Urocortin-1 (Ucn-1) is an endogenous peptide that protects heart from ischemia and reperfusion (I/R) injuries. Ucn-1 is known to prevent cardiac cell death, but its role in the transcription of specific genes related to survival signaling pathway has not been fully defined. The aim of this study was to investigate the molecular signaling implicated in the improvement of cardiac myocytes survival induced by Ucn-1.Ucn-1 administration before ischemia and at the onset of reperfusion, in rat hearts perfused in Langendorff system, fully recovered heart contractility and other hemodynamic parameters. Ucn-1 enhanced cell viability and decreased lactate dehydrogenase (LDH) release in adult cardiac myocytes subjected to simulated I/R. Annexin V-FITC/PI staining indicated that Ucn-1 promoted cell survival and decreased cell necrosis through Epac2 (exchange protein directly activated by cAMP) and ERK1/2  activation. We determined that Ucn-1 shifted cell death from necrosis to apoptosis and activated caspases 9 and 3/7. Furthermore, mini-array, RT-qPCR and protein analyses of apoptotic genes showed that Ucn-1 upregulated the expression of CD40lg, Xiap and BAD in cells undergoing I/R, involving Epac2 and ERK1/2 activation.Our data indicate that Ucn-1 efficiently protected hearts from I/R damage by increasing the cell survival and stimulated apoptotic genes, CD40lg, Xiap and BAD, overexpression through the activation of Epac2 and ERK1/2. Despite the considerable advances that have been made in the field of myocardial protection, ischemic heart disease represents a major public health problem and the main cause of mortality in the industrialized world . Percuta2) is known to enhance cAMP production [2+ handling in cardiac myocytes [Special interest has been made toward the endogenous protection elicited by the heart as a potent approach to limit heart lesions from I/R insult. In the last two-decade, urocortin peptides , which belongs to the corticotropin-releasing factor (CRF) family . In the oduction , which ioduction , cardiacoduction , and Ca2myocytes . Previoumyocytes . However2 (iPLA2) and protein kinase C epsilon (PKCε) [2+ handling, which included the recovery of cells excitability, the inhibition of diastolic Ca2+ increase and the regulation of Na+/Ca2+ exchanger [Different mechanisms are implicated in the cardioprotection afforded either by Ucn-1 or Ucn-2, involving the rapid activation of protective signaling pathways , calciumn (PKCε) , or ERK1n (PKCε) , among on (PKCε) , STAT3 [n (PKCε)  or p38MAn (PKCε) . We havexchanger .Herein, we explored the molecular pathway involved in Ucn-1 evoked heart protection with special emphasis on Epac and ERK1/2 on their role in cardiac myocytes survival. We also examined the effect of Ucn-1 on cell death pathways and its regulation of apoptotic genes, CD40lg, Xiap and BAD.All the experiments with animals were performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the local Ethics Committee on human Research of the “Virgen del Rocio” University Hospital of Seville and the Animal Research Committee of the University of Seville.Wistar rats weighing 250–350 g were heparinized (4 IU/g i.p.) and anaesthetized by intraperitoneal administration of an overdose of sodium thiopental (200 mg/Kg). The hearts were quickly removed, mounted on the aortic cannula of the Langendorff perfusion system apparatus and perfused with an oxygenated Krebs- Henseleit buffer  as described previously [Adult male eviously , 17. BefThe standard protocol of ischemia/reperfusion in perfused hearts was followed as described previously . Group 12, 0.5 MgCl2, 5.4 KCl, 22 glucose, 25 HEPES, 0.4 NaH2PO4, 5 NaHCO3; pH was adjusted to 7.4 with NaOH. Cardiac myocytes were plated in control solution containing 1.8 mM CaCl2 at 37°C, and were submitted to