The development of a sinus node preparation of almost microscopic dimensions allowing uniform voltage clamps created new possibilities. Three different groups discovered in this improved node preparation an hyperpolarization induced time‐dependent inward current, with a reversal potential positive to the resting potential, carried by a mixture of Na+ and K+ ions. A new current, If, or funny current was born. It is not the only pacemaker current. The following sequence of currents (membrane clock) has been proposed: diastole starts as a consequence of IK deactivation and If activation; followed by activation of the T‐type Ca2+ current, Ca2+‐induced Ca2+ release from the SR, and activation of sodium‐calcium exchange current with further depolarization of the membrane till threshold of the L‐type Ca2+current is reached. The release of Ca2+ can also occur spontaneously independently from a T‐type Ca2+current. The system acts then as a primary intracellular clock. The review is completed by description of an evolution in the direction of biological pacing using induced pluripotent stem cells or transcription factors.Initially, diastolic depolarization in Purkinje fibers was explained by deactivation of gK2 in the presence of inward current. Weakness of the hypothesis was a too negative reversal potential, sensitivity to external NaSee also:https://doi.org/10.14814/phy2.13860 & https://doi.org/10.14814/phy2.13861 Typical for the transmembrane electrical activity in the sinus and in Purkinje fibers is the existence of a slow diastolic depolarization. Different between the two tissues is their maximum diastolic potential and the upstroke at the end of the diastole. It is therefore not directly expected that the same current(s) will be found responsible for the two types of spontaneous depolarization.+ conductance consistent with the measured increase in impedance (2) an increase in Na+ conductance, (3) a decrease in Na+, K+‐pump activity. Notice that the last hypothetical possibility tacitly assumed an electrogenic Na+, K+‐pump. The first mechanism was called the gK‐decay hypothesis; it was selected by most electrophysiologists and has remained intact for more than 20 years. We will see later that a shift to the second hypothesis although in somewhat modified form  was made in the early eighties. In the meantime, the gK‐decay hypothesis was taken up in the first mathematical model for the Purkinje fiber cardiac action potential  The reversal was some 5–10 mV more negative than the EK estimated from the intracellular K+ concentration  a second delicate aspect was the disappearance of IK2 current in Na+‐free medium, rather unexpected for a pure K+ current. These two weak points in the theory would continue to bother Denis Noble.Three possible mechanisms to explain the process of diastolic depolarization in cardiac Purkinje fibers have been proposed by Weidmann : 1) a dal Noble . In this a dal Nompedance  an increEK was causing extracellular K+ concentration depletion in the narrow clefts between Purkinje fibers (Baumgarten and Isenberg How did the revolution start? What did provoke the change of mind? Two important evolutions: (1)The first was the accumulating evidence that application of voltage clamp hyperpolarizing pulses negative to the The first attempt to measure voltage clamp currents in the newly developed sinoatrial node (SAN) preparation was made by Irisawa using the double sucrose gap method Irisawa . I remem.” With the two‐microelectrode voltage clamp version  Weiss et al. , (2) Ma, (3) May+ ions suggests a pacemaker current by an ionic species with a positive reversal potential. The decrease in K+ conductance during pacemaker depolarization was considered too small to generate the diastolic depolarization. According to Hilary Brown's appreciation of the problems involved and their historical evolution and evaluation, Morad's group was the first to suggest activation of an inward current with a large positive equilibrium potential as the cause of the pacemaker depolarization. In her Physiological Reviews text, Brown  without narrow clefts and hopefully not subject to depletion‐accumulation problems, certainly not in a major form. Noble and DiFrancesco found that our findings were “amply” were excluded from playing a role. Their deactivation in combination with an inward background current could still participate in causing the early diastolic depolarization. The following ionic currents have been proposed to carry inward current during the slow diastolic depolarization: Isustained, ICaT and INCX. The sustained inward current  current has been described in atrial  by Ni2+.Apart from the L‐type Caial Bean  and vential Bean  cardiac ial Bean . The thrr et al.  that Ca2ent Fig. . The Ca22+ current, Ca2+‐induced Ca2+ release from the SR, and activation of INCX  is reached. The system is redundant.This brings us to the following sequence. At the beginning of the diastole, the membrane is depolarized as a consequence of IK deactivation and If activation; this leads to activation of the T‐type Ca2+ also occurs spontaneously under the form of sparks or openings of single RYR‐sensitive Ca2+ channels, independently from a T‐type Ca2+current  has the highest expression. HCN3 has not been detected in the SAN  and transform them into pacemaker cells with a minimally invasive intervention. Within a few days, ventricular myocytes transformed into SAN cells ., Protze et al. (Using this approache et al.  accordine et al. . EmbryoiThese examples show that interesting openings have been made. Progress is slow but the step for the test in the clinic is approaching.None declared.


Here, we report that SOCE, peak Ca2+ transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca2+ entry coincident with a modest decrease in total releasable Ca2+ store content. Importantly, this decrease in releasable Ca2+ store content observed after exercise was reversed by repetitive high-frequency stimulation, consistent with enhanced SOCE. The functional benefits of exercise on SOCE, constitutive Ca2+ entry and muscle force production were lost in mice with muscle-specific loss of Orai1 function. These results indicate that TT association with SR-stacks enhances Orai1-dependent SOCE to optimize Ca2+ dynamics and muscle contractile function during acute exercise.Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca While the precise physiological role(s) of SOCE in muscle remains incompletely defined, SOCE has been proposed to promote myoblast fusion/differentiation  fibers of wild type (WT) mice that involves: i) remodeling of the SR into stacks of flat cisternae; ii) elongation of the TT into the I band; and iii) increased co-localization of Orai1 with STIM1 in the I band  .In resting mammalian skeletal muscle, STIM1 proteins are found throughout the I band of the sarcomere . On the e I band . This exe I band . Importa, 2-APB) . As the 2+ dynamics, and muscle force production remain unknown. In fact, the precise degree to which CEUs formed after exercise promote SOCE, optimize Ca2+ dynamics and contribute to the adaptation of muscle function in response to acute exercise is unknown. Here, we addressed these issues using a combination of structural  and functional  approaches in EDL muscle and flexor digitorum brevis (FDB) fibers from WT and Orai1-deficient mice in the absence of exercise (control) or <1, 6, and 24 hr after acute treadmill exercise. Together, the results support the idea that TT association with SR stacks in the I band promotes Orai1-dependent SOCE needed to replenish releasable Ca2+ stores, maintain SR Ca2+ release, and maximize contractile force during repetitive stimulation of fast twitch skeletal muscle.In spite of these findings, the stability of CEUs formed following exercise, process for disassembly of the components, and impact on Orai1-dependent SOCE, CaSR-stacks)  . To dete-stacks) . Shortly-stacks) , the numacks)   and in the absence of pharmacological store depletion (-depletion). Although fura-2 emission was unaltered upon application of extracellular Mn2+ in non-depleted ﬁbers (2+ quench was significantly increased (~70%) in FDB fibers isolated from mice < 1 hr after treadmill exercise . While fura-2 quench did not occur upon addition of Mn2+ to fibers from non-exercised mice, a marked increase in quench rate was observed in fibers from mice < 1 hr after exercise , using mag-ﬂuo-4, a rapid, low-afﬁnity Ca2+ dye to maximize resolution of Ca2+ transient magnitude and kinetics . The results indicate that compared to fibers from control mice, resting total Ca2+ store content was significantly reduced (~25%) in fibers obtained from mice < 1 hr after exercise . Using this approach, the decay phase of the electrically-evoked transient is well-described by a double exponential fit in which the fast component primarily reflects Ca2+ binding to fast myoplasmic Ca2+ buffers while the slow component is dominated by SERCA-mediated SR Ca2+ reuptake  that generally lasted ~10–12 stimulus trains (left), maximal specific force increased significantly during stimulus trains 7–30 in EDL muscles from mice < 1 hr after exercise (middle), a potentiation that was lost by the end of the protocol (right). As a result, both peak-specific force , reduced Ca2+ store content and increased resting Ca2+, as well as sustained Ca2+ transient amplitude and force production during repetitive stimulation all increase/decrease in parallel with the time course of TT extension within the I band  correlates with reduced EDL muscle force decline during repetitive stimulation . Here, we I band , 5 and 6e I band ; and e) e I band . Togethe2+ dynamics (Two main findings of this study are: a) TTs retract from the CEU following exercise prior to SR-stack disassembly  and 2 andynamics , and maidynamics  declinesdynamics . Interesdynamics , one pos2+ quench in the absence of thapsigargin/cyclopiazonic acid-induced store depletion (constitutive Ca2+ entry) in FDB fibers isolated from mice < 1 hr after treadmill exercise (2+ entry in these fibers was rapidly blocked by application of BTP-2 (2+ entry observed <1 hr after exercise is absent in fibers from two separate mouse models deficient in Orai1 function (2+ entry is mediated by Orai1 channels (rather than TrpC or mechano-sensitive channels). D1ER measurements of ‘spatially-averaged’ free Ca2+ concentration in the SR were not significantly different after exercise . It is important to note, however, that D1ER localization studies indicate that the majority of the D1ER signal originated from the triad junction  after acute exercise resulting, at least in part, from enhanced SOCE activity promoting SR refilling and total releasable Ca2+ content to a level sufficient to augment Ca2+ flux.We investigated the mechanism by which increased SOCE following acute exercise increases peak Caed total  and consed total  SOCE actreuptake . On the  content  and restntration  were sigbump-phase of the repetitive stimulation contraction curve. The following observations are consistent with this bump-phase reflecting the contribution of Orai1-dependent SOCE: 1) it is reduced by interventions  that reduce SOCE  it iexercise , it will2+ entry to optimize myoplasmic Ca2+ dynamics in a manner that limits muscle force decline during sustained activation.In conclusion, the findings reported here support the idea that CEUs formed after acute exercise represent an adaptive response of skeletal muscle to exercise that augments Orai1-dependent Cafl/fl::ACTAMer-CreOrai1 or iOrai1) knockout mice  C57Bl/6 mice (4–6 months old) were housed in microisolator cages at 20°C on a 12 hr light/dark cycle