However, very little is known about the physiological significance of MC4R-induced [Ca2+]i since few studies measure MC4R-induced [Ca2+]i. High-throughput, read-out assays for [Ca2+]i have proven unreliable for overexpressed GPCRs like MC4R, which exhibit low sensitivity mobilising [Ca2+]i. Therefore, we developed, optimised, and validated a robust quantitative high-throughput assay using Fura-2 ratio-metric calcium dye and HEK293 cells stably transfected with MC4R. The quantitation enables direct comparisons between assays and even between different research laboratories. Assay conditions were optimised step-by-step to eliminate interference from stretch-activated receptor increases in [Ca2+]i and to maximise ligand-activated MC4R-induced [Ca2+]i. Calcium imaging was performed using a PheraStar FS multi-well plate reader. Probenecid, included in the buffers to prevent extrusion of Fura-2 dye from cells, was found to interfere with the EGTA-chelation of calcium, required to determine Rmin for quantitation of [Ca2+]i. Therefore, we developed a method to determine Rmin in specific wells without probenecid, which was run in parallel with each assay. The validation of the assay was shown by reproducible α-melanocyte-stimulating hormone (α-MSH) concentration-dependent activation of the stably expressed human MC4R (hMC4R) and mouse MC4R (mMC4R), inducing increases in [Ca2+]i, for three independent experiments. This robust, reproducible, high-throughput assay that quantitatively measures MC4R-induced mobilisation of [Ca2+]iin vitro has potential to advance the development of therapeutic drugs and understanding of MC4R signalling associated with human obesity.The melanocortin-4 receptor (MC4R), a critical G-protein-coupled receptor (GPCR) regulating energy homeostasis, activates multiple signalling pathways, including mobilisation of intracellular calcium ([Ca Secondly, the dye-loading and esterase-activity buffers, incubation times, and incubation temperatures were optimised in a step-by-step manner (2+]i (positive control). Thirdly, the method was validated for reproducible quantitative analysis of carbachol-induced M3-AChR mobilisation of [Ca2+]i and α-MSH-induced hMC4R and mMC4R mobilisation of [Ca2+]i.First, the speed and volume of vehicle injection onto HEK293 cell monolayer were optimised to eliminate stretch-receptor activation of i was quantitated following the protocol outlined for quantitation of [Ca2+]i in cell monolayers.The well volume (0.21–0.29 mL), injection volume (10–30 µL) and injection speed (100–150 µL/s) for the addition of vehicle or ligand was optimised for cell suspensions. Baseline, vehicle, and α-MSH (10The 96-well plates with cells were imaged using PheraStar FS (BMG LABTECH) at 37°C. Baseline fluorescence intensity for each well was recorded for 30 s using 340/380 nm excitation and 510 nm emission wavelengths . VehicleThe PheraStar FS plate reader was set up for imaging calcium with a settling time of 0.5 s before measurement of each well, and with a bi-directional horizontal reading direction to avoid activation of stretch-receptor calcium channels. The number of flashes (10) per well was set to average to one intensity value per well to improve accuracy. The kinetic window was set to 80 s, and the plate was set at the bottom optic to measure fluorescence. The focus and gain were adjusted prior to running each assay. The focal height adjustment of the optical system ensures best signal-to-noise ratio for the volume in every well. The gain adjustment optimises signal amplification so that maximum sensitivity was achieved.2+]i data to obtain EC50 values from three independent experiments. The quantitative [Ca2+]i data from three independent experiments were then pooled for comparison of maximum, minimum, or EC50 values between carbachol-induced and α-MSH-induced [Ca2+]i.GraphPad Prism 7.0 software (GraphPad Software Incorporated) was used to generate graphs and perform statistical analysis. Carbachol- and α-MSH-concentration-response curves were fitted to raw [Ca2+]i response in HEK293 cell monolayers  in two independent experiments consistently induced higher calcium signal with DMEM compared with CLB-HG . Data from two independent experiments showed that DMEM was optimal for Fura-2/AM loading. In contrast with CLB-HG, there was no vehicle-induced activation of stretch-receptor calcium response in the presence of DMEM. Furthermore, two doses of carbachol  in two independent experiments, consistently induced higher calcium signal in the presence of CLB-HG compared with HBSS or CLB-LG , calcium loading buffer-low glucose (CLB-LG), and CLB-HG were compared for optimal esterase cleavage of Fura-2/AM. Data from two independent experiments consistently showed no vehicle-induced stretch-activated calcium response with any of these buffers. However, three different doses of carbachol  consistently induced higher calcium signal when Fura-2/AM loading was performed at RT compared with 37°C  consistently induced higher calcium signal when Fura-2/AM esterase cleavage was performed at 37°C, compared with RT  consistently induced higher calcium signal when Fura-2/AM loading was conducted at RT for 60 min compared with other incubation times  consistently induced higher calcium signal when cells were incubated for 80 min compared with other times  consistently induced higher calcium signal when Fura-2/AM loading was conducted in the presence of pluronic acid compared with no pluronic acid  consistently induced higher calcium signal when probenecid was included both in Fura-2/AM loading buffer (DMEM) and Fura-2/AM esterase cleavage buffer (CLB-HG) . We questioned whether the probenecid interference of EGTA-calcium chelation could be due to esterase activity buffer used. Previous studies have used calcium-free buffers for EGTA chelation of calcium to determine Rmin .Fura-2i.Carbachol induces robust M3-AChR-activated mobilisation of [Ca0 values . Pooling2+]i in HEK293 cell suspensions . Therefore, we modified the assay to increase the number of cells viewed by the PheraStar FS aperture.Previously, α-MSH produced concentration-dependent curves for stably expressed mMC4R-activated mobilisation of [Ca2+]i. A 30 μL injection at 150 μL/s into a well containing 210 μL cell suspension consistently eliminated stretch-receptor-activated [Ca2+]i response in HEK293 while providing a robust carbachol-induced increase for [Ca2+]i (Supplementary Table 2). Using these optimised conditions, three independent experiments produced α-MSH-concentration-dependent curves for activation of hMC4R or mMC4R increasing [Ca2+]i, with similar EC50s for all experiments on each receptor  for α-MSH-activated MC4Rs-induced [Ca2+]i.We increased the number of cells viewed by the PheraStar FS aperture by loading cell suspensions into wells of a 96-well plate. We used stably expressed hMC4R or mMC4R in HEK293 cells, with Fura-2/AM loading and esterase activity taking place in cell suspensions. The buffers, incubation temperatures and incubation times used were those optimised for cell monolayers. The cells in suspension were then plated into a 96-well plate for imaging on PheraStar FS. The injection volume, injection speed, and volume of buffer in each well were optimised for use with cell suspensions in 96-well plates to eliminate stretch-receptor activation and maximise carbachol M3-AChR-induced [Careceptor . Data fr2+]i. The assay conditions were optimised to eliminate interference from stretch-activated receptor increases in calcium and to maximise ligand-activated MC4R-induced [Ca2+]i. The latter was achieved using carbachol-activated endogenous M3-AChR-induced calcium signal as a positive control. The cell monolayer assay lacked sensitivity to detect increases in [Ca2+]i following α-MSH-activated hMC4R transiently expressed in HEK293 cells. Therefore, a more sensitive assay was developed by plating cell suspensions into 96-well plates; this increased the number of cells monitored in each well compared with monolayers. The cell suspension assay was validated by showing reproducible α-MSH-concentration-dependent activation of stably expressed hMC4R or mMC4R in HEK293 cells inducing increases in [Ca2+]i, for three independent experiments. Stably expressing cells were used but we expect this method may also work for transiently transfected cells.We developed a novel, high-throughput assay for quantitative measurements of ligand-activated MC4R-induced increases in [Ca2+]i is a major problem when measuring ligand-activated GPCR-induced increases in [Ca2+]i in cell monolayers . However, this does not occur in the presence of probenecid; addition of EGTA either has no effect on fluorescence ratio or increases the fluorescence ratio. While it is unknown why probenecid interferes with EGTA chelation of calcium, probenecid is known to produce a profound and prolonged attenuation of transmitter release when co-administered with calcium chelator, BAPTA/AM  with an EC50 = 1.4 µM . The manual method showed a peak minus baseline α-MSH-activated mMC4R-induced increase in [Ca2+]i to ~100 nM with an EC50 = 0.5 nM  with an EC50 = 0.4 nM . The peak minus baseline α-MSH-activated hMC4R-induced increase in [Ca2+]i equal to 206 nM with an EC50 = 1.4 nM. The higher but less sensitive calcium response observed here for hMC4R compared with mMC4R, reflects a potential species difference for MC4R coupling to [Ca2+]i signalling pathway.We show good reproducibility for the assays developed here using either cell monolayers or cell suspensions in 96-well plates for quantitative measurement of [Ca0 values . Similar0 values  or mMC4R0 values  in cell 2+]iin vitro. This assay is reliable and reproducible from day-to-day and can therefore be used to compare quantitative transient changes in [Ca2+]i for different ligands on MC4R or different GPCRs, with assays performed on different days, and in different laboratories. The high-throughput assay to quantitatively measure ligand-activated MC4R-induced [Ca2+]iin vitro has potential to advance development of therapeutic drugs and understanding about MC4R signalling associated with human obesity. The utility of the assay in determining differences between Group V hMC4R variants and WT hMC4R remains to be determined.In summary, we have developed and validated a new high-throughput method to quantitate MC4R-induced mobilisation of [CaThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.Funding was provided from The University of Auckland postgraduate student fund.S S K and K G M conceptualisation ; S S K data curation and formal analysis; M-L W and K G M methodology; K G M supervision; S S K writing original draft; M-L W and K G M editing.