a protocol of I/R using a simulated ischemic solution (mM): 142 NaCl, 3.6 KCl, 1.2 MgCl2, 1.8 CaCl2, 20 Hepes, 20 Lactate-Na, 20 sucrose (pH 6.2). Cells were then placed during 30 minutes in an incubator of 1% O2 and 5% CO2. Afterward, cells were incubated in control solution in a 21% O2 and 5% CO2 incubator during 18 hours. All the experiments were performed on Ca2+-tolerant rod-shaped myocytes.The hearts were removed and mounted on a Langendorff perfusion apparatus. Ventricular myocytes were isolated using collagenase type II  as described previously . SubsequGroup 1, control: Untreated cardiac myocytes.Group 2, I/R: After stabilization in control solution, cells were exposed to simulated ischemia solution during 30 minutes followed by 18 hours of reperfusion with freshly control solution.Group 3, Ucn-1: Same as group 2, but Ucn-1 (10 nM) was applied 20 minutes before ischemia followed by reperfusion.Group 4, 8CPT: preconditioning with 8CPT (10 μM), specific agonist of Epac: Same as group 3 but 8CPT was applied instead of Ucn-1.Group 5 and 6: Cells treated with either ESI-05  or PD 098059  10 minutes before the addition of Ucn-1 (10 nM) following the same protocol as in group 3.The level of lactate dehydrogenase (LDH) was detected in supernatant of cells subjected to simulated I/R protocol according to the manufacturer’s instructions (Promega). Trypan Blue staining method was also used for detecting cell death in cardiac myocytes undergoing treatment with I/R ± Ucn-1 (10 nM). Briefly, after each treatment cells were washed three times with PBS, then cells were bathed in 0.5% of trypan blue for 10 minutes. Based on the classical cell counting, we acquired 4 random snapshots per each condition using 40x Objective of an Olympus microscope. Maximum projection was recorded and analyzed with “Image J” software to count the different proportion of trypan positive and negative cells by an investigator blinded to the treatment conditions. This process was repeated in triplicate for each condition and the experiment was replicated on 3 separate occasions. 100% indicates total number of trypan stained and unstained cells in each condition.Double staining with annexin V-FITC and propidium iodide (PI) was also performed to examine apoptosis and necrosis in cultured cardiac myocytes. During apoptosis, an early and ubiquitous event is the exposure of phosphatidylserine at the cell surface, which is detected with annexin V-FITC. PI is used to determine the population of cells that have lost membrane integrity, an indication of late apoptosis or necrosis. Briefly, cardiac myocytes were seeded in a 6-channel μ-Slide  from IBID. After treatments cardiac myocytes were incubated with 100 μl of the binding buffer supplied in the kit (Cat. 4830-01-K. Trevigen) and 1 μl of annexin-FITC reagent, during 60 minutes at 25°C. Then PI was added and incubated for 15 minutes and later washed before mounting the slides. Images were taken with confocal microscope Leica TCS SP2 microscopy (Leica). 5 snapshots per condition were acquired using a HCX PI Apo CS 40x objective in z-stacks intervals, and maximum projection was recorded and analyzed with “Image J” software to count the different proportion of cells that were labeled only with annexin (early apoptosis), or with PI and/or annexin and PI (necrosis or late apoptosis).To determine the caspase activity we used the Caspase-Glo assay kit system from Promega; TB323 for Caspase 3/7, TB332 for Caspase 8, and TB333 for Caspase 9. Protein extracts from cultured cardiac myocytes were quantified by Bradford (Sigma Aldrich) method and were diluted at 1.5 μg/μl of protein. 