while being provided free access to standard chow and water. Two previously described muscle-specific mouse lines deficient in Orai1 function were also employed: a) dominant-negative Orai1 (dnOrai1) transgenic mice  and b) tout mice . Four-mofl/fl::ACTAMer-CreOrai1 mice were provided ad libitum access to tamoxifen-infused mouse chow  for a period of 4 weeks, and then returned to regular chow for at least one additional week before being used for experiments. Semi-quantitative PCR of Orai1 transcript level was used to verify success of tamoxifen treatment in Orai1 ablation in muscle, as previously described (Two-three-month-old escribed .2+ quench/Ca2+ measurements) were removed and prepared for experiments at one of the following three time points: 1)<1 hr from the end of the in vivo exercise protocol (referred to as <1 hr after exercise), 2) 6 hr after exercise, or 3) 24 hr after exercise.A standardized acute exercise protocol was performed at room temperature using a running treadmill  on a flat surface (0° incline) as previously described . Briefly4 and 0.8% K3Fe(CN)6 for 1–2 hr followed by a rinse with 0.1M NaCaCO buffer with 75 mM CaCl2. Ultrathin sections (~50 nm) were cut using a Leica Ultracut R microtome  with a Diatome diamond knife  and double-stained with uranyl acetate and lead citrate. Sections were viewed in a FP 505 Morgagni Series 268D electron microscope , equipped with Megaview III digital camera  and Soft Imaging System at 60 kV (100 kV for TT staining).Intact EDL and FDB muscles were fixed at room temperature with 3.5% or 6% glutaraldehyde in 0.1M sodium cacodilate (NaCaCO) buffer (pH 7.2), and processed for EM acquisition as previously described . For TT 2 were determined from electron micrographs of non-overlapping regions randomly collected from transversal EM sections, as described previously : 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4. Muscles were then incubated in Ringer’s solution supplemented with 1 mg/ml collagenase A  for 60 min while rocking gently at 37°C to allow enzymatic dissociation. Single FDB fibers obtained by mechanical trituration were plated on glass-bottom dishes and allowed to settle for >20 min before experimentation (see below). Only fibers with clear striations and no signs damage were used for recordings.As healthy single fibers are readily isolated from FDB muscles, all Mn2+-free Ringer's solution containing (in mM): 145 NaCl, 5 KCl, 1 MgCl2, 0.2 EGTA, pH 7.4.To measure the maximal rate of Mn2+ quench, during fura-2 AM loading, fibers were also incubated with two SERCA pump inhibitors  to fully deplete SR Ca2+ stores (+depletion) and a skeletal muscle myosin inhibitor (30 μM N-benzyl-p-toluene sulfonamide [BTS]) to prevent movement artifacts (2+-free Ringer's and excited at 362 nm (isobestic point of fura-2), while emission was detected at 510 nm using a DeltaRam illumination system . After obtaining an initial basal rate of fura-2 decay (Rbaseline), fibers were exposed to Ca2+-free Ringer's supplemented with 0.5 mM MnCl2. The maximum rate of fura-2 quench in the presence of Mn2+ (Rmax) was then obtained from the peak differential of the fura-2 emission trace during Mn2+ application. The maximum rate of SOCE (RSOCE) was calculated as RSOCE = Rmax Rbaselineand expressed as dF/dt in counts/sec (FDB fibers were loaded with 5 μM fura-2 AM for 1 hr at 37° C in a Cartifacts . B) In aunts/sec .2+ transients were monitored in acutely isolated FDB fibers as described previously (0) were recorded at 10 kHz and analyzed using Clampfit 10.0 .Myoplasmic Caeviously . Briefly2+ transients were determined in mag-fluo-4-loaded FDB fibers isolated from both sedentary mice and mice < 1 hr after exercise. For these experiments, 10 electrically-evoked twitch transients (0.5 Hz) were collected both before and after delivery of 10 stimulus trains . The decay phase of each transient was fitted according to the following equation and used to generate average values for both before and after high-frequency stimulation:The decay kinetics of single electrically-evoked twitch Cat)=Afast x [exp(-t/τfast)] + Aslow x [exp(-t/τslow)]F(t) is the fluorescence at time t, Afast and τfast are the amplitude and time constants of the fast component, and Aslow and τslow are the amplitude and time constants of the slow component.where F every 250 ms (30 ms exposure per wavelength and 2 × 2 binning) using a monochromator-based illumination system . Fura-FF emission at 535 ± 30 nm was captured using a high speed, digital QE CCD camera . Total releasable Ca2+ store content was assessed from the difference between basal and peak fura-FF ratio (ΔRatio340/380) upon application of a Ca2+ release cocktail containing 10 µM ionomycin, 30 µM CPA, and 100 µM EGTA in a Ca2+-free Ringer’s solution (ICE). For a subset of experiments, total releasable Ca2+ store content was measured in fura-FF-loaded fibers isolated from control mice and mice < 1 hr after treadmill exercise following delivery of 10 consecutive high-frequency stimulus trains . To confirm that the peak fura-FF signal during ICE application was not saturated, maximal fura-FF responsiveness was assessed at the end of each experiment by subsequent application of Ca2+-containing Ringer’s solution. Analysis of peak ICE-induced change in fura-FF ratio (ΔRatio340/380) was calculated using Clampfit 10.0 .Total releasable Caeviously . Briefly2+ concentration was determined in FDB fibers loaded with 5 µM fura-2 AM for 30 min at room temperature in control Ringer’s solution followed by a 30 min washout with dye-free Ringer’s solution. Fura-2-loaded fibers were placed on the stage of an inverted epifluorescence microscope (Nikon Instruments) and alternatively excited at 340 and 380 nm (30 ms exposure per wavelength and 2 × 2 binning) using a monochromator-based illumination system with fluorescence emission at 510 nm was captured using a high speed, digital QE CCD camera . Fura-2 340/380 ratios from myoplasmic areas of interest were calculated using TILL visION software , analyzed offline using NIH ImageJ, and converted to resting free Ca2+ concentrations using a calibration curve for fura-2 generated as described previously (Resting myoplasmic Caeviously .2+ concentration in the SR lumen was assessed in FDB fibers expressing the D1ER cameleon Ca2+ sensor. D1ER was expressed in FDB fibers using an in vivo electroporation approach described previously (The free Caeviously . BrieflyYFP/FCFP) for each fiber was calculated offline with averages calculated both immediately before application of ICE and then again after reaching a minimum value in the presence of ICE.D1ER-expressing fibers from control mice and mice < 1 hr after exercise were bathed in control Ringer’s solution supplemented with 25 µM BTS on the stage of an inverted Nikon Eclipse TE-2000-U microscope . A relatively large rectangular region of interest of the fiber was excited at 436 nm (10 nm bandwidth). Fluorescence emission within the region of interest was split with a 515 nm dichroic mirror and collected at 480 nm (30 nm bandwidth) and 535 (30 nm bandwidth) using a photomultiplier counting system . In order to directly compare measurements across multiple fibers, all experiments used identical excitation and emission gains. Following subtraction of background fluorescence, YFP and CFP emission intensities were collected and digitized at 100 Hz first in the presence of control calcium-free Ringer’s solution supplemented with 40 µM BTS and then during perfusion of ICE supplemented with 40 µM BTS. The ratio of YFP to CFP emission , fibers were co-labeled overnight at 4°C using primary antibodies for GFP  and either α-actinin  or the type1 ryanodine receptor . All primary antibody incubations were in 2% BSA in 1X PBS-T. Following three 10 min washes in PBS-T, samples were incubated with a 1:500 dilution of Goat anti Rabbit Alexa fluor 488 (Molecular Probes Cat # A11034) and Goat Anti Mouse Alexa fluor 594 (Molecular Probes Cat # R37121) for 1 hr at room temperature. Following three 10 min washes in 1X PBS-T, samples were imaged using an Olympus FV1000 laser scanning confocal microscope  and a 100X, UPlanSAPO NA 1.4 oil immersion objective. Alexa 488 and 594 were sequentially excited at 488 and 559 nm and detected at 515 and 617 nm respectively.2PO4, 1 MgSO4, 2 CaCl2,10 glucose, and 24 NaHCO3. Before starting measurements, optimal stimulation level and muscle length (L0) were determined using a series of 1 Hz-twitch stimulation trains while stretching the muscle to a length that generated maximal force (F0). After establishing L0, muscles were first equilibrated using three tetani  given at 1 min intervals and then subjected to a standard force frequency protocol (from 1 to 250 Hz). After 3 min of rest, a single sustained high frequency tetanus . After an additional 5 min of rest, muscles were then subjected to a repetitive stimulation protocol . All muscle contractility experiments were carried out at 30°C. Muscle force was recorded using Dynamic Muscle Control software and analyzed using a combination of both Dynamic Muscle Analysis  and Clampfit 10.0  software. Specific force was calculated by normalizing the absolute force to the physiological cross sectional area as previously described . Amplitude histograms of maximal rates of Mn2+ quench were fit according to a single Gaussian distribution. In all cases, differences were considered as statistically significant at *p<0.05. All summary data were presented as mean ± SEM.Statistical significance was determined using either Student’s t-test for comparing two groups  and S3 a In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:2+ release and muscle twitch strength. Previous work has shown that after exercise the T tubule (TT) invaginations of the plasma membrane elongate and make close appositions to newly formed stacks of sarcoplasmic reticulum (SR), and that the presence of the STIM1 Ca2+ sensor and Orai1 calcium channels at these newly formed junctions may increase the loading of calcium into the SR to promote contractile force. Here the authors provide new evidence for this mechanism, showing that after exercise the appearance and disappearance of new SR stack-TT structures parallels the time course of augmented calcium entry through Orai, SR calcium content and release amplitude, and contractile force. The results thus support a functional link between increased muscle performance following exercise and the operation of store-operated Orai channels at newly formed SR-TT contacts. This work has important implications for understanding muscle function and morphological adaptations to exercise as well as the newly emerging roles of store-operated calcium channels in excitable cells.Following exercise, muscle fibers undergo membrane remodeling that results in increased CaDecision letter after peer review:[Editors’ note: the authors were asked to provide a plan for revisions before the editors issued a final decision. What follows is the editors’ letter requesting such plan.]2+ entry in skeletal muscle" for peer review at eLife. Your article is being evaluated by three peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Ronald Calabrese as the Senior Editor.Thank you for sending your article entitled "Transverse tubule remodeling enhances Orai1-dependent CaGiven the list of essential revisions, including new experiments, the editors and reviewers invite you to respond with an action plan and timetable for the completion of the additional work. We plan to share your responses with the reviewers and then issue a binding recommendation.2+ transients, and force production following exercise provides