrial Ca2+ accumulation further perturbed global Ca2+ handling, increasing the number of cells exhibiting spontaneous Ca2+ waves, shortening RyR refractoriness and decreasing SR Ca2+ content. In ex vivo optically mapped TAB hearts, kaempferol exacerbated proarrhythmic phenotype. On the contrary, incubation of cells with MCU inhibitor Ru360  normalized RyR oxidation state, improved intracellular Ca2+ homeostasis and reduced triggered activity in ex vivo TAB hearts. These findings suggest facilitation of mitochondrial Ca2+ uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca2+ homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca2+ accumulation can be protective.In a physiological setting, mitochondria increase oxidative phosphorylation during periods of stress to meet increased metabolic demand. This in part is mediated via enhanced mitochondrial Ca Abnormal activity of RyR leads to increased Ca2+ leak and promotes the generation of spontaneous Ca2+ waves (SCWs), that can subsequently propagate to trigger organ-wide arrhythmia  die suddenly due to the development of ventricular arrhythmia . Arrhythizations . These arhythmia .2+ homeostasis in physiological conditions  that transpires to a small and slow rise in mitochondrial Ca2+ concentration ([Ca2+]m), leading to enhanced energy production  channel complex is driven largely by the negative membrane potential across the inner mitochondrial membrane (2+/Li+-exchanger (NCLX)  .2+ release in the form of propagating Ca2+ waves that underlie increased triggered activity  in the myocyte, and while an increase in oxidative stress is a prerequisite for many cellular stress responses, excessive ROS production in cardiac disease contributes to ventricular arrhythmogenesis by altering the function of multiple ion channels and transporters . RyRs aractivity . Scavengor aging .2+ homeostasis remains an attractive therapeutic target , a condition characterized by mutations in the RyR macromolecular complex that renders channels hyperactive. Given mitochondria are in close proximity to SR Ca2+ release sites, it has been proposed that increasing mitochondrial Ca2+ uptake may improve buffering capacity . To achieve this goal, we utilized whole heart optical mapping, genetically encoded ROS and mitochondrial Ca2+ biosensors, confocal microscopy and biochemistry to dissect the influence of MCU enhancer kaempferol and NCLX inhibitor CGP-37157 on intracellular Ca2+ cycling, in both healthy and hypertrophic VMs. Our results suggest that enhancement of mitochondrial Ca2+ accumulation in either setting elevates mitochondrial ROS emission, increasing oxidation of RyR and aberrant spontaneous Ca2+ release. Attenuating mitochondrial Ca2+ uptake serves as an anti-arrhythmic treatment in hypertrophic hearts, whereby triggered activity was reduced by pharmacological inhibition of MCU with Ru360.In the present study, we aimed to determine the effects of pharmacological facilitation of m during workload, but after treatment with MCU enhancer kaempferol  was observed , while the time constant of transient decay, τ, was significantly increased after application of CGP-37157 . The time to peak of transient after application of CGP-37157 was also increased . This indicates that while pharmacological enhancers of [Ca2+]m do not increase overall mitochondrial Ca2+ loading in cultured control VMs during workload of 1 min pacing, they modify the time course for which those VMs accumulate or retain Ca2+. The finding that the rate of mitochondrial Ca2+ uptake in intact myocytes is only modestly accelerated with kaempferol and even slowed down with CGP-37157 suggest the existence of overriding feedback mechanisms to prevent mitochondrial Ca2+ overload that can cause cell death . Application of CGP-37157 had similar effects . The decrease in driving force due to the drop in Δψm may explain why kaempferol or CGP-37157 are not able to increase the amplitude of [Ca2+]m effectively limiting mitochondrial Ca2+ uptake, as measured with mtRCamp1h in Figure It was established that the excessive mitochondrial Cachannels . While ioverload . We usedm Figure . As in F2+ and Δψm, we next sought to establish the effects of modulating mitochondrial Ca2+ on cytosolic Ca2+ handling in VMs, as illustrated in Figure 2+ indicator Rhod-2 and we recorded cytosolic Ca2+ in the presence of β-adrenergic receptor agonist ISO (50 nmol/L), subjected to a burst-pace pause protocol . We assessed Ca2+ transient