Ca2+ is one of the key regulators of ATP production, and its uptake into the mitochondrial matrix occurs via the mitochondrial calcium uniporter. Fluorescent indicators are commonly used for detecting cytosolic Ca2+ changes. However, visualizing mitochondrial Ca2+ fluxes using similar methods is more difficult, as the fluorophore must be permeable to both the sarcolemma and the inner mitochondrial membrane. Our aim was therefore to optimize a method using the fluorescent Ca2+ indicator Rhod-2 to visualize beat-to-beat mitochondrial calcium fluxes in rat cardiomyocytes.Cardiomyocyte contraction requires a constant supply of ATP, which varies depending on work rate. Maintaining ATP supply is particularly important during excitation-contraction coupling, where cytosolic Ca2+ indicator Rhod-2 was reduced to di-hydroRhod-2 and confocal microscopy was used to validate mitochondrial compartmentalization. Cardiomyocytes were subjected to various pharmacological interventions, including caffeine and β-adrenergic stimulation. Upon confirmation of mitochondrial Rhod-2 localization, loaded myocytes were then super-fused with 1.5 mM Ca2+ Tyrodes containing 1 μM isoproterenol and 150 μM spermine. Myocytes were externally stimulated at 0.1, 0.5 and 1 Hz and whole cell recordings of both cytosolic ([Ca2+]cyto) and mitochondrial calcium ([Ca2+]mito) transients were made.Healthy, adult male Wistar rat hearts were isolated and enzymatically digested to yield rod-shaped, quiescent ventricular cardiomyocytes. The fluorescent CaP < 0.05). Beat-to-beat mitochondrial Ca2+ transients were smaller in amplitude and had a slower time to peak and maximum rate of rise relative to cytosolic calcium transients at all stimulation frequencies (P < 0.001).Myocytes loaded with di-hydroRhod-2 revealed a distinct mitochondrial pattern when visualized by confocal microscopy. Application of 20 mM caffeine revealed no change in fluorescence, confirming no sarcoplasmic reticulum compartmentalization. Myocytes loaded with di-hydroRhod-2 also showed a large increase in fluorescence within the mitochondria in response to β-adrenergic stimulation (2+ transients recorded from di-hydroRhod-2 loaded myocytes were distinct in comparison to the large and rapid Rhod-2 cytosolic transients, indicating different kinetics between [Ca2+]cyto and [Ca2+]mito transients. Overall, our results showed that di-hydroRhod-2 loading is a quick and suitable method for measuring beat-to-beat [Ca2+]mito transients in intact myocytes.Myocytes loaded with di-hydroRhod-2 revealed mitochondrial specific compartmentalization. Mitochondrial Ca Therefore, supply must be closely regulated to match the ATP demands of different work rates. When the heart is subjected to greater workloads, the rate of OXPHOS must increase to keep up with the high metabolic demands. There are two important processes that match ATP production to metabolic demand in cardiomyocytes: (i) mitochondrial ADP reuptake upon hydrolysis in the cytosol or (ii) an increase in cytosolic Ca2+ concentration . Cardiomyocyte excitation-contraction (EC) coupling is driven by cytosolic Calaxation . This pr2+ entry via voltage gated L-type Ca2+ channels. In turn, the L-type Ca2+ current rapidly induces release of Ca2+ from the sarcoplasmic reticulum (SR) during the “Ca2+ transient,” whereby cytosolic Ca2+ concentration ([Ca2+]cyto) reaches levels of up to 1 μM  reuptake back into the SR via the SR ATPase, (ii) extrusion through the sarcolemmal Na+/Ca2+ exchanger, or (iii) reuptake into the mitochondria , which is a low affinity transporter located on the inner mitochondrial membrane. The MCU complex is gated by Ca2+, and is regulated by its two subunits MICU1 and MICU2 , resulting in greater energy supply. Uptake of Ca2+ can occur either gradually or on a beat-to-beat basis, as cytosolic Ca2+ increases during EC coupling  the mitochondrial Na+/Ca2+ exchanger (3Na+:1Ca2+), (ii) the mitochondrial H+/Ca2+ exchanger (using the large pH gradient), or (iii) the mitochondrial permeability transition pore, which largely becomes active at high mitochondrial Ca2+ concentration and/or in the presence of increased oxidative stress  that target the inner mitochondria in cultured myocytes  with quenchers such as Mn2+ to minimize cytosolic contamination, based on the idea that Mn2+ would remain in the cytosol and not enter the mitochondria (2+ indicator such as Rhod-2 (in vitro dissociation constant (Kd) of 570 nM, which is probably higher once loaded into a cell. The positively charged Rhod-2 makes it suitable for mitochondrial compartmentalization, as the mitochondrial membrane is highly polarized. Using confocal microscopy, studies by 2+]mito transient kinetics due to temporal limitations during acquisition. Therefore, limitations in both aspects  have led to the uncertainty of how the [Ca2+]mito fluxes might differ from the [Ca2+]cyto kinetics.The importance of cytosolic Caterature , yet theof fuel” , as ther results . Isolatemyocytes . MitoCam+ uptake . In addir intact  or permer intact  with Ca2chondria . Other ss Rhod-2 . Rhod-2 2+ indicator Rhod-2. We then enhanced mitochondrial specific loading of Rhod-2 by combining the indicator with a reducing agent  dissolved in a small amount of methanol (+ borohydride reduced the indicator to non-fluorescent di-hydroRhod-2 (dhRhod-2), which was not only permeable to the sarcolemma, but also the mitochondrial membrane. Within the mitochondria, dhRhod-2 is oxidized back to Rhod-2, due to the presence of mitochondrial reactive oxidative species. This improves detection of mitochondrial Ca2+ signals. Any remaining dhRhod-2 in the cytosol is not oxidized and therefore would not respond to changes in [Ca2+]. 2+ sensitive mitochondrial dehydrogenases by monitoring [Ca2+]mito and [Ca2+]cyto over a long time scale . The purpose of our study was to measure mitochondrial Ca2+ changes in cardiomyocytes on a millisecond time scale to show beat-to-beat changes. This was done by loading cardiomyocytes with 5 μM dhRhod-2 for 1 h at 37°C. Loaded cells were excited at 542 nm and emission was collected at 581 nm. This allowed us to make dynamic measurements of mitochondrial Ca2+ fluxes in live, field stimulated cardiomyocytes. The aims of our study were therefore: (i) to confirm dhRhod-2 is giving a mitochondrial signal that is uncontaminated with cytosolic Ca2+, (ii) to test mitochondrial Ca2+ signals in isolated cardiomyocytes by exposing them to pharmacological interventions known to modulate energy demand and/or mitochondrial Ca2+ levels and (iii) to develop a technique that can be used for fluorometric mitochondrial Ca2+ measurements.Our group has employed a method that takes advantage of the low affinity/cationic nature of the fluorescent Camethanol . The com2+ Free Tyrodes buffer1CaType 2 Collagenase Protease Trypsin inhibitor Bovine Serum Albumin Constant pressure Langendorff Perfusion System2 stock solution1 M CaCl250 μg vial Rhod-2 2DMSO anhydrous 220% Pluronic + Borohydride 2Na50 μg vial Mitotracker Deep Red 2+ Tyrodes1.5 mM Ca1Ca2+ Free Tyrodes solution contains (in mM): 140 NaCl, 4 KCl, 10 Hepes, 1 MgCl2.6H2O, 10 Glucose. Adjusted to pH 7.4 with 5 M NaOH.2To make 1 mM di-hydroRhod-2 solution: Dissolve 1 μg vial of Rhod-2 with 22.25 μL DMSO. Add 22.25 μL of 20% Pluronic in DMSO. Once dissolved, add 10 μL of Na+ borohydride dissolved in 20 μL methanol. Wait until solution becomes colorless, add 5 μL to 1 mL of cell suspension for 5 μM working concentration.3100 nM mitotracker deep red: Dissolve 50 μg vial of mitotracker with 90 μL DMSO (brings stock concentration to 1 mM). Dilute further to 0.1 mM using DMSO then load 1 mL cells with 1 μL for 100 nM working concentration.2 as a carrier gas. Once unconscious, the rat was euthanized by decapitation, hearts excised at the aorta and placed in ice cold Ca2+ free Tyrode’s buffer (refer to section “For Cell Isolation”). Approval for this research was provided by the University of Auckland Animal Ethics Committee (AEC: 001929), in accordance with the Code of Ethical Conduct of The University of Auckland, and the New Zealand Animal Welfare Act 1999.Healthy, Wistar rats  were anesthetized using 2% isoflurane in 100% O2+ free Tyrode’s buffer for 5 min at 37°C using a gravity fed system. During this time blood should be cleared from the coronary circulation and effluent should run clear demonstrating adequate perfusion of the coronary circulation. Perfusion was then switched to 0.2 mM Ca2+ Tyrode’s containing enzymes: 275 U/mL Type 2 Collagenase  and 1.8 U/mL Protease . After 9–12 min of enzymatic digestion, ventricles were cut off and immersed in 0.15 mM Ca2+ Tyrode’s containing 0.1% BSA  and 0.05% Trypsin inhibitor . The ventricles were minced to yield isolated, quiescent myocytes and extracellular Ca2+ was gradually increased to 1.5 mM.Within 30 s after dissection, hearts were cannulated by the aorta, secured with suture, and Langendorff-perfused with oxygenated Ca2+ indicator which results in electrical potential-driven uptake into the mitochondria  prior to introducing it into the cardiomyocytes  was diluted to a 1 mM stock solution with DMSO and 20% Pluronic. For a working concentration of 10 μM, 10 μL of 1 mM Fura-2AM solution was added to 1 mL of cells. Cells were subsequently loaded for 20 min at room temperature then washed with 1.5 mM Ca2+ Tyrode’s for 10 min to allow de-esterification.As previously mentioned, Rhod-2 is a positively charged fluorescent Cachondria . The acemyocytes . The redrescence . DhRhod-refer to . To make2 (for 100 nM free [Ca2+]i), 5 NaCl, 140 KCl, 1 MgCl.6H2O, 5 Glucose, 10 Hepes, 15 butanedione monoxime and 5 EGTA. Myocytes were imaged as above, at baseline and after bolus application of 0.05% saponin, which