10 μl of protein extract and 10 μl of Caspase-Glo reagent were added in a 384 flat and white multi-well dish in quadruplicate replicates and incubated for 40 minutes at room temperature. Luminescent signal was detected in a Luminoskan ascent microplate luminometer (Thermo Scientific).Protein samples were extracted from cultured and treated cardiac myocytes and 40 μg of protein were subjected to SDS-PAGE (10% acrylamide) and electrotransferred onto PVDF membranes. Membranes were probed overnight at 4°C with specific primary antibodies in TTBS with 1% of BSA. CD40lg antibody was from sigma. BAD, phosphoBAD, Xiap, phosphoERK1/2 and ERK1/2 primary antibodies were from Cell Signaling. Detection was performed with the enhanced chemiluminiscence reagent ECL-plus (Amersham Bioscience) in the ImageQuant LAS 4000 mini . For quantification, the images were analyzed with “Image J” software using alpha-tubulin (Sigma) or GAPDH (Genetex) as housekeeping loading control.Total RNA was extracted from cultured cardiac myocytes using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. RNA purity and concentration were assessed by measuring absorption at 260 nm and 280 nm. 1 μg of RNA were retro-transcribed to cDNA with the RT quantitec kit (Qiagen). qRT-PCRs were performed with the use of a Prism 7900HT Sequence Detection System, Taqman primers and probes technologies (Applied Biosystems). Fold change in gene expression was calculated using the comparative cycle threshold CT (ΔΔCT) method.For apoptosis PCR array, we used plates from SA Biosciences (Cat. PAHS-012). Total RNA from Ucn-1 treated and no-treated cardiac myocytes (as described in cell treatment protocols) submitted to I/R was extracted and reverse transcribed into cDNA with the use of an RT2 First Strand Kit (SA Biosciences). The templates were combined with an RT2 SYBR Green qPCR Master Mix (SA Biosciences), and then equal aliquots of this mixture (25 μl) were added to each well of the same PCR Array plate that contained the pre-dispensed gene-specific primer sets. The qRT-PCR quantification was performed as described above by ΔΔCT method.Data analysis was carried out using SigmaPlot software, version 11.0. A sample size calculation was performed prior the start of this study. Group data are presented as mean ± S.E.M. Single or paired Student's t test was used to determine the statistical significance of the obtained data. The significance between multiple groups was evaluated using ANOVA followed by Tukey multiple comparison post hoc tests for comparing different groups. Data marked by *, **, *** were considered significantly different at p<0.05, p<0.01 and p<0.001 respectively.ex vivo Langendorff-perfused rat hearts submitted to I/R. The protective effects of Ucn-1 on heart contractility were analyzed in Furthermore To further evaluate the cardioprotection exerted by Ucn-1, we examined cardiac myocytes viability using several approaches. First, we estimated trypan blue staining in cardiac myocytes subjected to I/R. In addition, we used annexin V-FITC staining to label apoptotic cells in combination with red-fluorescent PI (propidium iodide) that stains late apoptotic/necrotic cells. Representative images and data analysis in We examined the contribution of ERK1/2 pathway in Ucn-1 cardioprotective effects. Because Epac activates the Ras1-ERK1/2 pathway , we alsoTo get more insight into the mechanism involved in Ucn-1 cytoprotection and apoptosis, we analyzed the expression of 84 key genes involved in apoptosis using a PCR based micro-array technique. Next, the analysis of protein expression showed that CD40lg, Xiap and Bad levels were not significantly affected by I/R, however the administration of Ucn-1 enhanced significantly the expression of CD40lg and Xiap protein in ERK1/2 dependent manner, since treatment with PD 098059 (5 μM) prevented Ucn-1 effects as shown in Clinical search for new heart protection strategies and new endogenous cardioprotector is still of