amplitude and SCW latency as an indication of the propensity for arrhythmogenic Ca2+ release. As shown in Figures 2+ transient amplitude in comparison to ISO alone . We observed a fivefold shortening in SCW latency after treatment with kaempferol (∗p < 0.001), as well a significant increase in the percentage of VMs exhibiting SCWs . A similar pattern was obtained in VMs treated with CGP-37157 . Changes in Ca2+ transient amplitude and the percentage of cells exhibiting waves were not significant on treatment with CGP-37157.Having demonstrated the effects of kaempferol and CGP-37157 on mitochondrial Ca Figures , whereby2+ accumulation exerts strong deleterious effects on intracellular Ca2+ handling we performed experiments using another MCU enhancer SB 202190 , a specific inhibitor of RyR. Figure 2+ transient amplitude and significantly reduces kaempferol-mediated shortening of SCW latency in ISO-treated VMs .Enhanced propensity to generate SCWs is often attributed to abnormally high activity of SR Cals, RyRs . To test2+ accumulation promotes generation of proarrhythmic SCWs by enhancement of activity of RyRs.Taken together, these data suggest that facilitation of mitochondrial Ca2+ release and proarrhythmic SCWs. Mitochondria is a major intracellular source of ROS and excessive RyR oxidation by mitochondria-derived ROS has been demonstrated in many models of HF and aging , confirming that facilitation of mitochondrial Ca2+ accumulation induces mitochondrial ROS release.RyR is well established as a ROS sensor, with increased oxidation and thus RyR activity associated with enhanced propensity for spontaneous SR Cand aging . Facilitransport . Therefo2+ accumulation leads to an increase in local ROS levels in the vicinity of RyR in cultured control VMs, measured using the ER-tuned redox-sensitive biosensor ERroGFP_iE , indicative of increased oxidation and ROS emission in close proximity of RyR , shown by increased DNP signal. These data demonstrate that in control VMs, enhanced mitochondrial Ca2+ accumulation leads to increased ROS in the closely situated SR. Subsequent increased RyR oxidation is likely the responsible mechanism for the shortened latency for proarrhythmic RyR-mediated SCWs in VMs with pharmacologically enhanced mitochondrial Ca2+ accumulation.To directly test the hypothesis that pharmacological enhancement of m modulation on Ca2+ handling and initiation of triggered activity at the whole organ level, using hearts from rats with TAB-induced hypertrophy, as illustrated in Figure 2+ transient amplitude and exacerbated the proarrhythmic phenotype, with incidences of PVCs and spontaneous VT/VFs with higher VF frequencies. The focal activity and/or transmural outbreak-like activation patterns were frequently seen during PVCs and VFs, suggesting enhanced triggered activity may underlie exacerbated ventricular arrhythmias by kaempferol. In contrast the MCU blocker, Ru360, suppressed spontaneous VT/VFs. The activation maps of pacing induced VFs showed that rapid pacing caused conduction block and reentry formation, suggesting that the protective effect of Ru360 is most likely through suppressing triggered activity. In recordings of intracellular Ca2+ transients, it was evident that kaempferol and NCLX inhibitor CGP-37157 had detrimental effects on both cultured control VMs (Figure 2+ release, indicative of increased activity of RyR in both cultured control and hypertrophic TAB VMs. The experiments with RyR inhibitor dantrolene demonstrating attenuation of kaempferol-induced disturbances in Ca2+ cycling (Figure 2+ release in this process.We assessed the effects of m, thereby improving redox homeostasis and protecting against arrhythmogenesis (2+ transients in VMs from diseased hearts in comparison to controls (Figure 2+ transient amplitude despite slowing down transient decay in TABs. It also did not improve but even worsened aberrant intracellular Ca2+ handling (Figure 2+ influx during diastolic period in mice with non-ischemic HF and posited that increased mitochondrial Ca2+ efflux drives activation of the sarcolemmal NCX and initiates EADs. In this work, inhibition of both influx and efflux were reported to have anti-arrhythmic effects. In direct contrast, the present study clearly demonstrates that inhibition of NCLX in both control and TAB VMs promotes proarrhythmic spontaneous SR Ca2+ release (Figures 2+ overload. Our results are in line with the latter and inhibition of NCLX with CGP-37157 