was used for cell permeabilization.In order to validate Rhod-2 localization in the mitochondria, cardiomyocytes were simultaneously loaded with 100 nM mitotracker deep red (refer to section “For Di-HydroRhod-2 Loading” subscript 3) and 5 μM Rhod-2 or dhRhod-2 . A perspex cell bath was fixed to the stage of an inverted laser scanning confocal microscope . Using a Zeiss 63x oil-immersion objective lens (NA 1.40), images with a 0.1 μm pixel resolution were captured using two lasers sequentially: 561 nm (Rhod-2) and 633 nm (Mitotracker) at 2% laser power. Images obtained for the validation experiments were a single-framed snapshot of the whole myocyte with 2 channels (dhRhod-2/Rhod-2 and Mitotracker), which were subsequently merged for analysis . Furthermore, cardiomyocytes loaded with dhRhod-2 (and Rhod-2) were electrically stimulated  with 5 ms pulses at 1 Hz (room temperature) before (baseline) addition of 20 mM caffeine . The response to caffeine was also measured by delivering a 20 mM bolus to the cardiomyocyte in the absence of electrical stimulation. This technique was used to detect potential loading of dhRhod-2 into the SR. The response to caffeine was then compared between myocytes loaded with dhRhod-2 and those loaded with Rhod-2. Using a similar method, the response to 1 μM isoproterenol  was recorded by collecting the emitted fluorescence from the whole cell over 2–3 min (1 frame/s). Cardiomyocytes loaded with dhRhod-2 were electrically stimulated with 5 ms pulses at 1 Hz (room temperature) before (baseline) and during addition of isoproterenol. This method was employed to determine whether dhRhod-2 signals were enhanced during beta-adrenergic stimulation. The response to 1 μM isoproterenol was also tested in cells that were pre-treated with 50 μM MCU inhibitor Ru265 for 30–60 min. Further evidence against cytosolic contamination was obtained by imaging permeabilized cardiomyocytes loaded with dhRhod-2. For cell permeabilization, cardiomyocytes were loaded with dhRhod-2 (as stated in section “Loading of Rhod-2 and Di-HydroRhod-2”), then washed in intracellular buffer containing (in mM); 2 CaCl2+ fluxes. Loaded cardiomyocytes were transferred into a perspex cell bath, which was fixed to the stage of an inverted fluorescence microscope. Cells were then externally field stimulated between 0.1 and 1 Hz (room temperature) and continuously super-fused with 1.5 mM Ca2+ Tyrodes containing 1 μM isoproterenol and 150 μM spermine . Spermine is an allosteric MCU agonist, which increases MCU affinity to Ca2+, enhancing its uptake into the mitochondria  from the whole cell.Fluorometric measurements were made from cardiomyocytes loaded with di-hydroRhod-2 to determine beat-to-beat changes in mitochondrial Cachondria . Therefot-tests to compare the overlap between the two indicators. The correlation index indicated the degree of mitochondrial localization, with a value of 1 representing mitochondrial compartmentalization and a value of 0 representing no compartmentalization  and analyzed using FIJI . The relationship in fluorescence intensity was measured by calculating the correlation index from a specific region of each cardiomyocyte co-labeled with Rhod-2/mitotracker and dhRhod-2/mitotracker. The correlation index, also known as Pearson’s coefficient, measures the overlap of pixels from two-channel images. The ImageJ Analysis Software Plugin “Just Another Co-localization Program” or “JACoP” , was uselization . In addi2+ transients: ΔF/F0, where ΔF is the transient amplitude and F0 the baseline fluorescence, maximum rate of fluorescence rise, time to peak fluorescence following stimulation, and time constant of fluorescence decay. Ca2+ transients were acquired once steady state was obtained for all stimulation frequencies measured, and the parameters from an average of three sequential transients were analyzed. Mean data from fluorometric measurements of Rhod-2 cytosolic Ca2+ transients and dhRhod-2 mitochondrial Ca2+ transients were analyzed using two-way ANOVA for multiple comparisons between groups and stimulation frequencies. Statistical significance was indicated by a P-value of < 0.05 for all data sets. All mean data  was statistically analyzed using Prism 9 Analysis software.In separate experiments, fluorometric measurements of cytosolic and mitochondrial Rhod-2 fluorescence were carried out using an Optoscan monochromator . Comparison of the following parameters were made from both the mitochondrial and cytosolic CaThe localization of the non-reduced indicator Rhod-2 was first investigated in isolated cardiomyocytes. n = 7 cells) was greater relative to the correlation index between Rhod-2/mitotracker labeling , further revealing dhRhod-2 mitochondrial localization.The localization of the di-hydroRhod-2 (dhRhod-2) loaded cells was then investigated and compared to that of Rhod-2. Myocytes were co-loaded with dhRhod-2 (green) and Mitotracker (red) as shown in 2+ Tyrodes and were imaged across a time series in the absence of stimulation. 2+ into the cytosol from the SR  at maximum response to caffeine. However, dhRhod-2 fluorescence did not change in response to caffeine .The first pharmacological intervention was used to determine potential loading of dhRhod-2 in the SR. m the SR . In basem the SR  relativem the SR . There wm the SR . The plo2+ uptake, myocytes loaded with dhRhod-2 were also subjected to the non-selective β-adrenergic agonist, isoproterenol . Myocytes were continuously stimulated at 1 Hz and imaged across a time series as stated in section “Live Cell Confocal Imaging.” Images were captured across three different time points during the response to ISO. n = 6 cells, P < 0.05). Pre-treatment of cells with MCU inhibitor Ru265, prevented the ISO-stimulated increase in dhRhod-2 fluorescence , however, there was no change in baseline fluorescence in the presence of Ru265 (P = 0.08).In order to increase mitochondrial Ca2+ transient relative to the cytosolic Ca2+ transient and (ii) to record beat-to-beat mitochondrial Ca2+ fluxes at different stimulation frequencies. 2+ transient superimposed with a dhRhod-2 mitochondrial Ca2+ transient, visually there are clear differences in both the transient amplitude and kinetics. 2+ transient with similar characteristics to a transient recorded from a representative myocyte loaded with the commonly used cytosolic Ca2+ indicator, Fura-2 (green).Fluorometric measurements were optimized and made following the protocols stated in section “Fluorometric Measurements in Live Myocytes” in order to: (i) determine the characteristics of the mitochondrial Ca2+ transients were recorded from cells subjected to stimulation frequencies of 0.1, 0.5, and 1 Hz. 2+]cyto transients (black) and [Ca2+]mito transients (red) recorded from representative myocytes loaded with Rhod-2 and dhRhod-2, respectively. 2+ transients at different stimulation frequencies. While there was no change in either group between frequencies, the Rhod-2 loaded myocytes had larger [Ca2+]cyto transient amplitudes relative to [Ca2+]mito transient amplitude at all stimulation frequencies (P < 0.001). 2+ transient kinetics, whereby Rhod-2 [Ca2+]cyto transients had faster maximum rates of rise and time to peak fluorescence relative to dhRhod-2 [Ca2+]mito transients at all stimulation frequencies (P < 0.001). The time constant of decay was not different between [Ca2+]cyto and [Ca2+]mito transients at any stimulation frequency  of 570 nM as opposed to cytosolic Ca2+ indicators , which has a Kd of 140 nM, therefore, Rhod-2 has a lower the affinity for Ca2+. This makes it useful for compartmentalization into areas with higher Ca2+ concentrations relative to the cytosol. The cytosol has a low diastolic [Ca2+] of 100 nM, which can increase up to 1 μM at the peak of the [Ca2+]cyto transient . The response to ISO was tested in order to determine whether MCU uptake was enhanced with β-adrenergic stimulation. tes, and , who reped cells . Ru265 ied cells , which ied cells . Figure dynamics , which m2+ uptake with β-adrenergic stimulation  recorded beat-to-beat mitochondrial Ca2+ fluxes at different stimulation frequencies. Fluorometric measurements were made from cardiac myocytes loaded with dhRhod-2 to determine beat-to-beat changes in mitochondrial Ca2+ fluxes. This could not be achieved using confocal microscopy as these events occur at a timescale beyond the temporal acquisition rate. Faster changes in signal can be obtained in a smaller region of the myocyte using line scans. However, line scanning only captures a single region of the myocyte, which is insufficient when measuring a small change in mitochondrial Ca2+ flux. Imaging of the whole myocyte can be done , but only with a slower acquisition rate. Any changes that might occur < 300 ms apart cannot be detected due to limited temporal resolution. Therefore, we have developed a technique for fluorometric measurements of [Ca2+]mito transients, which can capture changes that occur on a faster time scale . Using a spectrofluorometric system, we acquired rapid changes in mitochondrial Ca2+ on a beat-to-beat basis (see section “Fluorometric Measurements in Live Myocytes”). A window was fitted around the perimeter of a single cell, which allowed for [Ca2+]mito measurements across the whole cell .Using confocal microscopy, we were able to confirm localized dhRhod-2 loading . We weremulation . Next, w2+]mito transient superimposed with a single [Ca2+]cyto transient. The [Ca2+]mito transient had a smaller amplitude relative to the [Ca2+]cyto transient. Interestingly, the [Ca2+]mito also showed a slower time to peak fluorescence. When considering the kinetics of a [Ca2+]cyto transient, the time to peak fluorescence occurs approximately 30–60 ms after the stimulus (2+]cyto transient superimposed with a single Rhod-2 (non-ratiometric) [Ca2+]cyto transient. The kinetics between Rhod-2 and Fura-2 transients were almost identical, which suggested that Rhod-2 was a suitable measure of cytosolic [Ca2+] for comparison to dhRhod-2 [Ca2+]mito transients. 