some additional support for the idea that CEUs are involved in exercise-dependent effects on muscle contractility. However, to represent a significant advance beyond the previous work the reviewers all agreed that more quantitative experimental data are needed to establish a mechanistic link between increased SOCE and altered muscle function.All three reviewers thought the manuscript was an important extension of your earlier work on muscle calcium entry units (CEUs) that form after exercise. The time course of changes in SR-TT contacts, SOCE, Ca2+ transients and force. Increased SOCE cannot directly account for this because the flux is orders of magnitude smaller than the SR release flux. However, several non-exclusive mechanisms could contribute:The critical question is how increased SOCE leads to the enhancement of peak Ca2+ content leading to increased Ca2+ release flux might be responsible but is inconsistent with the 25% reduction in content that was reported. The method used to measure total SR content (Ca2+ release cocktail containing ionomycin) is not sufficiently quantitative and may also release Ca2+ from other compartments . To address this concern, a ratiometric Ca2+ indicator should be targeted to the SR to directly measure the store content during stimulation, and the Ca2+ release flux can be estimated from cytosolic transients assuming generally accepted cytosolic buffering parameters.1) An increase in SR Ca2+ enough to reduce fast buffering and thereby potentiate the effect of Ca2+ release. This could be assessed by a more detailed measurement of cytosolic Ca2+ between transients and estimating the effect of this increased Ca2+ on the buffering power.2) SOCE may elevate cytosolic Ca2+ transient decay rates at different times following exercise. Importantly, such measurements may reveal a time-dependent change in Ca2+ handling after exercise that is not directly related to the assembly and disassembly of CEUs.3) Finally, SERCA may be inhibited after exercise due to glycogen depletion. Reduction of SERCA activity could enhance the transient amplitude, as well as contribute to the partial depletion of the SR and tonic SOCE that was observed. SERCA activity should be measured from the Ca2+ entry in skeletal muscle" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Ronald Calabrese as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Eduardo Rios (Reviewer #2).Thank you for submitting your article "Transverse tubule remodeling enhances Orai1-dependent CaAfter the reviewers discussed the reviews with one another, the Reviewing Editor sent the list of essential revisions to you to verify that you could complete these within about two months. Your revision plan and timeline meet the requirements, so please proceed with the revisions and additional experiments. The complete review is printed below. Please note that additional comments have been added based on the revision plan you sent.Summary:2+ signaling and muscle contraction. This paper extends upon previous work by the authors describing the formation of SR stacks, TT extensions and increased STIM1-Orai1 colocalization  in the I band following exercise. The authors previously proposed that the CEUs promote resistance to fatigue during repetitive stimulation. Here the authors provide new functional evidence for this mechanism by showing that the time course of development and reversal of SR stack-TT structures parallels the timing of changes in SOCE, SR Ca2+ content, SR Ca2+ release transients, and contractile force following exercise. Using knockout mice, the authors also show that Orai1 is required for these changes, presumably through interaction with STIM1 and/or STIM2.Michelucci et al. present evidence that acute exercise promotes the association of sarcoplasmic reticulum (SR) stacks with extended transverse tubules (TT) in skeletal muscle, and that these contacts enhance store-operated calcium entry (SOCE) to optimize CaEssential revisions:2+ transients, and force production following exercise provides indirect support for the idea that CEUs are involved in exercise-dependent effects on muscle contractility. However, there was also strong agreement that more quantitative experimental data are needed to show how the observed changes in SOCE lead to altered muscle function during exercise. This could occur by several non-exclusive mechanisms, which need to be tested:All three reviewers thought the manuscript is an important extension of your earlier work on muscle calcium entry units (CEUs) formed after exercise. The time course of changes in SR-TT contacts, SOCE, Ca2+ content leading to increased Ca2+ release flux might be responsible. The free Ca2+ in the SR needs to be measured during stimulus trains to determine if it is elevated. The Ca2+ release cocktail method will indicate total stored Ca2+, not free Ca2+. Instead, a ratiometric Ca2+ indicator such as D1ER should be targeted to the SR to directly measure the free luminal Ca2+ during stimulation.1) An increase in SR Ca2+ enough to reduce fast buffering and thereby potentiate the effect of Ca2+ release. The effect on buffering could be assessed by a more detailed measurement of cytosolic Ca2+ between individual transients and after stimulus trains in control and exercised mice.2) SOCE may elevate cytosolic Ca2+ twitch transients (fast and slow components) can be used to estimate changes in SERCA activity and Ca2+ buffering from control and exercised mice before and after 10 stimulus trains. Importantly, such measurements may reveal a time-dependent change in Ca2+ handling after exercise that is not directly related to the assembly and disassembly of CEUs.3) SERCA may be inhibited after exercise due to glycogen depletion. Reduction of SERCA activity could enhance the transient amplitude, as well as contribute to the partial depletion of the SR and tonic SOCE that was observed in resting muscle after exercise. Kinetic analysis of cytosolic Ca4) The idea that CEUs are responsible for the constitutive SOCE after exercise is not well supported. In the Discussion section, the authors suggest that the new CEUs can account for the increased tonic SOCE after exercise. The supporting argument is that "non-depletion" quench after exercise (1200 cps) + TG/CPA quench before exercise (1700 cps) = TG/CPA quench rate after exercise (2800 cps). However, the quench after exercise is driven by only partial SR depletion, so TG/CPA should add more than 1200. This argument does not make quantitative sense to me. Unless it can be better substantiated, it should be deleted.2+ content peaks at the same time (<1 hour) as "non-depletion" SOCE, Ca2+ release transients and force production during trains. This complicates things. Because of tonic depletion, it is possible that pre-existing STIM and Orai complexes (activated by tonic store depletion) distinct from the new CEUs are being activated and contribute to augmented function. This possibility needs to be explicitly acknowledged, and the conclusions regarding CEU functions toned down.5) A related issue concerns the idea that CEUs are solely responsible for the SOCE that augments muscle function after exercise. The loss of SR Ca2+ entry in skeletal muscle" for further consideration at eLife. Your revised article has been favorably evaluated by Ronald Calabrese (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Transverse tubule remodeling enhances Orai1-dependent CaThe manuscript has been improved, and the new data have addressed several of the issues that were raised in the original review. However, there are some remaining issues that need to be addressed before acceptance, as outlined below:2+ Store Content is Reduced <1 Hour After Exercise, but Increased Following Repetitive, High-frequency Stimulation” and Discussion section). After exercise, the constitutive SOCE is greatly increased, but the new D1ER measurements show that resting free Ca2+ in the SR is not altered. Because STIM1 and SOCE are controlled by free Ca2+ in ER/SR, and not the total Ca2+ content, how can Orai activity be increased without a reduction of free SR Ca2+? The decrease in total SR Ca2+ that was observed cannot explain the appearance of constitutive SOCE. This conundrum needs to be clearly stated and possible explanations provided. While uncertainty in the D1ER measurement might mask a small depletion of free Ca2+, this is unlikely to be the whole explanation since the SOCE amplitude was so large .1)  The new twitch analyses are convincing, but it is not clear to anyone but an expert muscle physiologist how these were used to draw conclusions about Ca [Editors’ note: what follows is the authors’ plan to address the revisions.]2+ transients, and force production following exercise provides some additional support for the idea that CEUs are involved in exercise-dependent effects on muscle contractility. However, to represent a significant advance beyond the previous work the reviewers all agreed that more quantitative experimental data are needed to establish a mechanistic link between increased SOCE and altered muscle function.All three reviewers thought the manuscript was an important extension of your earlier work on muscle calcium entry units (CEUs) that form after exercise. The time course of changes in SR-TT contacts, SOCE, Ca2+ transients and force. Increased SOCE cannot directly account for this because the flux is orders of magnitude smaller than the SR release flux.The critical question is how increased SOCE leads to the enhancement of peak Ca2+transients and force during repetitive stimulation. Before describing our plan to address this issue, we would like to reiterate the backstory, motivation and objectives of the current study. Our initial report describing exercise-induced formation of CEUs in muscle  concluded that CEUs formed following exercise enhance SOCE to optimize Ca2+release for muscle force production during repetitive stimulation. However, the implication of CEUs on SOCE and Ca2+release was based on indirect evidence . Direct evidence to support a role for CEUs in promoting Orai1- dependent SOCE and Ca2+release were lacking. Thus, a primary objective of the current study was to quantify Orai1-dependent SOCE and myoplasmic calcium transients during repetitive stimulation under conditions that result in CEU formation and disassembly. EM studies (in both EDL and FDB muscles) revealed that CEUs form < 1 hour after exercise and that TTs retract from the CEU earlier than SR stacks disassemble. We took advantage of this time course to assess changes in SOCE (Mn quench), calcium (mag-fluo4), and force (ex vivo EDL contractility) at different times during the assembly/disassembly of CEU component parts. Our results provide the first direct evidence that Orai1-dependent SOCE and Ca2+ release during repetitive stimulation are increased when TTs are associated with SR stacks. Unexpectedly, we found that acute exercise also results in substantial constitutive Orai1-dependent SOCE . These findings provide critical needed evidence to support the assertion that acute exercise promotes formation of CEUs to augment Orai1-dependent SOCE in order to optimize Ca2+ release and muscle contractility during repetitive stimulation.We thank the Reviewing Editor and three reviewers for their acknowledgment of the importance of the current work in supporting the conclusion that calcium entry units (CEUs) are involved in exercise-dependent effects on muscle contractility. We also appreciate the question raised regarding how increased SOCE after exercise enhances peak Ca2+transient during repetitive stimulation. Since we did not have direct