significantly increased ROS emission in cultured control VMs (Figures 2+ handling in both cultured control (Figure During pathological mitochondrial Ca pathway . Pharmac pathway . Howeverogenesis . In agres Figure . Howeverg Figure . Xie et  Figures , 12. Imp Figures . This wa Figures , resultil Figure  and TAB 2+ flux is closely associated with ATP generation and ROS emission, it has also been suggested that mitochondria may act as buffers that can shape global Ca2+ transients during EC coupling (2+ (Kd ∼ 10–20 μmol/L Ca2+; 2+ transients, mitochondrial Ca2+ uptake could occur at high local [Ca2+] near SR Ca2+ release sites (2+ between the two has been reported in striated muscle (2+ transport may be altered due to changes in expression levels of scaffolding proteins including Mfn1 and 2 (2+. Indeed, in a mouse model of CPVT, enhancement of mitochondrial Ca2+ influx reduced frequency of arrhythmogenic Ca2+ waves and incidences of VT/VF (While mitochondrial Cacoupling . Althougse sites . One endse sites , and is se sites  with strse sites . This fad muscle . In cardn1 and 2 . We testn1 and 2 . Mitochoof VT/VF . Also, Z2+ release events are an important driver of [Ca2+]m, as the total mitochondria Ca2+ flux is small, the overall ability of mitochondria to shape intracellular Ca2+ dynamics remains debated (2+ buffers in the myocyte, and enhancement or increased expression of SERCa has been shown to significantly improve Ca2+ buffering capacity and attenuate arrhythmogenic spontaneous Ca2+ release (2+ influx significantly increases the propensity for SCW in both cultured control and TAB VMs (Figures 2+]m modulated the velocity and propagation of SCWs (Figure 2+ leads to profound approximately fourfold decrease in the amplitude of pacing-induced mitochondrial Ca2+ transient (Figures 2+ modulates mitochondrial function, mitochondria do not serve as a significant buffer of intracellular cytosolic Ca2+ (2+ homeostasis are likely overwhelming and cannot be compensated for by an increase in local Ca2+ buffering capacity by mitochondria.Although SR Ca debated . Troponi release . In rece Figures , 11, 12,s Figure . Importa Figures . These dlic Ca2+ . The eff2+ accumulation produces deleterious effects on Ca2+ homeostasis under β-adrenergic stimulation. It promotes excessive ROS that enhances RyR activity thereby proarrhythmic spontaneous Ca2+ release. In cardiac hypertrophy, where ROS defenses are weakened, it exacerbates the proarrhythmic alterations in Ca2+ handling. Inhibition of mitochondria Ca2+ uptake is protective because it reduces emission of ROS by mitochondria.In conclusion, our data suggest that pharmacological enhancement of mitochondrial CaSH, B-RC, JO-U, GC, and DT participated in the study design. SH wrote first draft of manuscript. SH, RT, TYK, PB, RC, JO-U performed the experiments. SH, RT, TYK, B-RC, DT conducted data interpretation and analyses. SH, RT, TYK, PB, RC, JO-U, GC, B-RC and DT reviewed the manuscript submitted for publication. All authors revised and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ca2+ takes part in the regulation of neuronal excitability, and the imbalance of intracellular Ca2+ is a trigger factor for the occurrence of epilepsy. Several anti-epileptic drugs target voltage-dependent calcium channels (VDCCs). Intracellular Ca2+ levels are mainly controlled by VDCCs located in the plasma membrane, the calcium-binding proteins (CBPs) inside the cytoplasm, calcium channels located on the intracellular calcium store (particular the endoplasmic reticulum/sarcoplasmic reticulum), and the Ca2+-pumps located in the plasma membrane and intracellular calcium store. So far, while many studies have established the relationship between calcium control factors and epilepsy, the mechanism of various Ca2+ regulatory factors in epileptogenesis is still unknown. In this paper, we reviewed the function, distribution, and alteration of VDCCs and CBPs in the central nervous system in the pathological process of epilepsy. The interaction of VDCCs with CBPs in the pathological process of epilepsy was also summarized. We hope this review can provide some clues for better understanding the mechanism of epileptogenesis, and for the development of new anti-epileptic drugs targeting on VDCCs and CBPs.As an important second messenger, the calcium ion (Ca Alterations of VDCC functions can cause abnormality in cellular events, leading to pathological consequences. In neurons, VDCCs