major relevance. There is continuous interest in developing novel therapeutic targets that potentially protect the heart against I/R injuries and complications , 22. SevOur experimental data from Langendorff-perfused rat hearts showed that Ucn-1, given both before and after a global ischemic insult, was able to recover heart contractility, to preserve vascular resistance, and to improve the ventricular diastolic pressure, indicative of its prevention of hypercontracture and rigor. Ucn-1 effectively sustained rat hearts hemodynamics properties and protected it from I/R injury consistent with the effect of Ucn-2 described in previous studies , 25.The degree of cardiac dysfunction after I/R injury reflects the level of cardiac myocytes lesion related to subsequent cell death, scare formation and a worse long-term outcome. Here, we demonstrated that Ucn-1 exerted significant cytoprotective effects as it efficiently decreased trypan blue cell staining, inhibited LDH release, and enhanced cell viability by the modulation of apoptosis and necrosis. Ucn-1 administration decreased significantly necrotic cells but it increased apoptotic cells number. In fact, while Ucn-1 increased cell viability it shifted cell death from necrosis to apoptosis, a more programmed and controlled cell death. Ucn-1 promotion of apoptosis in detriment of necrosis is supposed more beneficial to reduce the impact of I/R lesions as discussed earlier . ApoptosMoreover, we demonstrated that Ucn-1 implicated Epac2 and ERK1/2 in its cardioprotective actions. ERK1/2 role as a survival pathway in cardiac cells is widely accepted –12, neveIn conclusion, we demonstrated that the administration of Ucn-1 before ischemia and during the first minutes of re-oxygenation produced significant long-lasting protective effects, mediated by a complex signaling pathway that involve Epac2 and ERK1/2. Ucn-1 promotion of cell survival and apoptosis might be beneficial since apoptosis is considered a highly regulated energy-consuming process, required for controlled and programmed cell death, which will limit cardiac cell loss and posterior inflammation processes in I/R. Together with the other cardioprotective actions of urocortin including vasorelaxation of human coronary artery , the posS1 FigGraph shows caspase 8 activity analyzed from cells exposed to ischemia and reperfusion  ± urocortin-1 . Ucn-1 (10 nM) was also applied in presence of 5 μM PD 098059 (Ucn-1+PD). Data are given as means ± s.e.m. n = 5 cultures. “ns” indicates that values are not significantly different from control.(TIF)Click here for additional data file.S2 FigGraph shows summary data from untreated cells (control), cells exposed to I/R (30 minutes/18 hours), cells exposed to I/R in presence of 5 μM PD 098059 (I/R+ PD) or 10 μM ESI-05 (I/R + ESI). “*” indicates significance between control and different treatment at p<0.05. “ns” indicates that values are not statistically different between I/R and I/R+ESI or I/R +PD. Values are means ± s.e.m. n = 4 cultures.(TIF)Click here for additional data file.S3 FigPCR-based array was performed in samples from cardiomyocytes exposed to I/R and treated or not with Ucn-1 (10 nM) as described in cell treatment protocols in material and methods.(TIF)Click here for additional data file.S4 FigA) Bar graph shows the analysis of phospho-BAD level from western blot experiments.Protein samples were from cells exposed to ischemia and reperfusión  ± urocortin-1 . Ucn-1 (10 nM) was also applied in presence of 5 μM PD 098059 (Ucn-1+PD) and 5 μM ESI-05 (Ucn-1+ESI). 8CPT (10 μM) was applied instead of Ucn-1 to activate Epac B) Analysis of total BAD and non phosphorylated free BAD in the same experiments as in “A”. Values are means ± s.e.m. from 3 different experiments.(TIF)Click here for additional data file.