2+]cyto transient has peaked, then it begins to decline at approximately 70–80% of the [Ca2+]cyto transient decay. This is evidence that MCU uptake begins when intracellular [Ca2+] is highest  and then begins to decline as cytosolic Ca2+ approaches diastolic levels. This supports original findings from 2+]mito occurred 40 ms after the start of systole and a subsequent decline in [Ca2+]mito 95 ms after the start of systole. Studies using adenoviral probes targeted to the mitochondria also reported similar findings. For example, 2+]cyto peaked at 50–100 ms and [Ca2+]mito peaked 100–150 ms after. Furthermore, in cultured rat adult myocytes, 2+]mito transients at a cytosolic [Ca2+] of 0.9 μM, which is equivalent to the peak [Ca2+]cyto transient at systole. These data are in contrast to a study by 2+]mito preceded peak [Ca2+]cyto in isolated guinea pig myocytes. However, 2+ transient events. However, we were able to compare the kinetics of Rhod-2 [Ca2+]mito to [Ca2+]cyto transients in separate myocytes as we used the same indicator, which allowed direct comparison without variability in the fluorophore binding kinetics. Collectively, our findings provide further evidence that mitochondrial Ca2+ fluxes occur during EC coupling, and are therefore able to regulate mitochondrial metabolism on a beat-to-beat basis.stimulus . This wa2+ transient, the next aim was to record beat-to-beat mitochondrial Ca2+ fluxes at different stimulation frequencies. As mentioned above, we were able to do this using a spectrofluorometric system (see section “Fluorometric Measurements in Live Myocytes”). Cells were super-fused with 1.5 mM Ca2+ Tyrodes containing 1 μM ISO and 150 μM spermine (MCU agonist) and externally stimulated at frequencies of 0.1, 0.5, and 1 Hz until steady state was achieved. Fluorescence traces from representative myocytes in 2+]mito and [Ca2+]cyto transients occurred in response to each applied stimulus. Cytosolic Ca2+ transient amplitudes were larger, with faster maximum rates of rise and time to peak fluorescence  compared to that of the present study. However, they acknowledge difficulties in measuring [Ca2+]mito transient kinetics as aequorin systems cannot detect changes with high temporal resolution. Therefore, the use of spectrofluorometric systems has the advantage of detecting rapid changes in [Ca2+]mito transient kinetics that might have been misinterpreted in past studies due to acquisition limitations.Once we were able to determine the characteristics of a single mitochondrial Carescence  relativeeratures . In the quencies . However2+]mito transients were enhanced by the presence of ISO and spermine, as baseline [Ca2+]mito transients could not obtained. This could have suppressed the response of [Ca2+]mito to increasing stimulation frequencies, as the SR may have already been at capacity. However, it is known that the MCU is mostly active at higher cytosolic [Ca2+]  as it is a low affinity transporter, therefore basal MCU activity is limited at resting levels of cytosolic Ca2+ .M-LW and AP conceived the study. AK performed the experiments and data analysis. AK and M-LW drafted the article. AK, AP, and M-LW performed the critical revision of the article for important intellectual content. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Faculty mentoring programs in academic medicine have benefits that range from the individual to the institution and are important to maintain especially during challenging times. Some benefits of faculty mentoring include an enhanced collegial academic culture , 2, imprThroughout the COVID-19 pandemic, both mentors and mentees faced unprecedented uncertainty. As priorities shift and stressors become chronic, the mentoring relationship risks marginalization. However, during these times mentoring needs to remain an important, proactive activity that should not function as crisis management but rather adopt new meaning and adapt to increasing complexities. How does a mentor continue to serve as an effective role model when prior experience from which to draw upon does not exist? Both mentors and mentees have been forced to manage new relational dynamics in an attempt to maintain fidelity to the purpose of the mentoring process, and understanding the psychological responses to stress in both the mentor and mentee can frame how these dynamics may shift in a beneficial manner.In this commentary, we consider changes within mentoring in academic medicine during the COVID-19 pandemic. We outline several facets of the mentoring process that have been affected by COVID-19, and describe ways mentoring relationships may evolve to respond to these issues. Since the beginning of COVID, several articles have been published offering guidance on how to improve mentoring techniques, such as the importance of maintaining nurturing and consistent mentor-mentee relationships, displaying transparency regarding personal struggles, maintaining active and open communication, acknowledging challenges, and engaging in reflective practices –17. The Across the globe, people have been surrounded by uncertainty and submerged in anxiety as a result of the pandemic. Individuals react to stress in various ways, with some finding it challenging to adapt to change and others adjusting to uncertainty more easily. The stresses unique to the health care setting during the pandemic have resulted in shifts within the mentor and mentee relationship, and those changes have been complicated by the psychological and behavioral reactions of each individual to increased stress.Given the global anxiety inherent to a pandemic, one can expect that there might be a temptation and a tendency to discuss more personal details, both from the mentee and the mentor, especially if it is reinforced with positive response from either participant. This can result in significant role diffusion, demonstrated by confusion of previously well-defined social roles between individuals. Professionals in the mental health field could potentially be especially vulnerable to such role diffusions, since interpersonal principles and ethics in psychiatry and psychology can sometimes be unique compared to other medical specialties . For insUsually, one of the greatest benefits of the mentoring relationship to the mentee is the advanced vantage point mentors have in their professional careers and the resulting experience they share. However, the pandemic has created uncertainty and ambiguity about the future, so our traditional perspectives on career development may be shifting. Both mentor and mentee may find themselves experiencing role reversal, which occurs when individuals acquire each other’s distinct social roles for a time, experiencing and attempting to understand the other person’s perspective. In a time of stress, when role reversal occurs without awareness, it threatens the integrity of the relationship. As mentors are expected to be honest and trustworthy, difficulty acknowledging they lack a certain skill set during the pandemic can threaten this characteristic. For instance, traditionally, the mentee looks at the mentor for guidance and acquisition of skills . However, the mentor may find themselves in the pupil’s role as the mentee helps them navigate through the nuances of technology associated with telehealth hardware and software. This can be particularly challenging if there is rigidity to change within the mentor, the mentee, or the system . AdditioGiven the potential threats to effective mentoring engendered by the pandemic, it is important to consider ways in which the mentoring relationship can change and evolve to ensure continued success (see Table Recognize your own comfort with vulnerability in order to address and deal with emotions during this time of stress. As humans, we all have vulnerabilities, although these may have been hidden behind boundaries in the past within the mentoring relationship. Mental health professionals have training in recognition and management of emotions, which could imply they should be more efficient at recognizing their and other’s vulnerabilities. However, simultaneously some mental health professionals may struggle with knowing how to express their emotions overtly without violating what in the past have been held as clear limits. The experience of chronic, universal stress requires us each to consider our level of comfort with vulnerability. Allow space to process emotions, and this will enhance the honesty and trust within the mentoring relationship. Vulnerability is strength, and most of all, understanding one’s own level of comfort, awareness, and expression of vulnerability is strength. Flexibility and adaptability are essential as mentoring relationships allow for consideration of unexpected topics of conversation and processing of emotional reactions to the current state. However, it remains incumbent upon mentors to monitor their level of vulnerability and not impart this too heavily on mentees. All of us should be familiar with how to complete a basic “risk assessment” with our mentees, and how to suggest or encourage additional intervention if someone appears to be struggling. Being aware of your own comfort with emotional content also can inform an approach to asking about your mentee’s emotional health. In times of significant stress, recognizing signs of poor coping or mental health needs becomes even more important. Take time to learn local available resources for