evidence for a specific mechanism, we refrained from speculating on this in the Discussion section of the manuscript. If provided an opportunity to submit a revised version of the manuscript, we would be happy to consider these potential mechanisms and their implications in the Discussion section. We have also initiated new experiments (outlined below) in hopes of being able to provide at least some evidence to support/exclude these possibilities. If successful, we are prepared to include the results on these studies in a revised version of the manuscript. However, given the importance and extent of the results/contributions of the study outlined above (none of which were questioned in the Editor’s summary of reviewer comments), we would hope that publication of our study will not be inexorably linked to our providing compelling evidence for an answer to this question.We agree with the Editor and reviewers that the next important question to address is to explain how increased SOCE after exercise optimizes the myoplasmic Ca2+ transients during repetitive stimulation. Our discussion would include each of the three points below raised by the Editor and reviewers. Provided sufficient time, we also compete the series of experiments below designed to provide supportive evidence for one or more of these explanations.We plan to revise the Discussion section of the manuscript to provide a comprehensive description of potential reasons for how increased SOCE function after acute exercise could result in enhanced peak CaHowever, several non-exclusive mechanisms could contribute:2+ content leading to increased Ca2+ release flux might be responsible but is inconsistent with the 25% reduction in content that was reported. The method used to measure total SR content (Ca2+ release cocktail containing ionomycin) is not sufficiently quantitative and may also release Ca2+ from other compartments . To address this concern, a ratiometric Ca2+ indicator should be targeted to the SR to directly measure the store content during stimulation, and the Ca2+ release flux can be estimated from cytosolic transients assuming generally accepted cytosolic buffering parameters.1) An increase in SR Ca2+ store content after acute exercise were conducted on resting non-stimulated fibers, whereas the increase in Ca2+release during repetitive high-frequency stimulation was only observed after 8-10 consecutive stimulation trains. In addition, the increase in peak Ca2+transient amplitude in fibers from exercised mice developed during the time course of each 500 ms stimulus . Thus, the evolution of the increase in evoked Ca2+ release is dynamic, making resting steady state store content measurements irrelevant Ca2+ regard to SR Ca2+ available for release at the end of the 10th stimulus train. Thus, one possibility is that in fibers from exercised mice, increased SOCE during each stimulus train enhances store Ca2+such that Ca2+load is increased to a level sufficient to augment Ca2+flux during the 10th and subsequent trains. We are planning experiments where we will measure either total C Ca2+ a store content (using ionomycin/CPA/EGTA or ICE as before) or just RYR1-releasable store content (using 4-chloro-m-cresol) after delivery of 10 stimulus trains. Since the longitudinal SR (with SERCA) and CEU junctions (with STIM1-Orai1) lie outside the triad and lack RYR1 release channels, we expect that the ICE assay will provide a more accurate assessment of the impact of SOCE on total Ca2+ store content. However, it will be interesting to see if the RYR1-releasable Ca2+ pool following 10 tetani is increased in fibers from exercised mice. Given the extent of SR and amount of SR Ca2+binding proteins in skeletal muscle, the peak ICE response in experiments is dominated by Ca2+within the SR compartment. Consistent with this, we previously used the Ca2+dependent UV absorbance spectra of BAPTA to show that the vast majority (~80%) of total Ca2+in EDL muscle resides within the SR . In addition, the peak ICE response in fura-2ff-loaded FDB fibers is markedly reduced (~90%) in fibers from CASQ1 KO mice  introduced via electroporation.To clarify, our measurements of a ~25% reduction in total Camice see  below. T2+ enough to reduce fast buffering and thereby potentiate the effect of Ca2+ release. This could be assessed by a more detailed measurement of cytosolic Ca2+ between transients and estimating the effect of this increased Ca2+ on the buffering power.2) SOCE may elevate cytosolic Ca2+levels were increased in FDB fibers from exercised mice . Thus, the increased SOCE during repetitive stimulation could further elevate myoplasmic Ca2+levels enough to reduce fast Ca2+ buffering  during subsequent stimuli. In fact, this could in part explain the time-dependent increase observed in the peak of the Ca2+transient during a given stimulus train in fibers from exercised mice . To further test this idea, we plan to quantify resting Ca2+levels in fibers from control and exercised mice immediately after 10 consecutive high-frequency stimulus trains.We agree. Consistent with our observation of significant constitutive SOCE after exercise, resting Ca2+ transient decay rates at different times following exercise. Importantly, such measurements may reveal a time-dependent change in Ca2+ handling after exercise that is not directly related to the assembly and disassembly of CEUs.3) Finally, SERCA may be inhibited after exercise due to glycogen depletion. Reduction of SERCA activity could enhance the transient amplitude, as well as contribute to the partial depletion of the SR and tonic SOCE that was observed. SERCA activity should be measured from the Ca2+transients measured using the low affinity Ca2+dye, mag-fluo-4. The decay of the Ca2+transient is well-described by a double exponential fit in which the fast component primarily reflects binding of Ca2+to fast Ca2+buffers  while the slow component reflects SERCA-mediated Ca2+ reuptake. The relative contribution of the slow (Aslow) and fast (Afast) amplitudes will be calculated by dividing the absolute value of each component by the sum of the two components . This kinetic analysis will be completed on twitch Ca2+transients measured in FDB fibers from control and exercised mice both immediately before or after delivery of 10 consecutive high frequency stimulation trains. Comparison of the fast and slow amplitudes (absolute and relative) and the time constants of the two kinetic components will provide an index of the effects of exercise and repetitive stimulation on fast Ca2+buffering and SERCA activity.This is an interesting suggestion that we had not considered previously. To obtain an estimate of SERCA activity in intact fibers from control and exercised mice before and after repetitive stimulation, we will fit the decay phase of electrically-evoked twitch Ca[Editors’ notes: the authors’ response after being formally invited to submit a revised submission follows.]Essential revisions:2+ transients, and force production following exercise provides indirect support for the idea that CEUs are involved in exercise-dependent effects on muscle contractility. However, there was also strong agreement that more quantitative experimental data are needed to show how the observed changes in SOCE lead to altered muscle function during exercise. This could occur by several non-exclusive mechanisms, which need to be tested:All three reviewers thought the manuscript is an important extension of your earlier work on muscle calcium entry units (CEUs) formed after exercise. The time course of changes in SR-TT contacts, SOCE, Ca2+ content leading to increased Ca2+ release flux might be responsible. The free Ca2+ in the SR needs to be measured during stimulus trains to determine if it is elevated. The Ca2+ release cocktail method will indicate total stored Ca2+, not free Ca2+. Instead, a ratiometric Ca2+ indicator such as D1ER should be targeted to the SR to directly measure the free luminal Ca2+ during stimulation.1) An increase in SR Ca2+ store content after acute exercise were conducted on resting non-stimulated fibers, whereas the increase in Ca2+ release during repetitive high frequency stimulation was only observed after the seventh stimulation train. In addition, the increase in peak Ca2+ transient amplitude in fibers from mice <1 hour after exercise developed progressively over time during the stimulation trains . Thus, the evolution of the increase in evoked Ca2+ release is dynamic during repetitive stimulation, making resting steady-state Ca2+ store content measurements irrelevant with regard to SR Ca2+ available for release during repetitive stimulation.To clarify, our measurements of a ~25% reduction in total Ca2+ store content such that the total releasable Ca2+ load is increased to a level sufficient to augment Ca2+ flux during the seventh and subsequent stimulus trains. To test this idea, we conducted new experiments in fura-FF-loaded fibers to assess total releasable Ca2+ store content (using ionomycin/CPA/EGTA or ICE as before) after delivery of 10 stimulus trains . Results from these experiments are quite informative as both myoplasmic resting and total releasable Ca2+ store content were increased significantly after 10 stimulus trains in fibers from acutely exercised mice compared to that observed for fibers from both sedentary mice and <1 hour after exercise in the absence of stimulation .Thus, one possibility is that in fibers from mice <1 hour after exercise, increased SOCE during each stimulus train progressively enhances total Cation see . These r2+ binding proteins in skeletal muscle, the peak response to application of Ca2+ free solution containing ionomycin/cyclopiazonic acid/EGTA (ICE) is dominated by Ca2+ within the SR compartment. Consistent with this, we previously used the Ca2+ dependent UV absorbance spectra of BAPTA to show that the vast majority (~80%) of total Ca2+ in EDL muscle resides within the SR . Importantly, the peak ICE response in fura-FF-loaded fibers is markedly reduced (~90%) in FDB fibers from calsequestrin-1 knockout (CASQ1-null) mice, as expected since CASQ1 is the primary SR Ca2+ binding protein in muscle (see 2+ within mitochondria and other compartments contributes <10% of the ICE response in these experiments.Given the extent of SR and level of SR Cascle see . These d2+probe (D1ER). However, it is important to point out upfront that the D1ER probe reports the “free Ca2+concentration” within the SR/ER lumen, not the total releasable Ca2+content as assessed in our fura-FF studies. For these experiments, the D1ER probe was expressed in FDB fibers via electroporation . As reported previously, the D1ER probe localizes nicely to both triad junctions and longitudinal extensions of the SR, which are plentiful within the I band of the sarcomere . Using identical excitation and emission gains, the D1ER YFP/CFP ratio was not significantly different in fibers from control mice and mice <1 hour after exercise both under resting conditions with a full SR Ca2+ store and after depletion of SR Ca2+ with ICE . Thus, the spatially-averaged Ca2+ concentration within the SR lumen is not significantly altered by acute exercise . These findings are consistent with the strong buffering power in the SR in skeletal muscle and prior observations  that the free Ca2+ concentration in the SR lumen (~500 μM) remains largely unaltered in face of marked changes in total SR Ca2+ content. These results are included in the revised manuscript .Nevertheless, we conducted experiments using a SR-targeted Ca2+ enough to reduce fast buffering and thereby potentiate the effect of Ca2+ release. The