Caffeine induces a release of calcium from intracellular stores. We tested whether caffeine modulated GABAergic transmission by increasing [Ca2+]i. A brief local puff-application of caffeine to hippocampal CA1 pyramidal cells transiently suppressed GABAergic inhibitory postsynaptic currents (IPSCs) by 73.2 ± 6.98%. Time course of suppression and the subsequent recovery of IPSCs resembled DSI (depolarization-induced suppression of inhibition), mediated by endogenous cannabinoids that require a [Ca2+]i rise. However, unlike DSI, caffeine-induced suppression of IPSCs (CSI) persisted in the absence of a [Ca2+]i rise. Intracellular applications of BAPTA and ryanodine  failed to prevent the generation of CSI. Surprisingly, ruthenium red, an inhibitor of multiple calcium permeable/release channels including those of stores, induced metaplasticity by amplifying the magnitude of CSI independently of calcium. This metaplasticity was accompanied with the generation of a large inward current. Although ionic basis of this inward current is undetermined, the present result demonstrates that caffeine has a robust Ca2+-independent inhibitory action on GABAergic inhibition and causes metaplasticity by opening plasma membrane channels.GABAergic inhibition plays a critical role in the regulation of neuron excitability; thus, it is subject to modulations by many factors. Recent evidence suggests the elevation of intracellular calcium ([Ca Caffeine is a methylxanthine that acts as a nonspecific phosphodiesterase inhibitor . It is wμM range) i in response to caffeine were plotted against time.After establishing a whole-cell recording, the cells were held at −70 mV for 10 min before imaging in order for dyes to be diffused and equilibrated in the cytosol. Ca2+ indicators were calibrated according to Grynkiewicz et al. [2+]i . However, Kd measured in buffered solution (reported by Molecular Probes) could be different when the same dye was introduced to the cytosol of intact cells. In our experiments, Rmin and Rmax were measured in situ as follows: CA1 pyramidal cells were whole-cell patch clamped with a pipette that contained 2 mM BAPTA and fura-2 or fura-FF in the recording pipette solution. Five pairs of 380/360 measurements were taken 5 min and 10 min after the break-in. Extracellular ACSF was then switched to a nominally Ca2+-free ACSF. The replacement of the extracellular ACSF was checked by the disappearance of eIPSCs (evoked every 3 s). When eIPSCs became undetectable, which occurred in 15–20 min, an additional 5 pairs of 380/360 measurements were taken. These last pairs were averaged and used to calculate Rmin. Subsequently, 20 mM CaCl plus 100 μM ionomycin was pressure-ejected for 120 s from a micropipette (2 μm in diameter) that was placed close to the soma. Measurements of fluorescence at 380 and 360 nm were made continuously during the entire ejection period. The 380/360 ratio decreased to 10–16% of the original values within 30 s and remained at that value until the end of the ejection. Rmax was calculated from the lowest ratio value observed. The ratio recovered to 42–50% of the original within 25–30 s after the ejection ended, showing that the decrease was attributable to the rapid perfusion of cell interior with a high [Ca2+]i instead of the loss of recording.Ratiometric Caz et al.  to estimμM) and APV (100 μM), while stimulating the stratum radiatum at 0.2 Hz. Caffeine (100 μM–100 mM) was pressure-ejected for 1.5 s from a glass pipette positioned immediately above the recording neuron. Caffeine induced robust instantaneous suppression of IPSCs. The recovery of IPSCs was immediate upon the termination of caffeine application, suggesting that caffeine directly interacted with GABAergic synapses without involving a series of intermediary molecules  completely blocked IPSCs during the application . The recCSI was accompanied with an increase of cytosolic calcium to 300 nM when measured with an intracellular application of fura-2 . AlthougμM), cADPR (100 μM), and ruthenium red (10 μM) intracellularly by dissolving these compounds in the recording pipette solution. cADPR is an agonist of the ryanodine receptor and facilitates a release of calcium from ryanodine-sensitive stores to empty them. Inclusion of cADPR in the recording pipette exhibited a decrease in [Ca2+]i in response to caffeine in 15 min after break-in  . In addie stores  and by ae stores . Additio2+]i, caffeine competitively binds to multiple regulatory sites of the GABAA receptor and interferes with GABAergic transmission [2+]i in modulation of GABAergic IPSCs. Multiple actions of