more formal intervention and share these with your mentees.Respond to issues as they arise, recognizing that this may temporarily change mentoring priorities. Mentoring relationships that are built around a specific goal may feel strained during the pandemic as one or both parties have a shift of priorities or experience feelings of futility around their original goal. Be ready to adjust priorities as needed. Spend time completing new goal setting exercises that do not lose the mentee’s ultimate long-term aspirations but modify short- and mid-term goals to accommodate new problems. Maintain respect for the mentor’s experience and reputation within the mentoring relationship, even if the focus shifts around one topic temporarily. For example, even though certain biological, psychological, and social circumstances of the world may have changed for a mentee during a pandemic, the mentor may still be the relative authority on the bio-psycho-social model.Explore role reversal openly or consider other forms of mentoring relationships for a time if necessary. Unprecedented and uncertain times may be the ideal opportunity to explore different types of mentoring paradigms. Several distinct styles of mentoring have been reviewed in the literature . HistoriUse technology effectively while also acknowledging the fatigue and isolation resulting from a transition to a predominantly virtual life. Virtual meetings have allowed a mechanism for continued mentoring in the midst of the pandemic, although many feel a sense of lost human connection due to the limited ability for face-to-face, in-person encounters. The use of videoconferencing in the mentoring relationship has several advantages, as well as limitations. It allows interactions and opportunities for mentorship between individuals in geographically distanced locations that otherwise might not have been possible. It eliminates travel to allow for meetings to take place in a narrower window of time. However, the loss of in-person contact and limitations with nonverbal communication challenge the mentor-mentee dynamic. Exclusively virtual communication may lead to lower satisfaction and poorer perceptions of the interaction . DecreasRecognize that pre-existing societal burdens and inequities have become exacerbated during this time. Historically, underrepresented groups and women have struggled with various aspects of faculty development, including but not limited to a sense of family and/or community obligations, feelings of isolation, and lack of suitable mentors –27. DuriRemember the basics of mentoring. Success in mentoring involves active engagement and initiative. Both parties must actively listen to one another, with a willingness to incorporate feedback. Historically, mentoring success often revolved around initiative rather than reactivity. However, it becomes difficult to think ahead in a situation requiring constant change and reaction. Intense stress can lead to defensiveness and challenges the ability to remain in the moment and focused, let alone open-minded. Acknowledge that we are all finding a new process of moving forward. We need to discover ways to remain connected to our compassion and to set new expectations for what is possible right now . As mentAs with any time of increased stress, the strain of a global pandemic requires those in academic medicine to consider their coping strategies and resiliency. Within the mentor and mentee relationship, dynamics have shifted as a result of COVID-19. We encourage academic medicine faculty to remember the importance of mentoring during these times and to focus on adjusting expectations and approach in order to accommodate increased pressure, excess demands, and emotional responses. By utilizing the mentoring relationship as an opportunity to increase our emotional awareness and practice increased patience and grace with ourselves and others, we create an environment that allows both the mentor and the mentee to evolve through crisis and to integrate the benefits of the mentoring relationship as permanent changes that transcend well beyond COVID-19.


MLL/AF9 AML cells. Metformin could significantly repress the proliferation of MLL/AF9 AML cells by inhibiting oxidative phosphorylation.Acute myeloid leukemia is a group of metabolic heterogeneous cancers, of which the long-term overall survival is still poor, especially in elderly patients. Targeting metabolic reprogramming in leukemic cells is becoming a promising strategy. The aim of our research was to explore the relation of genetic mutations with the metabolic phenotype and potential therapeutics to target metabolic pathway dependence. We confirmed the metabolic heterogeneity in AML cell lines and found the high dependence on oxidative phosphorylation in MLL/AF9 have upregulated mitochondrial activity and mainly depend on OXPHOS for energy production. Furthermore, we show that metformin repressed the proliferation of MLL/AF9 AML cells by inhibiting mitochondrial respiration. Together, this study demonstrates that AML cells with an MLL/AF9 genotype have a high dependency on OXPHOS and could be therapeutically targeted by metformin.Acute myeloid leukemia (AML) is a group of hematological cancers with metabolic heterogeneity. Oxidative phosphorylation (OXPHOS) has been reported to play an important role in the function of leukemic stem cells and chemotherapy-resistant cells and are associated with inferior prognosis in AML patients. However, the relationship between metabolic phenotype and genetic mutations are yet to be explored. In the present study, we demonstrate that AML cell lines have high metabolic heterogeneity, and AML cells with  Acute myeloid leukemia (AML) is one of the most common and lethal leukemias in adults, with around 120,000 new cases every year worldwide ,4. RecenCancer cells rewire their metabolism to maintain a high proliferation rate and to avoid death-inducing signals. This goal is fulfilled by metabolism reprogramming in cancer cells to rapidly produce energy and intermediates to provide building blocks for the synthesis of nucleotides, amino acids, and fatty acids. Meanwhile, metabolites generated from metabolic pathways also contribute to signaling functions, remodeling the epigenome, and altering the expression of specific sets of genes that can initiate and support cancer development . AccumulMLL/AF9 genotype had a higher dependence on OXPHOS than non-MLL/AF9 AML cells. We also found that the inhibition of OXPHOS by metformin significantly inhibited the proliferation of MLL/AF9 positive AML cells.Human cells meet energy requirements through glycolysis and OXPHOS pathways. Determining metabolic pathway dependence in AML individuals by metabolic phenotype technology such as Seahorse XF analyzer-based investigations could provide a new potential horizon in leukemia diagnosis and treatment. The metabolic phenotype of leukemia cells could represent actionable therapeutic vulnerabilities and may be utilized to explore therapeutic options. In the present study, we determined the metabolic pathway dependence of various human AML cell lines and showed that AML cells with an 2 at 37 °C, and all media were supplemented with 100 U/mL penicillin, and 100 mg/mL streptomycin .All cell lines were obtained from the DSMZ . The hum2 incubator. On the day of the experiment, the optimized numbers of cells were plated in a Poly-D-Lysine (PDL) -coated XF96 microplate  using 180 µL of XF DMEM base medium supplemented with glucose, glutamine, and/or sodium pyruvate  and temperature conditions (37 °C) required for the assay. Three measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were recorded at the basal levels and after sequential injections of inhibitors into Seahorse ports , and rotenone + antimycin A; inhibitors for ECAR measurement: oligomycin, 2-DG). The concentration of inhibitors was optimized for all the cell types  was used to perform the Seahorse Mito Stress Test and Glycolysis Stress Test. The measurements were performed according to the manufacturer’s instructions. In brief, cells were maintained at log phase growth in their respective medium. Seahorse XF96 cartridges were hydrated overnight at 37 °C in a non-COpyruvate . After sll types . The meaMLL-AF9 and helper plasmid pCL-ECO using CaCl2 as a transfection agent [MLL-AF9 was a gift from Daisuke Nakada [MLL/AF9 by spin infection using polybrene  as a transfection reagent [HEK293T cells were co-transfected with transfer plasmids pMIG or pMIG-FLAG-on agent . pMIG wae Nakada  .Murine lineage negative (Lin-) cells were retrovirally transduced with MLL/AF9, followed by GFP+ sorting. The GFP+ cells were cultured for a few days and transplanted into lethally irradiated (7 + 3Gy) C57BL/6 mice to induce AML .MLL/AF9 or the BM from control mice or MLL/AF9 AML mice were used to measure MMP and mitochondrial number by flow cytometer according to the manufacturer’s instructions. Briefly, 1 × 106 K562TRBSR cells or mouse BM cells stained with anti-mouse CD117-BV421  were incubated with 50 nM tetramethylrhodamine ethyl ester  or 50 nM MitoTracker Deep Red FM  for 20 min at 37 °C in a CO2 incubator. After washing with 1 mL PBS one time, the fluorescence emissions of stained cells were collected, and median fluorescence intensity (MFI) values were determined after excluding doublets and debris, gating target cell population, and subtraction of the background fluorescence.MMP and mitochondrial number detection: K562TRBSR cells transduced with pMIG or MLL/AF9 AML mice were stained with anti-mice CD117-BV421, and c-kit+/GFP+ blast cells were isolated by fluorescence-activated cell sorting.Cell sorting: Total BM from All cells were maintained at log phase growth in their respective medium