effect on buffering could be assessed by a more detailed measurement of cytosolic Ca2+ between individual transients and after stimulus trains in control and exercised mice.2) SOCE may elevate cytosolic Ca2+ levels were significantly elevated in FDB fibers isolated from mice <1 hour after acute exercise. Importantly, we now show (see panel B in 2+ in FDB fibers after acute exercise (where SOCE activity is increased) was even greater immediately after delivery of 10 stimulus trains . Thus, the elevation in resting Ca2+ after acute exercise could conceivably reduce fast buffering enough to potentiate the effect of Ca2+ release. However, detailed quantitative analysis of twitch Ca2+ transient decay kinetics before and after 10 stimulus trains does not support a significant change in either fast Ca2+ buffering or SERCA-mediated Ca2+ reuptake . We updated the Discussion section of the revised manuscript to include a consideration of these.As shown above, resting myoplasmic Ca2+ twitch transients (fast and slow components) can be used to estimate changes in SERCA activity and Ca2+ buffering from control and exercised mice before and after 10 stimulus trains. Importantly, such measurements may reveal a time-dependent change in Ca2+ handling after exercise that is not directly related to the assembly and disassembly of CEUs.3) Finally, SERCA may be inhibited after exercise due to glycogen depletion. Reduction of SERCA activity could enhance the transient amplitude, as well as contribute to the partial depletion of the SR and tonic SOCE that was observed in resting muscle after exercise. Kinetic analysis of cytosolic Ca2+ transient decay using a low affinity Ca2+ dye (mag-fluo-4) before and after delivery of 10 consecutive stimulus trains  in FDB fibers from both sedentary mice and mice <1 hour after exercise. Using this approach, the decay phase of the electrically-evoked mag-fluo-4 twitch transient is well-described by a double exponential fit in which the fast component primarily reflects Ca2+ binding to fast Ca2+ buffers while the slow component is dominated by SERCA-mediated SR Ca2+ reuptake . For these experiments, we first recorded 10 electrically-evoked twitch transients (0.5 Hz) in FDB fibers from either sedentary mice or mice <1 hour after exercise before delivery of 10 stimulus trains . After the 10 stimulus trains, a second set of 10 electrically-evoked twitch transients (0.5 Hz) were recorded. Despite a significant increase in the peak Ca2+ transient amplitude during the 10th stimulus train (1.51 ± 0.07) compared to the first stimulus train (1.32 ± 0.09) only in fibers from mice <1 hour after exercise, no significant changes in Ca2+ transient decay kinetics were observed compared to that obtained for fibers isolated from sedentary mice. These results are included in the revised manuscript .We thank the reviewer for this excellent suggestion. We completed the suggested experiments to quantify the kinetics of electrically-evoked twitch Ca4) The idea that CEUs are responsible for the constitutive SOCE after exercise is not well supported. In the Discussion section, the authors suggest that the new CEUs can account for the increased tonic SOCE after exercise. The supporting argument is that "non-depletion" quench after exercise (1200 cps) + TG/CPA quench before exercise (1700 cps) = TG/CPA quench rate after exercise (2800 cps). However, the quench after exercise is driven by only partial SR depletion, so TG/CPA should add more than 1200. This argument does not make quantitative sense to me. Unless it can be better substantiated, it should be deleted.As suggested, we removed this speculation from the Discussion section of the revised manuscript.2+ content peaks at the same time (<1 hour) as "non-depletion" SOCE, Ca2+ release transients and force production during trains. This complicates things. Because of tonic depletion, it is possible that pre-existing STIM and Orai complexes (activated by tonic store depletion) distinct from the new CEUs are being activated and contribute to augmented function. This possibility needs to be explicitly acknowledged, and the conclusions regarding CEU functions toned down.5) A related issue concerns the idea that CEUs are solely responsible for the SOCE that augments muscle function after exercise. The loss of SR CaWe thank the reviewer for pointing out this possibility, which is now explicitly acknowledged in the Discussion section of the revised manuscript.The manuscript has been improved, and the new data have addressed several of the issues that were raised in the original review. However, there are some remaining issues that need to be addressed before acceptance, as outlined below:2+ Store Content is Reduced <1 Hour After Exercise, but Increased Following Repetitive, High-frequency Stimulation” and Discussion section). After exercise, the constitutive SOCE is greatly increased, but the new D1ER measurements show that resting free Ca2+ in the SR is not altered. Because STIM1 and SOCE are controlled by free Ca2+ in ER/SR, and not the total Ca2+ content, how can Orai activity be increased without a reduction of free SR Ca2+? The decrease in total SR Ca2+ that was observed cannot explain the appearance of constitutive SOCE. This conundrum needs to be clearly stated and possible explanations provided. While uncertainty in the D1ER measurement might mask a small depletion of free Ca2+, this is unlikely to be the whole explanation since the SOCE amplitude was so large .1) . It is important to note, however, that our D1ER localization results indicate that the majority of the D1ER signal is in the triad junction . Thus, the D1ER data primarily reflect the free Ca2+ concentration within the triad junction, which is not different after exercise. These results suggest that the constitutive SOCE observed after exercise does not originate from Orai1 channels in the triad junction. Rather, the constitutive Ca2+ entry more likely arises from Orai1 channels localized in the elongated TT within the I band region of the sarcomere that couples with SR-stack membranes forming CEUs, that are not well-reflected in the spatially-averaged D1ER signal. These observations support the idea that CEUs represent the site of constitutive Ca2+ entry after exercise. An alternative explanation is that exercise could produce a signal that directly activates Orai1 independent of store depletion (even within the triad).The D1ER measurements indeed show that the “spatially-averaged” free CaWhile admittedly too superficial, we attempted to address this issue in the following sentence from the Discussion section of the prior version of the revised manuscript:2+ entry could be due to either a fraction of the SR store being partially depleted  or generation of a direct activator of Orai1 channels after exercise.""Thus, the constitutive Ca2+ concentration. However, we appreciate the reviewers point and realize that our attempt at brevity missed several important salient points (discussed above). Therefore, we further revised this part of the Discussion section to more explicitly consider these issues.By “fraction of the SR being partially depleted” we were indirectly referring to CEUs formed after exercise. We also left open the possibility that exercise could produce a direct activator of Orai1 that does not require a reduction in the SR Ca2+ binding and SERCA activity. In Subsection “Total Releasable Ca2+ Store Content is Reduced <1 Hour After Exercise, but Increased Following Repetitive, High-frequency Stimulation”, the statement that kinetics of twitch Ca2+ transient decay do not change needs to be explained; why was this experiment done, and what is the interpretation? In the Discussion section, it would help to discuss the possibilities that were brought up in review, including saturation of buffers and depletion of glycogen and effects on SERCA, and how the new results argue against these alternative explanations. An example of a transient should be shown along with the analysis in Figure 5—figure supplement 3 to illustrate the fast and slow components and how they were fitted.2) The new twitch analyses are convincing, but it is not clear to anyone but an expert muscle physiologist how these were used to draw conclusions about Ca2+ transient decay kinetics were analyzed . We revised the manuscript to include this rationale in the Results section, just before these data are presented . As suggested, we also now include example twitch Ca2+ traces with superimposed two-exponential fits in Figure 5—figure supplement 3. We now also discuss potential effects of saturation of Ca2+ buffers, depletion of glycogen and SERCA activity in the Discussion section of the revised manuscript.We apologize for not providing a clear rationale in the Results section for why the twitch Ca


The study showed that the use of mirrors may represent a low-cost useful tool to stimulate the expression of natural behaviors when rabbits are bred in small groups in a free-range system. The use of mirrors appeared to simulate the condition in which rabbits were allowed to have visual contacts with the other groups, improving important natural behaviors . This is important in the view of adopting welfare-friendly techniques in rearing of farm animals, since consumers demand for high quality animal products is changing in a way that includes in the concept of home-made products also alternative rearing systems complying with the conditions of welfare.The aim of this research was to evaluate the possible usefulness of mirrors in improving rabbit behavior in a free-range breeding system. Three groups  were compared: isolated, isolated with mirrors and separated by a wire mesh . Rabbits allowed to have a visual and olfactory contact showed a significantly higher expression of important natural behaviors  compared to the isolated rabbits (with or without mirrors); while rabbits in the mirror group showed higher allo-grooming activity than those isolated and no different locomotion activity than those separated by wire mesh. Thus, mirrors seemed to be able to modify the behavioral repertoire of isolated rabbits by acting on social perception in rabbits reared in small groups in a free-range system. Nevertheless, despite it being advisable to use a combination of different indicators in order to assess the stress level of an animal, the accuracy of serum cortisol, as well as of some secondary stress markers assay, appeared to be limited in this type of breeding. Public opinion strongly asks for adopting welfare-friendly techniques in rearing of farm animals. Also, consumer demand for high quality animal products is increasing , in partEighty-one 45 day-old New Zealand White x California male rabbits from the same farming industry in Avellino, Italy, were included in the trial. Rabbits were clinically healthy, with a mean body weight of 1422.6 ± 15.6 g. Each rabbit was identified by using an ear tag. Animals were moved from the cages and divided into three groups (27 rabbits as nine replicates of three rabbits per group). Each group was stabled into nine pens  having the following features: face to face (F2F) group—the rabbits were allowed to see each other as well as the surrounding space and to have olfactory contact, blind group—they were not allowed to look around by the use of blackout plastic panels, mirrors group—they were not allowed to see either the surroundings nor the other groups but two mirrors (1.2 × 0.4 m) were placed on two adjacent walls in each box. As depicted in The panels were made of hard plastic, 0.5 cm thick, odorless and had a flat surface. Every day at 10 am the pens were