caffeine on GABAergic synapses and homeostasis suggest caffeine's powerful “affinity” for successful access to fundamental mechanisms in inhibitory neurotransmission.Postsynaptically, independently of [Casmission . Caffeinsmission . Taketo smission  reported+ currents including Ca2+-activated K+ currents , inwardly rectifying K+ current, M-current, and the Ca2+-activated Cl− current. These currents might directly or indirectly influence neuronal membrane resistance and thus modulate cell's excitability, which could affect GABAergic inhibitory transmission. Caffeine-induced inward current that we observed in the present study may be similar to the inward current revealed as a consequence of the blockade of M-current by muscarine (muscarine-sensitive K+ current) [2+ such as Ca2+-release activated Ca2+ entry (CRAC). Ruthenium red is known to interact with various calcium-permeable channels and transporters including ryanodine receptors , TRIP channels , calcium homeostasis modulator 1 (CALHM1), calcium pumps (Ca2+-ATPase), mitochondrial Ca2+ uniporter, and Ca2+ binding proteins including calmodulin [We acknowledge that our findings on the caffeine-induced inward current and its amplification in the presence of ruthenium red are preliminary. Generation of inward currents by a brief topical application of caffeine has not so far been established. In the available pool of literatures, caffeine is suggested to interact with various types of Kcurrent) . On the lmodulin –28. Furt


In this study dendritic Ca2+ entry was investigated using two photon excitation fluorescence microscopy and whole-cell patch-clamp of juvenile (P4-11) motoneurons. Neurons were filled with both Ca2+ Green-1 and Texas Red dextrans, and line scans performed throughout. Steps were taken to account for different sources of variability, including (1) dye filling and laser penetration, (2) dendritic anatomy, and (3) the time elapsed from the start of recording. First, Ca2+ Green-1 fluorescence was normalized by Texas Red; next, neurons were reconstructed so anatomy could be evaluated; finally, time was recorded. Customized software detected the largest Ca2+ transients (area under the curve) from each line scan and matched it with parameters above. Overall, larger dendritic diameter and shorter path distance from the soma were significant predictors of larger transients, while time was not significant up to 2 h (data thereafter was dropped). However, Ca2+ transients showed additional variability. Controlling for previous factors, significant variation was found between Ca2+ signals from different processes of the same neuron in 3/7 neurons. This could reflect differential expression of Ca2+ channels, local neuromodulation or other variations. Finally, Ca2+ transients in SOD1G93A motoneurons were significantly smaller than in non-transgenic motoneurons. In conclusion, motoneuron processes show highly variable Ca2+ transients, but these transients are smaller overall in SOD1G93A motoneurons.Previous studies of spinal motoneurons in the SOD1 mouse model of amyotrophic lateral sclerosis have shown alterations long before disease onset, including increased dendritic branching, increased persistent Na Many disruptions have been found in both patients and animal models that could negatively affect motoneuron health, including altered protein degradation  channels. Though Cav1.3 channels failed to show higher expression levels in adult SOD1 motoneurons using immunohistochemistry  in SOD1 motoneurons remain relatively unstudied. Voltage-gated Ca2+ currents measured from wild type spinal motoneurons include P/Q (Cav2.1), N (Cav2.2), R (Cav2.3), L (Cav1.2 and 1.3), and T-type (Cav3) currents  of hypoglossal motoneurons  To compare the patterns of these transients in wild type motoneurons to those of SOD1G93A motoneurons.Therefore, in this study there were two main goals: (1) To examine normal anatomical patterns of high-voltage activated CaExperiments were performed in accordance with the United States National Institutes of Health Guide for Care and Use of Laboratory Animals. Approval of the Northwestern University's Animal Care and Use Committee was obtained for all experiments performed in this study. All efforts were made to minimize animal suffering and to reduce the number of animals used.G93A gene  were identified using standard PCR techniques , decapitated and eviscerated. The lumbar spinal cord was removed and embedded in 2.5% w/v agar . The agar block was then superglued with Loctite 401  to a stainless steel slicing bath and 350 μm transverse slices are made using the Leica 1000 