before experiments. Cell number and viability were measured at 72 h after metformin (Sigma-Aldrich) treatment; the cell suspension was mixed at a 1:1 ratio with Trypan Blue Solution (Sigma-Aldrich) before the measurement.Immunoblot was performed according to standard procedures ,17. BrieMitochondrial DNA (mtDNA) content was quantified by real-time PCR as described . The prit-test, * p < 0.05, ** p < 0.01, and *** p < 0.001. Statistical analysis was performed using Prism version 6.0c .All data are represented as mean ± standard deviation. Samples were compared using two-tailed, unpaired Student’s MLL/AF9 rearrangement have a significantly higher OCR/ECAR ratio, indicating a preference for OXPHOS as a primary metabolic energy pathway  or MLL/AF9, and Seahorse Extracellular Flux analyzer was performed  were more sensitive to metformin treatment than other cell lines. Moreover, an additive effect of metformin with cytarabine (Ara-C) was also observed in the THP1 and MOLM13 cells  [MLL/AF9 cells are probably mediated by increased NRF1 levels. About 43% of MLL–rearranged leukemias are EVI1 positive, and a recent study showed EVI1 activated mitochondrial respiration in MLL/AF9 leukemia, which could be another explanation of our observation [NRF1 and EVI1) involved in metabolic reprogramming in MLL/AF9 AML by the omics analysis. In addition, from murine AML models we found that mitochondrial activity was significantly upregulated in MLL/AF9 blast cells. However, the heterogeneity with respect to mitochondrial respiration and especially respiration capacity was observed, which probably resulted from the acquisition of new mutation during AML development in mice. Collectively, MLL/AF9 AML has a mainly metabolic dependence on OXPHOS, but metabolic heterogeneity is observed in MLL/AF9 AML mice transplanted with the same donor-derived transformed cells. Future technological advances similar to flow cytometry to identify metabolic phenotype would benefit AML patients on the stratification of diagnosis and target therapy.In the present study, we characterized the metabolic phenotypes of various human AML cell lines. We found that AML cell lines with an r (Tfam) . Therefoervation ,29. TherMLL/AF9 mutation included in the study. This could be addressed in future studies by the analysis of a large population of AML patients to explore the relation of metabolism with genetic mutations. A recent study of AML patients found that genetic signatures of shorter survival are enriched in OXPHOS and mitochondrial metabolism gene sets [MLL gene is one of the major driver mutations in acute leukemia (AML and acute lymphoid leukemia (ALL)), accounting for up to 10% of cases across all age groups [MLL-rearrangement is an independent poorer prognostic factor in both ALL and AML [MLL/AF9 mutation in AML only indicates an intermediate prognosis [Nonetheless, the primary limitation of the study is the small sample size of human AML cell lines, especially only two cell lines with ene sets . Unfortue groups . MLL-rea and AML , but therognosis ,32. HoweAs accumulating data show that mitochondrial respiration plays a vital role in many cancers, including AML, clinically applicable OXPHOS inhibitors have been developed. Several studies found that old drugs such as IDH inhibitors, BCL2 inhibitor (venetoclax), and biguanides (including metformin and phenformin) showed indirect or direct effects on mitochondrial respiration, but the mechanism has yet to be fully illustrated ,34. A reMLL/AF9 AML cells, we determined the response of various AML cell lines to metformin and found that THP1 and MOLM13 cells were more sensitive to metformin treatment. Furthermore, by knock-in of an MLL/AF9 gene into murine HPCs and human K562TRBSR cells, we validated that MLL/AF9 cells were highly susceptible to the anti-proliferative effects of metformin and the OXPHOS inhibitor; this was further supported by our ex vivo observation in MLL/AF9 blast cells from AML mice. In agreement with previous studies, we showed that metformin significantly decreased OXPHOS in all cells, but MLL/AF9 cells showed a higher sensitivity to metformin treatment. Together, metformin could inhibit the proliferation of MLL/AF9 AML cells by repressing OXPHOS, but whether other pathways such as AMPK/mTOR also contribute to the sensitivity of MLL/AF9 AML to metformin needs to be further explored. Many clinical trials have been conducted to investigate the effect of metformin in solid tumors and have achieved varying degrees of success, but few trials were undertaken in leukemia patients [MLL/AF9 mutation. Furthermore, future studies should explore whether AML with other MLL-rearrangements and ALL with MLL-rearrangements share the same metabolic phenotype and sensitivity to metformin as MLL/AF9.Metformin is a relatively safe drug widely used to treat diabetes with manageable side effects. It has been reported that metformin can induce cancer cells apoptosis by inhibiting mitochondrial complex I, thus leading to a decrease in OXPHOS ,22,36. Cpatients . TherefoMLL/AF9 AML cells have higher mitochondrial activity and metabolic dependence on OXPHOS. We further elucidated that MLL/AF9 AML cells were more sensitive to metformin and the OXPHOS inhibitor treatment mediated by OXPHOS inhibition. It will be of further interest to explore whether leukemias with other MLL-rearrangement share the same metabolic phenotype and sensitivity to metformin treatment with MLL/AF9 AML.This study showed that AML cells attribute high metabolic heterogeneity due to a multitude of genetic alterations.


SOCE is triggered by a reduction of Ca2+ content (i.e. depletion) in intracellular stores, i.e. endoplasmic or sarcoplasmic reticulum (ER and SR). In skeletal muscle the mechanism is primarily mediated by a physical interaction between stromal interaction molecule-1 (STIM1), a Ca2+ sensor located in the SR membrane, and ORAI1, a Ca2+-permeable channel of external membranes, located in transverse tubules (TTs), the invaginations of the plasma membrane (PM) deputed to propagation of action potentials. It is generally accepted that in skeletal muscle SOCE is important to limit muscle fatigue during repetitive stimulation. We recently discovered that exercise promotes the assembly of new intracellular junctions that contains colocalized STIM1 and ORAI1, and that the presence of these new junctions increases Ca2+ entry via ORAI1, while improving fatigue resistance during repetitive stimulation. Based on these findings we named these new junctions Ca2+ Entry Units (CEUs). CEUs are dynamic organelles that assemble during muscle activity and disassemble during recovery thanks to the plasticity of the SR (containing STIM1) and the elongation/retraction of TTs (bearing ORAI1). Interestingly, similar structures described as SR stacks were previously reported in different mouse models carrying mutations in proteins involved in Ca2+ handling  or associated to microtubules (MAP6 knockout mice). Mutations in Stim1 and Orai1  genes have been associated to tubular aggregate myopathy (TAM), a muscular disease characterized by: (a) muscle pain, cramping, or weakness that begins in childhood and worsens over time, and (b) the presence of large accumulations of ordered SR tubes  that do not contain myofibrils, mitochondria, nor TTs. Interestingly, TAs are also present in fast twitch muscle fibers of ageing mice. Several important issues remain un-answered: (a) the molecular mechanisms and signals that trigger the remodeling of membranes and the functional activation of SOCE during exercise are unclear; and (b) how dysfunctional SOCE and/or mutations in Stim1, Orai1 and calsequestrin (Casq1) genes lead to the formation of tubular aggregates (TAs) in aging and disease deserve investigation.In the last decades the term Store-operated Ca After the initial discovery, it was shown that Ca2+ release-activated calcium (CRAC) channels were involved in the mechanism  of the plasma membrane (PM) , which uses external Ca2+ to induce release from internal stores in cardiac and smooth muscle , are directly linked to the Ca2+ release channels of the SR, the ryanodine receptors type-1 (RYR1)  coupling, the mechanism linking the depolarization of PM to SR Ca Fabiato ; and mec2+ independent, the role of extracellular Ca2+ in skeletal muscle function and contractility has been neglected for many years. Evidence for a Ca2+ entry pathway independent from DHPRs was reported in the 90′s  receptors in the SR membrane with transient receptor potential cation channels (TRPC) in the PM  reduced the ability of skeletal muscle to maintain contractile force during prolonged stimulation  following Ca2+ store depletion  one pool that mediates rapid SOCE activation located at the triad; and (b) a reserve pool at the I-band that would produce graded SOCE following store depletion  type II fibers, while loss-of-function mutations in STIM1 have been also associated to severe muscle hypotonia  has been recently characterized and manifested a multi-systemic phenotype, but no formation of TAs was reported  are unusual membranous structures, which represent an important indicator of human myopathies, and that have been also found in aging mice, where they preferentially assemble in fast twitch fibers , a protein that acts as the main CaStim1 and/or Orai1 induced formation of stacks of ER cisternae joined together by electron-dense small linkers, with ORAI1 clustered in specialized domains of the PM. It is worth emphasizing that overexpression of Stim1 in this study induced the formation PM invaginations (structures similar to primitive TTs of muscle) associated to ER stacks  studies proposed ER-PM junctions that form following depletion of intracellular stores as