cleaned, and water and feed were provided always by the same operator. A commercial feed for meat rabbits (crude protein (CP) 15.6, ether extract (EE) 3.6, crude fiber (CF) 19.0% as feed) was used all along the trial. The feed intake was recorded for the entire period of the trial by weighing the administered, refused and scattered feed. The environmental temperature and humidity were recorded along the trial.All animals were free to move inside the pen, the duration of the trial was of 45 days (including seven days for adaptation), which is considered as the standard fattening period of rabbits reared for meat production in Italy.After one week of adaptation, a 24 h recording was performed every seven days by using an AP-320S C&Xanadu camera for each pen. During the recording nobody had access to the pens to avoid interfering with the normal behavioral repertoire adopted by the animals. Recordings were watched by a single operator and analyzed for the following behaviors: drinking (minutes), feeding (minutes), olfactory investigation (minutes), gnawing (minutes), nose to nose (seconds), scratching (minutes), alarm (seconds), allo-grooming (minutes), kennel stay (number), stretching (seconds), locomotion (minutes), inactivity (minutes) and cecotrophy (number). For each pen, how many times and for how long the rabbits showed each of the above-mentioned behavioral repertoire was measured.At 90 days of age, blood samples were collected fasting from an ear vein of each rabbit by the same veterinary practitioner in plastic tubes with and without K3-EDTA. K3-EDTA were used for packed cell volume (PCV) assay by using an automatic counter . The other aliquot was centrifuged at 2000× g for 15 min and serum was used for determination of cortisol, glucose, lactate and total protein. An AIA-360 automated immunoassay analyzer and reagents from Tosoh  were used to assay cortisol. Glucose, lactate and total protein were assayed by means of an automatic biochemical analyzer AMS AUTOLAB  using reagents from Spinreact . All the procedures used in the study were approved by the Ethical Animal Care and Use Committee of the University of Naples Federico II and received formal institutional approval in accordance with local and national law, regulations and guidelines .Results were expressed as means + root mean squared error (RSME) and analyzed for groups  and for daytime .Data were analyzed by means of ANOVA . Data concerning the stress markers were compared by ANOVA for one-way according to the following model:p ≤ 0.01.The data concerning the ethogram were processed by two-way ANOVA according to the model:No stereotypic behavior was observed among rabbits. The rabbits’ ethogram is reported in p < 0.01) and spent much more time in gnawing  and olfactory investigation . By contrast, the mirrors and blind groups showed more similar behaviors in terms of time spent on olfactory investigation, gnawing, alarm and stretching, but always lower than F2F group. Compared to the other groups, the blind one spent more time in terms of number of kennel stay . The mirrors group showed the higher % of time, after inactivity time and feeding, in allo-grooming  compared to both the other groups. Moreover, the time spent on the mirrors was higher during the dark period (time slot 3) compared to the other time slots.The rabbits kept in pens F2F appeared more active compared to the other groups, they spent daily only 135.6 min to rest (p < 0.01), in time slot 2 in olfactory investigation gnawing, nose to nose (p < 0.05) and less time inactive (p < 0.01). Interaction between group x time resulted significantly different (p < 0.05) only for locomotion, the differences are depicted in Despite no significant differences were detected among groups for drinking and feeding , some differences were detected in terms of time slots. In particular, rabbits spent significantly more time drinking (64.0 min/day) from 4:00 pm to 12:00 pm (time slot 2) and less time feeding (89.5 min/day) from 8 am to 4 pm (time slot 1). In time slot 1, rabbits spent more time inactive (In p < 0.01) in F2F group . Total protein resulted in significantly different values (p < 0.01) for mirrors and blind . Lactate resulted significantly higher (p < 0.05) in F2F group .Glucose resulted significantly lower  preference of the rabbits bred in pens for mirrors during the active (dark) period.Batchelor  noted thWhen the animals are not in good conditions of well-being they undergo to different degrees of stress. Stress behaviors arise when the animal perceives a threat that leads it to have an abnormal, physiological and emotional state . From thIn order to assess the welfare state of an animal, it is advisable to use a combination of different stress indicators to avoid interpretation problems that arise when using a single observation method . It is nHowever, the latitude of the response is not the same among the species. Cortisol and corticosterone are influenced by social stress and constitute good stress indicators in rabbits as reported by Szeto et al. . No diffp < 0.001). Also, some authors [The behavioral patterns suggested that the use of mirrors may be effective in ameliorating rabbit welfare. Among the three groups, the F2F one showed a greater incidence, respect to the blind group, of important behaviors such as allo-grooming, OI, gnawing, alarm and scratching that reveal a more natural behavioral repertoire, thus, more respectful of rabbit behavior. Despite a similar time spent in carrying out behaviors such as: OI, gnawing, alarm and stretching of the blind group compared to mirrors one, this last group showed more time spent to allo-grooming and less in kennel stay, suggesting greater activity of the rabbits belonging to the mirrors group due to the higher stimulation caused by the mirrors in the pens. Thus, mirrors may represent a useful, economic and not invasive tool to reduce the stress of rabbits kept in captivity. The importance of establishing a kind of visual social relationship has been also underlined for the disposition of the animals in cage rows, as in the industrial systems . Those a authors ,34,35 obIn nature, rabbits spend most of their time with breed conservation activities  while in breeding conditions rabbits are mainly bored and if the gnawed sticks are not available, they gnaw the cage, the power supply or even one of the others . AccordiInterestingly, the activity behaviors  were higher in F2F group respect to the other two groups. Thus, the “visual” isolation from the other animals and the environment modified rabbits’ behavior, but the difference was not significant for locomotion between F2F and mirrors. This may indicate that mirrors may partially reduce the effects of isolation, but such hypothesis need to be further investigated. Importantly, such result may also have effects on energy balancing and, as a consequence, on breeding’ productive performance.In conclusion, mirrors may represent a useful, low cost environmental enrichment in the free-range breeding system by acting on the behavioral repertoire with potential effects on animal welfare and productive performance.


The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes. This contrasts with reports using whole-cell recordings that describe this channel as displaying either selectivity for anions or being relatively non-selective amongst the major physiological monovalent ions.TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca+-selective currents revealed an outwardly rectifying conductance activated by a high intracellular Ca2+ concentration in all three species. These currents appeared after 5–6 minutes and were blocked by CaCCinh-A01, properties typical of TMEM16F. Ion substitution experiments showed that the underlying conductance was predominantly Cl–-permeable in rat megakaryocytes and HEL cells, yet non-selective between monovalent anions and cations in mouse megakaryocytes. In conclusion, the present study further highlights the difference in ionic selectivity of TMEM16F in platelet lineage cells of the mouse compared to other mammalian species. This provides additional support for the ionic “leak” hypothesis that the scramblase activity of TMEM16F does not rely upon its ability to conduct ions of a specific type.We have studied TMEM16F expression and channel activity in primary rat and mouse megakaryocytes and the human erythroleukemic (HEL) cell line that exhibits megakaryocytic surface markers. Immunocytochemical analysis was consistent with surface TMEM16F expression in cells from all three species. Whole-cell recordings in the absence of K Such s systems ,15. This systems , thus ar2+-activated TMEM16F channels in freshly isolated primary rat and mouse MKs and the human erythroleukemia (HEL) cell line. HEL cells express a number of megakaryocytic glycoproteins o and/or [Na+]o were reduced by equimolar substitution with gluconate− or NMDG+, respectively. Internal solutions contained 150mM NaCl, 1mM MgCl2, 10mM glucose, 10mM HEPES, 50µM Na2-GTP, 1mM EGTA . [Ca2+]i was set at ≈5nM  or 100µM (by addition of CaCl2), calculated using Maxchelator (http://web.stanford.edu/~cpatton/webmaxcS.htm). The effect of CaCCinh-A01  was compared with vehicle (DMSO) control. Statistical analysis was by two-way ANOVA .rrection . Bath soTMEM16F expression in HELs and rat and mouse MKs was assessed by immunocytochemistry with an antibody previously used in mouse dendritic cells . Fluores+ currents activated by depolarisation and/or intracellular Ca2+ [2+]i (EC50 of ≈100μM) [+-free bath and pipette salines were used to abolish K+ currents prior to the predicted activation of TMEM16F. Voltage ramps from −120 to +120mV were applied at regular intervals and [Ca2+]i set at either ≈5nM or 100µM. At 100µM [Ca2+]i, outwardly rectifying currents developed at similar timepoints in cells from all species: 439 ± 25s (n = 5) in HELs, 418 ± 18s (n = 5) in rat MKs and 368 ± 20s in mouse MKs (n = 4). This current was not observed at ≈5nM [Ca2+]i and its activation time course is consistent with reports of TMEM16F [2+]i induced large outwardly rectifying currents that reversed (Erev) at −3.3 ± 0.5mV  ,14. Ther ± 0.5mV  and were ± 0.5mV . The conV by A01 . For mou+ and Cl− is 0mV and thus Ca2+-activated currents may reflect movement of cations and/or anions. Following reduction of external chloride (gluconate substitution), 100µM [Ca2+]i evoked large A01-sensitive currents in each species (rev shifted in HELs (+37.2 ± 2.1mV) and rat MKs (+33.5 ± 2.7mV), whereas mouse MK currents continued to reverse close to 0mV  and HELs  would depolarise rather than hyperpolarise the cell. This argues against a role for TMEM16F in promoting Ca2+ entry via regulation of the membrane potential.The ionic selectivity of Ca anionic ,9–12, ca anionic ,15 and n anionic ,14. Hereouse MKs . Althoug cations ,31 or an cations ,15, comptivation ,15. We wng force . Howeverchannels ,30,32 whnd TRPC6 ,30,33. S2+ concentrations used in the present work [−/− mice [Although we demonstrate an interspecies difference of ion selectivity for MK TMEM16F-like channels, further work is required to understand the functional significance of the resultant ionic movements for lipid scrambling and/or other functional events in this cell type. Furthermore, the possible relevance of expression of other members of this scramblase/channel family should also be examined with knock-down studies. It is worth noting however that a transcriptomic analysis of purified platelets detected <2% expression of other TMEM16 family members compared to TMEM16F . Furtherent work . A studyent work . Further−/− mice ,8 and Sc−/− mice .