initiate synaptic transmission [Neuronal intracellular calcium increase plays an important role in the triggering and propagation of seizure activity ,2. Ca2+ ne cells ,3,4. It ne cells ,11,12,13smission ,15. Enhasmission ,17,18,19smission  suggestssmission , VDCCs asmission ,23,24.2+ dependent-signaling cascades are largely mediated by calcium-binding proteins (CBPs) [2+-dependent or Ca2+-independent signaling mechanisms [2+-dependent signaling transduction pathways and regulate Ca2+ influx via the VDCCs in Ca2+-dependent feedback mechanisms [2+ binding motifs are verified to regulate high voltage-activated VDCCs [2+ kinetics directly through the regulation of VDCC properties [2+ in these neurons is controlled by the synergy of VDCCs and CBPs. With the availability of human genetic databases and advanced molecular technologies, accumulated evidence suggest that dysfunctions in CBP-mediated VDCC regulation may be one of the mechanisms leading to human diseases [2+, and protect neurons from over-excitation and neuronal damage [Cas (CBPs) ,26, and chanisms ,28. CBPschanisms . CBPs coed VDCCs ,32,33,34operties ,32,33,34operties ,36, and diseases . CBPs arl damage .2+ influx and may be modulated by CBPs [The pathological process of epilepsy includes three periods: an acute period with initiating factors , including status epilepticus, a latent period , and a chronic stage with spontaneous recurrent seizures. VDCCs and CBPs have been reported to be involved in all stages of the pathogenesis of epilepsy. Epileptogenesis is mainly related to the latent period . Interictal spikes, as the omen of seizure activity, present during this period ,39,40. I by CBPs ,43,44, i2+ currents designated as L-, N-, P/Q-, R-, and T-type, based on the long-lasting current and intermediate voltage dependence, which were first recorded in Purkinje neurons and cerebellar granule neurons that were resistant to subtype-specific organic and peptide Ca2+ channel blockers, and transient current, respectively [v1.1), Cav1.2, Cav1.3, and Cav1.4. P/Q-, N-, and R-type are corresponding to Cav2.1, Cav2.2, and Cav2.3, respectively. T-type VDCCs include Cav3.1, Cav3.2, and Cav3.3. Electrophysiological studies reveal different Caectively ,46. The v1.2 and Cav1.3, shape neuronal firing and activate Ca2+-dependent pathways involved in the control of gene expression [v3.1, Cav3.2, and Cav3.3 are important for the repetitive firing of action potentials in rhythmically firing cells, and could be activated and inactivated more rapidly and at more negative membrane potentials than other VDCCs mentioned above [L-type VDCCs members, Capression . N-type,ed above . T-type ed above .The physiological function, and distribution of VDCCs are summarized in the 2+ concentrations might trigger seizures in the brain tissues [2+ may indicate at least in part, the Ca2+ influx through neuronal VDCCs [The evidence to show VDCCs in epileptogenesis has been reported four decades ago, which indicated that decreases in extracellular free Ca tissues ,73,74. Wal VDCCs . Pieces 2+ concentration in the cytosol, and participate in numerous cellular functions by acting as Ca2+ transporters across cell membranes or as Ca2+-modulated sensors, i.e., decoding Ca2+ signals. The members of intracellular CBPs with EF-hand [2+ binding domains [Intracellular CBPs of the EF-hand superfamily are involved in the regulation of cell function and they contribute to the control of Ca EF-hand ,26,105,  domains , have im domains . 2+ sensor in all types of cells, it is assumed that CaM may be involved in many pathological processes, such as epilepsy [Among the EF-hand CBPs, the CaM family is very large and has been extensively characterized. CaM was firstly discovered in the bovine  and rat epilepsy ,111, PD,In the PV family of CBPs, PVs play an important role in cells by checking on calcium switching . BecauseCR, as a member of the EF-hand CBPs family, was first identified in the retina. It is involved in intracellular calcium buffering, messenger targeting, cell cycle arrest, and apoptosis . CR play2+ buffer [2+ transients and evoke Ca2+ signals in most neuronal groups [2+ mediated neurotoxicity by buffering intracellular Ca2+.As a high-affinity CBP, CB is expressed in the mammalian kidney, intestine, placenta, brain, peripheral nervous system, parathyroid gland, bone, and pancreas ,118. Whi+ buffer ,120, age+ buffer ,123,124.l groups ,125, and2+ concentration in the cytosol and participate