the SOCE sites  come in close contact with each other. CRUs are the specialized intracellular junctions allowing the mechanical coupling between DHPRs, or CaV1.1 L-type Ca2+ channel, with the SR Ca2+ release channels, the RYR1  into myotubes. While myotubes grow in size, the nuclei that are initially placed approximately in the center of myotubes, move toward the periphery to leave space for the contractile, metabolic, and Ca2+ sensor of SR membranes, and ORAI1, a Ca2+ release activated channel (CRAC) placed in external membranes, either the PM of its invaginations, i.e. the TTs . In the triadic junction RYR-feet are in direct contact with one another, corner-to-corner, forming regular arrays  muscles from pre-exercised animals were more resistant to fatigue than control muscles during repetitive stimulation in presence of 2.5 mM external Ca2+ quenching of Fura-2 fluorescence, the state-of-the-art technique to measure entry of divalent cations from the extracellular space. Results of those experiments pointed to a significantly augmented entry of Mn2+ both following depletion of intracellular stores (+ depletion) and even in absence of depletion (- depletion) in FDB fibers of pre-exercised animals compared to controls. These findings were complemented by the demonstration that this Ca2+ entry pathway was dependent on presence of ORAI1, as the exercise-induced adaptations were absent in two different models of Orai1-knockout: (a) dominant-negative Orai1 transgenic mice  to support SOCE during repetitive muscle activity. This conclusion is also reinforced by immunofluorescence and immunogold labeling in Boncompagni et al. In conclusion, the increased rate of Mn2+-binding protein located in the lumen of the SR terminal cisternae  exhibit reduced SR Ca2+ content and undergo rapid and deep SR Ca2+ depletion  during sustained, high-frequency stimulation  the recovery of Ca2+ ions via SOCE served to promote enhanced maintenance of peak Ca2+ transient amplitude, and increased maintenance of contractile force during repetitive, high-frequency stimulation.Skeletal muscles from mice lacking calsequestrin-1  TTs retracts from SR stacks within 6 h following exercise; b) SR stacks disappear within 24 h. The fact that SOCE function is enhanced when TTs extension and contacts with SR stacks are increased, but returns to control levels once TTs retract from the I band, points to a central role of TTs in the assembly of functional CEUs. The mechanism underlying TT elongation and the reason of a higher plasticity of TT vs. SR stacks remain elusive and deserve further investigation  caused a drastic reduction on SOCE activation in C2C12 cells . In the EM images published by Ko et al. irregular triad junctions.Junctophilins (JPs) are a family of proteins that contribute to the formation of junctional membrane complexes linking the PM (or its invaginations) with the ER and SR in non-excitable and excitable cells  was performed at the time. An SR stack in double Casq-null muscles in shown in Fig. Casq1-null mice contain a great amount of CEUs and their presence correlates with enhanced SOCE  in slow- and fast-twitch skeletal muscles using knockout mice , independent of SR store depletion, also received considerable attention in the first decade of this millennium. ECCE is based on a retrograde signal from RYR1 to CaV1.1, the two main players in EC coupling, which activates a slow Ca2+ entry pathway, not required for EC coupling itself . Unfortunately, in those papers a detailed analysis of TTs, the second element required for the assembly of functional CEUs, is missing. Hence, a final conclusion about the presence of fully assembled CEUs cannot be drawn. See Other Unresolved Issues (Is assembly of SR stacks in different mouse models underlined by a common mechanism?) for additional detail. To understand more about the molecular mechanisms underlying membrane remodeling future efforts should be focused in studying the role that proteins known to be involved in TT biogenesis and membrane-bending, such as bridging integrator-1 (BIN1) also known as amphiphysin-II, caveolin-3 (CAV3), myotubularin-1 (MTM1), junctophilins (JPs) , now widely recognized as signaling molecules in muscle function.Decrease in ATP/ADP ratio and increase in levels of inorganic phosphate, whose concentration can increase rapidly during intense fatigue.Increase in temperature, due to generation of heat during aerobic metabolism and break down of ATP.2 consumption during aerobic ATP production.Reduction in partial pressure of oxygen (hypoxia), due to increased 0Repetitive shortening and relaxation of contractile elements, which is sensed by mechano-sensors in contractile filaments and cytoskeleton , that preferentially assemble in fast twitch fibers  which should be involved in permanent clusters with ORAI1 at the triad  peak of Ca2+ transient, and (iii) rate of Mn2+ quench  SR Cairregular triad junctions, in our eyes clearly SR stacks) accompanied by defective Ca2+ signaling (reduced SR Ca2+ store and defective operation of SOCE).Ko et al. On the other hand the following two papers do not contain experimental evidence to support the hypothesis of a common mechanism underlying formation of SR stacks:2+ content were reported    may represent a positive adaptation of muscle, triggered by SR depletion, aiming to create a preferential pathway to recover extracellular Ca2+, replenish the SR, and possibly delay the onset of (or reduce) muscle fatigue. This hypothesis is supported by findings in Casq1-null muscle, where the constitutive presence of CEUs in Casq1-null muscle is likely an adaptation of fibers to the chronic reduced amount of Ca2+ in the SR. With this in mind we could hypothesize that CEUs creates an open door for prompt access to extracellular Ca2+ supply when internal Ca2+ stores cannot provide sufficient ions from muscle function.The mechanisms that underlie muscle fatigue are very complex (Allen et al.


Mlkl−/− hematopoietic stem and progenitor cells released reduced amounts of G-CSF while retaining their capacity for CSF3 (G-CSF) mRNA expression, G-CSF protein translation, and G-CSF receptor signaling. MLKL associates with early endosomes and controls G-CSF release from intracellular storage by plasma membrane pore formation, whereas cell death remained unaffected by loss of MLKL. Of note, MLKL expression was significantly reduced in AML patients, specifically in those with a poor-risk AML subtype. Our data provide evidence that MLKL controls myeloid differentiation in AML by controlling the release of G-CSF from leukemic progenitor cells.The blockade of cellular differentiation represents a hallmark of acute myeloid leukemia (AML), which is largely attributed to the dysfunction of lineage-specific transcription factors controlling cellular differentiation. However, alternative mechanisms of cellular differentiation programs in AML remain largely unexplored. Here we report that mixed lineage kinase domain-like protein (MLKL) contributes to the cellular differentiation of transformed hematopoietic progenitor cells in AML. Using gene-targeted mice, we show that MLKL facilitates the release of granulocyte colony-stimulating factor (G-CSF) by controlling membrane permeabilization in leukemic cells.  Acute myeloid leukemia (AML) is characterized by the accumulation of genetic aberrations in hematopoietic stem/progenitor cells , 2. GeneProgrammed cell death represses leukemogenesis by killing transformed cells and, in the case of inflammatory forms of cell death, also by eliciting an inflammatory response . The infNecroptotic cell death provides a substantial inflammatory stimulus, which is largely mediated by damage-associated molecular patterns (DAMPs) released from dying cells , 8 eventMLKL activation and translocation to the plasma membrane represents the final stage of the necroptotic pathway . Yet, MLHere, we report that MLKL mediates the release of granulocyte colony-stimulating factor (G-CSF) via controlling plasma membrane permeabilization, thereby effectively promoting myeloid progenitor differentiation during conditions of inflammation as well as leukemogenesis.MLKL/−− mice have been described previously [® 576408) for 7 days; after that cells were detached with 0.5 mM EDTA/PBS for about 2 min on ice for reseeding. To block membrane pore formation, polyethyleneglycols  with different molecular weights  were diluted in the medium with a final concentration at 100 mM. One day after treatment the cells and supernatant were harvested for further analysis.The eviously  and wereeviously . AnimalsTM GF M3434 medium (STEMCELL Technologies Inc.). GFP+/Lin− (green fluorescent protein positive/lineage negative) BM cells were sorted and seeded at a density of 2500 cells/plate and after 10–14 days the colonies were counted by light microscopy. The FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), mixed lineage leukemia-eleven nineteen leukemia translocation (MLL-ENL), and AML-ETO (RUNX1/RUNX1T1 translocation) retroviral plasmids have been previously described [TM Pro transfection, and the pMIG empty plasmid served as a control [For colony formation assays, all duplicate cultures were performed in 35 mm Petri dishes with MethoCultescribed . Retrovi control .6 cells/ml. Then cells were pre-incubated with Fc-block and subsequently stained with fluorescently labeled antibodies as listed in the antibody list  in PBS , then permeabilized with 0.1% Triton-X100 for 5 min in room temperature and blocked with 3% FBS. Cells were incubated 1 h with the indicated primary and secondary antibodies. The supernatant cytokine measurement cytometric bead array  was used according to the manufacturer’s instructions. Each assay was performed in triplicate.Standard