SOCE is mediated by the assembly of Stim and Orai proteins at endoplasmic reticulum (ER)-plasma membrane junctions upon depletion of the ER Ca2+ store. Additionally, Stim and Orai proteins underpin constitutive Ca2+ entry in a growing number of cancer cell types due to the partial depletion of their ER Ca2+ reservoir. Herein, we investigated for the first time the structure and function of SOCE in primary cultures of colorectal carcinoma (CRC) established from primary tumor (pCRC) and metastatic lesions (mCRC) of human subjects. Stim1-2 and Orai1-3 transcripts were equally expressed in pCRC and mCRC cells, although Stim1 and Orai3 proteins were up-regulated in mCRC cells. The Mn2+-quenching technique revealed that constitutive Ca2+ entry was significantly enhanced in pCRC cells and was inhibited by the pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3. The larger resting Ca2+ influx in pCRC was associated to their lower ER Ca2+ content as compared to mCRC cells. Pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 prevented ER-dependent Ca2+ release, thereby suggesting that constitutive SOCE maintains ER Ca2+ levels. Nevertheless, pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 did not affect CRC cell proliferation and migration. These data provide the first evidence that Stim and Orai proteins mediate constitutive Ca2+ entry and replenish ER with Ca2+ in primary cultures of CRC cells. However, SOCE is not a promising target to design alternative therapies for CRC.Store-operated Ca The Ca2+ response to CPA in the absence of extracellular Ca2+ (0Ca2+) is a widely established tool to monitor changes in ER Ca2+ levels i which reflects SOCE activation [2+]i in pCRC cells  reduced in the presence of either Pyr6   -derived endothelial colony forming cells (IH-ECFCs), but not normal ECFCs (N-ECFCs), despite the fact that there is no difference in the expression levels of Stim and Orai proteins. The constitutive activation of the SOCE machinery is maintained by the chronic underfilling of the ER CaIH-ECFCs . Constitll types , 35–37. protocol , 30, 38,+ levels . Subsequtivation , 30, 38.s Figure , while is Figure . Neverthtivation , 22, in 2+ store , 35–37.3-sensitive ER Ca2+ pool. ATP may accumulate within tumor microenvironment ER) (i.e. ≈500 μM) i so that no global elevation in [Ca2+]i occurs, as also described in RBL-1 cells stimulated with low doses of thapsigargin i that was attenuated by the genetic deletion of Stim1, Orai1 and Orai3. This finding is somehow in line with the constitutive activation of SOCE in CRC cells: accordingly, 2-APB evoked a transient increase, followed by a slight reduction, in store-operated currents recorded from HEK 293 cells transfected with Stim1, Orai1 and Orai3 i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed “ratio”). An increase in [Ca2+]i causes an increase in the ratio [CRC cells were loaded with 4 μM fura-2 acetoxymethyl ester  in PSS for 1 hour at room temperature. After washing in PSS, the coverslip was fixed to the bottom of a Petri dish and the cells observed by an upright epifluorescence Axiolab microscope , usually equipped with a Zeiss ×40 Achroplan objective . ECFCs were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. A first neutral density filter  reduced the overall intensity of the excitation light and a second neutral density filter  was coupled to the 380 nm filter to approach the intensity of the 340 nm light. A round diaphragm was used to increase the contrast. The excitation filters were mounted on a filter wheel . Custom software, working in the LINUX environment, was used to drive the camera  and the filter wheel, and to measure and plot on-line the fluorescence from 10 up to 40 rectangular “regions of interest” (ROI). Each ROI was identified by a number. Since cell borders were not clearly identifiable, a ROI may not include the whole cell or may include part of an adjacent cell. Adjacent ROIs never superimposed. [Cahe ratio , 88. Rat2 incubator until confluence was reached. After removing culture medium, a couple of washing with 1 mL PBS have been performed and then the monolayer was scratched by using a sterile 0.1 - 10 μL pipette tip to create a wound-like gap; floating cells were removed by washing another time in PBS solution and finally cells were treated with experimental conditions. The assay was performed on pCRC and mCRC monolayers exposed to the culture medium alone, as a control, and on pCRC and mCRC monolayers grown in the presence of the standard cultre medium supplemented with Pyr6 , La3+  or G418 as well as on siStim1, siOrai1, siOrai3 mCRC monolayers grown to confluence with the culture medium. Cells were left to migrate for 24 hours and the width of the wound space was measured at wounding and at the end of treatment, by using microscope equipped with a camera. Measurements of wound width were made for each wound at randomly chosen points. Wound closure rates were determined as the difference between wound width at 0 and 48 hours. All the experiments were carried out on pCRC and mCRC cells isolated from 4 different donors . As a control for immortalized CRC cells, the effect of Pyr6  and La3+  was assessed on SW480 cells (obtained from ATCC). SW480 cells were grown in RPMI 1640 supplemented with 10% FCS.Cell migration assay was performed with the scratch wound healing analysis. Briefly, cells were seeded in 12-well plates to form a confluent pCRC/mCRC monolayer and were maintained at 37°C with 5% CO2+ data have been collected from at least 4 different pCRC and mCRC cells harvested from different patients . Pooled data are given as mean±SE and statistical significance (P<0.05) was evaluated by the Student’s t-test for unpaired observations. The amplitude of Ca2+ release in response to either CPA or ATP was measured as the difference between the ratio at the peak of intracellular Ca2+ mobilization and the mean ratio of 1 min baseline before the peak. The magnitude of SOCE evoked by either CPA or ATP upon Ca2+ restoration to the bath was measured as the difference between the ration at the peak of extracellular Ca2+ entry and the mean ration of 1 in baseline before Ca2+ readdition. The rate of Mn2+ influx was evaluated by measuring the slope of the fluorescence intensity curve at 400 sec after Mn2+ addition [All the Caaddition .t test. In the proliferation assays, results are expressed as percentage (± SE) of growth compared to controls (given as 100% growth), obtained from 4 different sets of experiments, each performed in duplicate. Differences were assessed by the Student t-test for unpaired values. All statistical tests were carried out with GraphPad Prism 4.As to mRNA and protein analysis, all data are expressed as mean ± SE. The significance of the differences of the means was evaluated with Student’s As to cell migration assays, the effect of the pharmacological or genetic treatments was evaluated as the percentage of cell migration inhibition under the designated treatments. Statistical analysis was carried out with GraphPad Prism 4.


The accelerated metabolism of tumor cells, inevitable for maintaining high proliferation rates, is an emerging target for tumor therapy. Increased glucose and lipid metabolism as well as mitochondrial activity have been shown in solid tumors but also in leukemic cells. As tumor cells are able to escape the blockade of one metabolic pathway by a compensatory increase in other pathways, treatment strategies simultaneously targeting metabolism at different sites are currently developed. However, the number of clinically applicable anti-metabolic drugs is still limited. Here, we analyzed the impact of the anti-diabetic drug metformin alone or in combination with two non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac and diflunisal on acute myeloid leukemia (AML) cell lines and primary patient blasts. Diclofenac but not diflunisal reduced lactate secretion in different AML cell lines  and both drugs increased respiration at low concentrations. Despite these metabolic effects, both NSAIDs showed a limited effect on tumor cell proliferation and viability up to a concentration of 0.2 mM. In higher concentrations of 0.4–0.8 mM diflunisal alone exerted a clear effect on proliferation of AML cell lines and blocked respiration. Single treatment with the anti-diabetic drug metformin blocked mitochondrial respiration, but proliferation and viability were not affected. However, combining all three drugs exerted a strong cytostatic and cytotoxic effect on THP-1 cells. Comparable to the results obtained with THP-1 cells, the combination of all three drugs significantly reduced proliferation of primary leukemic blasts and induced apoptosis. Furthermore, NSAIDs supported the effect of low dose chemotherapy with cytarabine and reduced proliferation of primary AML blasts. Taken together we show that low concentrations of metformin and the two NSAIDs diclofenac and diflunisal exert a synergistic inhibitory effect on AML proliferation and induce apoptosis most likely by blocking tumor cell metabolism. Our results underline the feasibility of applying anti-metabolic drugs for AML therapy. Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by the accumulation of myeloid blasts in the bone marrow and blood as well as an arrest in differentiation. AML represents the most common acute leukemia in adults and is still a mostly incurable and lethal disease in the majority of patients. Induction therapy with cytarabine and anthracyclines is highly effective in killing leukemic cells and has largely remained unchanged since the 1970s . DespiteNew studies expanded the knowledge on AML heterogeneity and identified new molecular groups. These advances are now translated into several new targeted therapies . MutatioIn solid tumors it is well-established that oncogenes reprogram tumor metabolism and accelerate glucose metabolism. This phenomenon, known as the “Warburg effect,” is associated with an aggressive phenotype and poor prognosis in many tumor entities . Ju et aIn line, inhibition of glycolytic metabolism by 2-DG increased the efficacy of Aurora kinase inhibitors in AML cell lines  and poteMany hypotheses tried to explain the therapeutic resistance in AML patients. A linkage between reduced sensitivity to chemotherapy and accelerated glycolysis has been described in a murine AML model. Here, chemotherapy-resistant AML exhibited increased lactate production and fewer mitochondria . In contin vitro and suppresses xenograft tumor formation -signaling seems to be associated with changes in metabolism and chemoresistance. Blocking VEGF receptor 2 (VEGFR2) induced mitochondrial biogenesis and increased the vulnerability of leukemic cells . VEGF-C ormation . Moreoveormation .These results suggest that AML cells are heterogeneous in their metabolic profile and use different metabolic pathways to fuel proliferation and acquire resistance to chemotherapy. Here we investigated the impact of three inhibitors which target glycolysis, COX, and OXPHOS alone and in combination on AML cell lines and primary human AML blasts. Our study illustrates that simultaneous targeting of different metabolic pathways may represents a powerful therapeutic strategy for AML patients.All drugs were purchased from Sigma-Aldrich  and dissolved in water, unless otherwise indicated. The sodium salt of diclofenac  and metformin hydrochloride (Sigma-Aldrich) were dissolved in culture medium. Diflunisal  was dissolved in 20 mM arginine containing water and cytarabine  in 0.11 mM sodium lactate solution.The cell lines U937  and THP-1  were cultured in RPMI 1640, 10% fetal calf serum , 2 mM glutamine, 50 U/mL penicillin/50 μg/mL streptomycin  at 5% CO2 and 37°C.5 cells/mL medium were seeded in 24-well plates at 5% CO2 and 37°C. Every 3–4 days, medium was changed.Primary AML blasts were obtained from patients after written informed consent. The study was approved by the Institutional Ethics Committee of the University Hospital of Regensburg and designed and conducted in accordance with the Declaration of Helsinki (ethic vote 05-097). AML blasts were cultured in RPMI 1640, 10% fetal calf serum (both from PAN), 10% human AB serum, 2 mM glutamine, 50 U/mL penicillin/50 μg/mL streptomycin , 20 ng/mL IL-3, G-CSF as well as TPO . 