in numerous cellular functions by acting as Ca2+ transporters across cell membranes or as Ca2+-modulated sensors, i.e., decoding Ca2+ signals [Neuronal intracellular calcium increase plays an important role for the triggering and propagation of seizure activity . CBPs co signals . Therefo2+ are frequently mediated via its interaction with CaM, and strong evidence indicate that the effects of Ca2+/CaM are often achieved through the regulation of conductance of ion channels and the protein phosphorylation process [+ channel (Nav) intracellular C-terminal domains (CTDs), and can limit persistent Na+ current and accelerate inactivation across the Nav family. Mutations located in Nav1.2 channel CTDs can reduce CaM binding and result in increased persistent Na+ current and the occurrence of epilepsy [+ current and decreased neuronal activity. Meanwhile, CaM knockdown reduced the KCNQ2 current and increased neuronal activity. It suggests that CaM may regulate neuronal excitability by KCNQ2, and it could be a target of gene therapy in epilepsy [The effects of Ca process . A recenepilepsy , suggestepilepsy . CaM oveepilepsy . These depilepsy . CaM mayepilepsy . In variepilepsy ,132,133.epilepsy . These depilepsy . In the epilepsy . These repilepsy ,138,139 epilepsy ,141 are epilepsy  and betaepilepsy  subunits2+ to reduce the overload-induced excitotoxicity and prevent the pathological process of epilepsy. The loss of CB from granule cells markedly increased the Ca2+-dependent inactivation (CDI) of VDCCs currents, thereby diminishing Ca2+ influx during repetitive neuronal firing in epilepsy [By a gene knockout study, the vulnerability of hippocampal CA1 neurons to seizure-induced injury was shown to be modified by CB . In GEPRepilepsy  which maepilepsy . -/- mice show impaired LTP induction following tetanic stimulation of hippocampal inputs, which could be restored by using bicuculline (a GABA receptor blocker) [In the cerebellum, granule cells produce CR to excite Purkinje cells through parallel fibers. In vivo electrophysiological recording indicated that knocking out CR induced dramatic alterations in motor coordination and Purkinje cell firing . In acutblocker) . At a ceblocker) ,156. CR-blocker) ,158,159.blocker) ,164,165.blocker) ,161, andblocker) ,175,176 blocker) ,163,177.blocker) . Based o2+ buffering and GABA release regulation are two pathways through which PV regulates neuronal excitability and epileptogenesis , facilitation (CDF), and Ca2+-independent regulation (CIR) of the channels. Pathogenesis of human diseases may occur once CBPs-mediated VDCCs are impaired [v2.1 CDF was blocked in the familial hemiplegic migraine type 1(FHM-1). This disruption of Cav2.1 CDF may cause the cerebellar ataxia-associated FHM-1 due to an imbalance between the excitatory and inhibitory inputs to the cerebellar Purkinje cells [CBPs such as CaM do not act as solely Caimpaired . The besimpaired . CaM binimpaired ,215,216 impaired ,34,217. je cells . Therefov2.1 in vitro, co-expression of CR inhibits CDI and enhances CDF via a direct interaction with Cav2.1. The direct modulation of Cav2.1 by CR affects intracellular Ca2+ signaling, and probably also the neuronal excitability, via a mechanism that is different from Ca2+ buffering function [2+ entry into neurons  and the lack of isoform-specific inhibitors of VDCCs, clinical application of antagonists of VDCCs in patients with epilepsy has been compromised due to universal severe side effects. Recent studies have indicated that the redistribution of VDCCs may play an important role in the pathological process of epilepsy, understanding the trafficking mechanism of VDCCs becomes very important and may provide novel therapeutic targets for seizure control or prevention of epilepsy. CBPs  as Ca2+ buffers, can reduce Ca2+ overload and protect neurons from excitotoxicity, and regulate neuronal excitability through CDF and CDI of VDCCs, or other pathways. Further study to understand the molecular mechanisms of the interaction between CBPs and VDCCs in the pathological process of epilepsy will greatly facilitate the discovery of promising targets for the development of new antiepileptic drugs. By overexpression of CBPs to investigate their gain-function in reducing epileptogenesis, novel approaches in controlling or prevention of the pathological process of epilepsy may be developed. As important pathways for Ca


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