FACS staining protocol was followed as previously described . In short Table S. PropidiMLKL/−− BM cells were seeded in the chamber slide in the density of 1 × 106/ml for 2 days , for immunofluorescence staining, the cells were fixed with 4% paraformaldehyde (pH = 7) in PBS in room temperature for 10 min, then permeabilized with 0.1% Triton-X100 for 5 min in room temperature and blocked with 3% FBS. After that cells were incubated for 1 h with the indicated antibodies and then with secondary antibodies (Table SThe WT or ®RNA (MACHEREY-NAGEL), following the manufacturer’s instructions. RNA concentration and purity were determined using the NanoDrop spectrophotometer (NanoDrop Technologies). One µg total RNA was reverse transcribed to 20 µl cDNA by SuperScript II Reverse Transcriptase (Life Technologies), The qPCR was performed using LightCycler® 480 (Roche) Real-Time PCR System, the reaction protocol was 95 °C for 10 min, followed by 40 cycles of 92 °C for 15 s and 62.5 °C for 1 min. Every sample was in triplicate and normalized to the reference gene expression using the 2−ΔΔCt method, all the primers in Table SRNA isolation was performed by NucleospinPrimary AML samples were obtained from patients treated at the III. Medical Department at the Technical University of Munich after approval of the local ethics committee . Informed consent was obtained from patients at study entry. For IHC eligible patients had received a diagnosis of de novo AML, which had been confirmed by means of a cytologic examination of blood and BM. Cases were re-evaluated using hematoxylin and eosin (H&E), Giemsa, AS-D chloroacetate esterase and standard diagnostic IHC . The infiltration rate of AML blasts was >50% in most cases. Biopsies were fixed in 4% formaldehyde, EDTA decalcified, and paraffin-embedded. IHC was performed using an automated immunostainer with the VIEW DAB detection kit  according to the company’s protocols for open procedures. Biopsies were stained with anti-MLKL  according to the manufacturer’s instructions and counterstained with hematoxylin. Appropriate positive controls were used to confirm the adequacy of the staining. Specific MLKL staining was localized to the cytoplasm. Human BM samples were collected according to the institutional guidelines and written informed consent was obtained from all patients in accordance with the Declaration of Helsinki. All use of patient material was approved by the Local Ethics Committees.t-test (two-tailed) or one-way ANOVA with posthoc (Dunnett’s multiple comparisons test) analysis. And *P < 0.05, **P < 0.005, ***P < 0.0005 were calculated by SPSS Statistics  or GraphPad Prism . The level 3 Cancer Genome Atlas (TCGA) acute myeloid leukemia (LAML) datasets containing 173 patient samples were downloaded for analysis, where “count” refers to RNA-Seq by expectation-maximization (RSME)-normalized transcript read count [http://software.broadinstitute.org/gsea (Version 3.0) [Data are presented as the mean ± SD or SEM and analyzed using a Student’s ad count . The uniad count . The GSEad count –32. For ion 3.0) .MLKL in human AML samples in 542 human AML BM samples and 73 healthy bulk BM controls (GSE13204). We found that MLKL expression was significantly reduced in AML BM   and healthy controls, its expression was significantly repressed in AML patients  Fig. , which wnts Fig. .Fig. 1SiMLKL expression based on the clinical AML risk category. Subdividing patients from the TCGA AML dataset (TCGA-LAML) according to their clinical risk classification, we found that patients with a favorable risk (n = 32) had significantly higher MLKL expression compared to patients in the intermediate (n = 103) or poor (n = 36) risk  compared to more differentiated AML subgroups (FAB M3–M5) or BM samples from healthy individuals  failed to show any coding mutations in the MLKL gene, only one mutation was found in the TCGA dataset for AML in the PanCancer cohort and no mutational hotspot was identified across other cancer types  myeloid cells or myeloid progenitor cells (Lin− Sca1− c-Kit+)  and observed no relevant difference in the viability of Mlkl−/− or WT cells  ex vivo. Upon retroviral transduction, we generated ls Fig. S.Our data revealed that loss of MLKL resulted in an expansion of progenitor populations in primary murine BM Figs. , S2D, whMlkl−/− BM might be explained by the inability of Mlkl−/− progenitors to differentiate into a specific myeloid lineage, we tested their colony-forming capacity. In line with the data obtained by FACS, Mlkl−/− colonies showed an accumulation of the myeloid progenitor population as measured by an increase in the CFU-GEMM colonies  compared to WT colonies  [MLKL expression and several inflammatory response pathways  including 173 AML patient samples, we selected the samples with the top 10% highest and the top 10% lowest h group) . We thenays Fig. . Specifiays Fig. , Table 1MLKL expression (AML MLKLhigh) compared to AML patients with the lowest MLKL expression (AML MLKLlow)  gene expression was observed in AML MLKLlow patients and vice versa   Table S. We idennts Fig. . Specifirsa Fig. . This waL) Fig. S.MLKL negatively correlated with CSF3 (G-CSF) expression.Collectively, these data showed that high expression of Mlkl−/− AML cells expressed significantly elevated levels of CSF3 mRNA (encoding for G-CSF)  Fig. . This waCSF3 mRNA into G-CSF protein by measuring the intracellular amount of G-CSF by FACS. We identified a significantly elevated level of G-CSF in AML cells from Mlkl/−− mice when compared to WT mice  to activate G-CSF receptor signaling irrespective of the levels of endogenously released G-CSF. In addition, we tested the effect of an α-mG-CSF blocking antibody with respect to the inhibition of G-CSF receptor signaling. Moreover, we treated the cells with an IgG isotype to check for the extent of differentiation solely based on the presence of endogenously secreted G-CSF.Based on our finding that MLKL facilitates G-CSF release, we hypothesized that MLKL influences myeloid progenitor differentiation in AML by limiting the release of G-CSF. We initially tested the ability of malignant myeloid progenitors from WT or We found significant repression of myeloid differentiation as measured by an expansion of primitive myeloid progenitors, when the cells were treated with α-mG-CSF antibody for all three tested AML subtypes Figs. , S5B. ThMlkl/−− genotypes  population . These data suggested that necroptotic pores measure approx. 4 nm in size [+ BM cells and found that PEG8000 effectively reduced the release of G-CSF only in WT FLT3-ITD+ BM cells but not in Mlkl/−− FLT3-ITD+ BM cells  had no impact on the release of G-CSF, while larger PEG (PEG6000 and PEG8000) had a size-dependent inhibitory effect, an effect also observed in AML-ETO and MLL-ENL-transformed cells  in necroptosis, specifically ESCRT-III, supports this finding by showing that ESCRT-III sustains the plasma membrane integrity of cells when MLKL activation is incomplete or limited by alternative factors . We, theIn leukemic cells, the amount of activated MLKL in response to oncogenic signaling  might beAdditional functions of MLKL have been reported that are not primarily based on cell death induction. These include MLKL in endosomal trafficking , insulinMLKL resulted in a significant upregulation of mRNA of cytokines such as CSF1 (encoding M-CSF), CSF2 (encoding GM-CSF), and IL-8 amongst others [CSF3 (G-CSF) was not reported in their study, the data supported the notion that low/reduced levels of MLKL contribute to a reduction in released cytokine levels and a subsequent failure of a negative feedback mechanism keeping cytokine mRNA expression in check [Additional support for a role of MLKL in cytokine release comes from a study by Yoon et al. that reported that deletion of t others , Using tin check . A publiin check .It is interesting to note that the main differences in cytokine release were observed for G-CSF in our study, whereas alternative cytokines such as TNF or IL-6 differed to a lower extent. The type of oncogenic driver, the time points of analysis, or the propensity of primary murine BM-derived cells to release G-CSF might cause this difference. Irrespective of alternative cytokines, our data support a critical contribution to MLKL-mediated G-CSF release for the process of cellular differentiation in AML. This is of specific interest as we utilized three clinically relevant AML oncogenic drivers that together represent the majority of cytogenetically normal AML patients.A substantial body of evidence shows that G-CSF instructs lineage choice and proliferation of hematopoietic progenitor cells during steady-state hematopoiesis –55, as wMlklD139V), elevated the plasma levels of several pro-inflammatory cytokines (including G-CSF) [+ cellular infiltrates in MlklD139V/D139V mice, yet no clear correlation to exaggerated cell death [Instead of using a loss-of-function model of MLKL as in our study, Hildebrand et al. recently reported that an auto-active mutant of MLKL (g G-CSF) . The repll death . It is rIn summary, we show that the differentiation blockade in AML partly results from the failure of AML cells to release G-CSF from intracellular storage. Our findings thereby expand the molecular mechanism of differentiation blockade in AML to auto- or paracrine G-CSF-mediated signaling. Our data provide a pathophysiological context for the observation that pore formation in the plasma membrane and/or endosomal trafficking can be blocked or delayed without necessarily proceeding to cell death.Supplementary legendsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6Supplementary Table S1Supplementary Figure S7Supplementary Table S2Supplementary Table S3Supplementary Table S4