5 × 104 cells/0.2 mL medium were seeded into flat-bottom 96-well plates with indicated concentrations of diclofenac, diflunisal, metformin and cytarabine. To analyze immediate anti-proliferative effects, 0.5 μCi/0.2 mL 3H-thymidine  was added after 2 h and 3H-thymidine incorporation was determined after 24 h. In a second set of experiments, cells were labeled after 24 h and cultured for another after 24 h .To measure proliferation of cell lines, 3 × 105 cells/mL medium were treated with diclofenac, diflunisal, metformin and cytarabine for 24 h or 48 h. After treatment, cells were stained with Annexin-V-FITC and 7-aminoactinomycin D (7-AAD)  according to the manufacturer’s instructions. Flow cytometric analyses were performed on a FACSCalibur (BD Biosciences) using BD CellQuestPro for data acquisition and analysis. Final processing and analysis was performed with FlowJo v9.5.3 software .For analysis of apoptosis, 3–5 × 105 cells/ mL medium with or without diclofenac, diflunisal, metformin and cytarabine. After 24 or 48 h, lactate levels in cell culture supernatants were determined with a Dimension Vista  using reagents from Roche  at the Department of Clinical Chemistry, University Clinic, Regensburg, Germany.Cells were seeded into 24-well plates at a concentration of 5 × 10® Reader  is a 24-channel oxygen and pH meter. The optical oxygen (OxoDish®) sensor is integrated at the bottom of each well of a 24-well multidish. The sensors are luminescent dyes embedded in an analyte-sensitive polymer. The luminescence lifetime of these dyes depends on the amount of analyte. The sensors are read out non-invasively through the bottom of the multidish by the SensorDish® Reader. The resulting signal is converted automatically to the respective parameter (dissolved oxygen) using calibration parameters stored in the software. 5 × 105 cells/mL medium were incubated with or without diclofenac, diflunisal, metformin and cytarabine. The SensorDish® Reader was used in the incubator for the whole duration of the 16-h cultivation period and measurements were performed in 30–60 s intervals.The SDR SensorDishpost hoc by Holm-Sidak’s multiple comparisons test, ∗∗∗p < 0.001; ∗∗p < 0.01; and ∗p < 0.05.All results represent mean ± standard error (SEM) of at least three independent experiments. Significance was determined by ANOVA and Tumor metabolism supports tumor growth and is associated with chemotherapy resistance. Targeting tumor metabolism is an attractive option to support conventional tumor therapy. Here we investigated the impact of anti-metabolic drugs alone or in combination on human AML cell lines and primary AML blasts.in vitro and in clinical trials, however, mainly in solid tumors (The anti-diabetic drug metformin lowers OXPHOS via inhibiting complex I of the electron transport chain in the mitochondria. Several studies addressed the anti-cancer potential of metformin d tumors . Metformd tumors . In parad tumors . Diclofed tumors  significd tumors . Anotherd tumors . Howeverd tumors . In compd tumors ; OXPHOS d tumors .Next we analyzed whether metabolic targeting modulates cell growth of different AML cell lines. Even though metformin significantly blocked respiration at doses of 1-10 mM, no significant effect on proliferation was detected for THP-1, U937, and KG-1 AML cell lines Figures –C. This Next we tried to combine low physiological doses of the drugs to augment the effect compared to the single treatment. Combination of 0.1 mM of diclofenac and 0.1 mM diflunisal, clearly suppressed the proliferation of U937 but not in THP-1 and primary AML blasts Figures –C. AdditAs the salicylic acid derivative diflunisal can be administered to patients in 10-fold higher concentrations compared to diclofenac, we increased the diflunisal dose and combined it with low concentrations of diclofenac and metformin Figures –I. As shAnalysis of the metabolic effects of the drugs on AML blasts revealed similar results compared to the leukemia cell lines. Metformin accelerated lactate secretion whereas diclofenac alone or in combination with diflunisal reduced lactate secretion Figures . CombinaTo distinguish proliferation arrest from the induction of cell death, we incubated THP-1 cells with the drugs alone and in combination and stained with annexin-V/7-AAD to determine apoptosis. Low concentrations of diclofenac but not diflunisal and metformin induced apoptosis, however, high diflunisal concentration of 0.8 mM had a strong effect on cell viability Figure . The comIn contrast to THP-1 cells, AML blasts did not respond to single drug treatment Figure . CombinaFinally, we analyzed the impact of low dose standard chemotherapy with cytarabine alone and in combination with anti-metabolic treatment. In THP-1 cells and in primary blasts, cytarabine dose-dependently inhibited proliferation and the effect of cytarabine was more pronounced in primary blasts Figures . Triple Cellular proliferation requires nutrient uptake, synthesis of building blocks and energy. Oncogenes that drive tumor cell proliferation directly or indirectly lead to metabolic changes; c-MYC and other oncogenes upregulate metabolite transporters and enzymes required for glucose metabolism . In lineMany classical anticancer agents inhibit nucleotide metabolism, but some chemotherapeutic drugs have also been shown to target tumor energy metabolism. Cyclophosphamide caused a drop in the glycolytic rate in a murine fibrosarcoma model . SorafenHere, we analyzed the efficacy of drugs targeting glycolysis, COX, and OXPHOS alone and in combination on AML cell lines and primary human AML blasts.Our data show that the glycolytic inhibitor 2-DG and low concentrations of the NSAID diclofenac both limit glycolysis and decrease lactate secretion of AML cell lines. Furthermore, both drugs modulated respiration; in low concentration diclofenac and diflunisal increased respiration. However, higher concentrations of diflunisal and 2-DG decreased oxygen consumption. In line, several other NSAIDs impair mitochondrial activity indicating that OXPHOS inhibition is a COX-related effect . But thein vitro studies use concentrations above 1 mM 2-DG, this indicates that effective 2-DG concentrations will most likely not be reached in patients. Therefore novel compounds that can target glycolysis in patients are warranted. In our experiments 0.1 mM diclofenac was even more effective than 10 mM 2-DG in decreasing lactate secretion, a concentration which is achievable in patients (in vitro and in murine tumor models may in part relate not only to COX inhibition but also to glycolysis inhibition (The glycolytic inhibitor 2-DG has already been tested in clinical trials with limited efficacy. patients . Based ohibition .Diflunisal is a difluorophenyl derivative of salicylic acid which has been shown to be an effective and well-tolerated analgesic for long-term use. In contrast to diclofenac it has a long half-life and prolonged intake of 500 mg b.i.d. for 11 days results in mean peak plasma levels of 190 ± 33 μg/mL (about 0.8 mM, Metformin is commonly used for the treatment of type 2 diabetes and epidemiological studies indicate that tumor incidence is lower in diabetic patients taking metformin . In the Targeting OXPHOS has also been suggested by In summary, there is increasing evidence that not only solid tumors but also leukemia, exhibit changes in metabolism which could be used for metabolic targeting. Beside AMLs with increased glycolytic activity  other reThis study has shown that anti-metabolic treatment can be used to inhibit AML cell line and primary blast proliferation and induce apoptosis in physiologically achievable concentrations. These findings suggest that targeting tumor cell metabolism is an option not only for solid tumors but also hematological malignancies.KR and MK designed the experiments and wrote the manuscript. KS and KP analyzed the data and wrote the manuscript. AnS, NK, IU, PS, S-MD, AsS, KH, MF, CB, and CBrum were involved in experiments and data collection. SK, ST, and EG designed the protocols and discussed data.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.


The occurrence of Langerhans cell histiocytosis (LCH) and acute myeloid leukemia (AML) in the same case has been reported occasionally. We report a new case of AML with t/AML-ETO in an adolescent after LCH. To our knowledge, this is the first description of AML with t/AML-ETO after LCH diagnosis and therapy.9/L; hemoglobin, 60 g/L; and platelet count, 12x109/L. Bone marrow biopsy showed 69% myeloblasts, and Auer rods were found. The immunophenotype profile of the blast cells was CD34 (++), CD13 (+), CD33 (++), CD117 (++), CD38 (+), CD15 (+), HLA-DR (++), MPO(+). Cytogenetic analysis revealed 46, XY, t[20]. The AML-ETO and WT1 genes were positive. The patient responded well to induction chemotherapy. Standard DA chemotherapy (daunorubicin and cytarabine) was given and the boy achieved complete response (CR) after one cycle. After an additional cycle of DA consolidation chemotherapy, he received an HLA-identical sibling allogeneic hematopoietic stem cell transplant (HSCT). He received a conditioning protocol composed of busulphan and cyclophosphamide, and he was given fluconazole and acyclovir as infection prophylaxis and cyclosporine and mycophenolate mofetil as graft-versus-host disease prophylaxis. Up to 30 March 2019, the patient was in a state of persistent CR for 16 months after the diagnosis of the AML, and the AML-ETO and WT1 genes were negative.A 15-year-old boy was diagnosed with LCH in October 2010. He presented with a 1-year history of a skull mass. After 9 cycles of ifosfamide, vincristine, etoposide, and prednisone, the skull mass disappeared. Two years later, the patient presented to the Hematology Department of Beijing Friendship Hospital with progression of his disease in the form of lumber fracture. The mutation BRAF V600E was negative. After relapse of LCH, he received 6 cycles of etoposide and prednisone and 1 cycle of etoposide, prednisone, cyclophosphamide, and vincristine. On 12 March 2013, he received an autologous hematopoietic stem cell transplant. When he came to the clinic with complaints of dizziness on 20 November 2017, a routine blood examination was performed with the following results: white blood cell count, 6.3x10There is an association between LCH treatment and subsequent development of AML; however, the reason why AML develops in patients treated for LCH is not entirely understood. The chemotherapy for LCH and genetic predisposition may be explanations. Previously, 27 such cases have been reported , and lymAdditionally, previous studies have suggested common neoplastic precursors for LCH and AML . Recent In our case, successful allogeneic HSCT not only controlled the patient’s AML but also had a long-lasting effect on his relapsed LCH. For these patients, induction chemotherapy combined with allogeneic HSCT is a good choice.