vibratome  as described previously . After cutting, the slices were incubated for >1 h at 30°C in incubating solution containing (mM) NaCl 126.0, KCl 2.5, CaCl2 · 2H2O 2.0, MgCl2 · 6H2O 2.0, NaHCO3 26.0, glucose 10.0, pH 7.4 when bubbled with 95% O2/5% CO2 .For this study, 60 juvenile mouse pups were used between postnatal day (P) 4-11. Transgenic B6SJL mice overexpressing the human SOD13, 1.10 KH2PO4, 1.26 MgSO4, 2.52 CaCl2, and 11.1 glucose. The solution was oxygenated with 95% O2 and 5% CO2 and the perfusion rate was 2.5–3.0 ml/min. Patch electrodes contained (in mM) 138 K-gluconate, 10 HEPES, 5 ATP-Mg, 0.3 GTP-Li and Texas Red dextran and Ca2+ Green-1 dextran . The Kd of Ca green-1 varied between lots, and ranged from 406 to 737 nM.Whole cell patch clamp was performed on motoneurons from the lumbar segments using 2–4 MΩ glass electrodes pulled from glass capillary tubes  with a Flaming-Brown P-97 . Electrodes were positioned using a Sutter Instrument MP-285 motorized micromanipulator (Sutter Instrument Company). Whole-cell patch clamp measurements were performed at room temperature using the Multiclamp 700B amplifier  and Winfluor software . Briefly, slices were perfused with a modified Ringer's solution containing (in mM): 111 NaCl, 3.09 KCl, 25.0 NaHCO2+ channels in the dendrites. Depolarizing the soma to threshold for a single spike elicited a fairly robust Ca2+ signal in the soma and proximal dendrites. However, it was not clear whether single spikes would be strong enough to spread electrotonically back as far as 300 um or more when more distal processes were examined. Conversely, limiting the duration of the stimuli was necessary to minimize the exposure time (and thus any damage) to the processes by the laser. With 50, 100, and 200 ms pulses, we avoided damaging dendrites with prolonged laser exposure yet produced a depolarization robust enough to measure even in distal dendrites. As shown in Figure 2+ signals from the three events were averaged unless otherwise noted.During line scans, neurons were subjected to three depolarizing steps each at 50, 100, and 200 ms to evoke 1 action potential, 3–5 action potentials, and ~10 action potentials per step, respectively. Stimulation protocols were developed to assess activity of high voltage gated Ca2+ green-1. Based on these spectra, the laser was tuned to 820 nm. The average laser power at this wavelength was about 2 W, though it is expected that some power was lost as the beam passed through the Bio-Rad Radiance modulators and the objective. Since emission of these fluorophores is well-separated, two Bio-Rad 2100 MPD photomultiplier tubes : Green PMT (500–550 nm) and Red PMT (570–650 nm) were used to collect emission in the red and green channels. Unidirectional line scans were performed along the length of the dendrites with a dwell time of 10 μs/pixel. Precautions were taken to reduce noise in the images, including very low ambient light levels during experiments and a heavy curtain surrounding the rig. Fortunately, in the spinal cord of young mice at the ages used in these experiments, the auto-fluorescence of tissue is very low compared to the adult cord, probably due to incomplete myelination.An Olympus BX-51WIF microscope fitted with an Olympus 40x/0.8NA water-dipping objective lens was used for visualizing the patched neuron. Two photon excitation fluorescence microscopy (2PEF) was performed with a galvanometer-based Coherent Chameleon Ultra II laser . To choose an appropriate wavelength for excitation of both fluorophores, two photon excitation spectra were run for both Texas Red and CaThe ventrolateral motoneuron pools could be easily visualized in the slice preparation  and quantified with Neurolucida Explorer. Neuronal reconstructions, z stacks, and line scan reference images were utilized to determine the path distance from the soma to the line scanned processes, along with the mean diameter of the sampled segments.2+ transient. In brief, custom software for MATLAB  was developed by our laboratory and used for precision measurement of Ca2+ signal along each line scan. For each line scan, a single position along the dendrite was retained, corresponding to the pixel that generated the largest Ca2+ signal for that line scan. The exact 0.2–0.3 μm region of the dendrite (after applying a moving average of range 2.6 μm) which gave rise to that peak signal was then assessed for (1) diameter and (2) path distance to the soma. The code is included as a Supplemental File.All signals throughout this manuscript are measured from the area under the curve of the averaged Ca2+ Green and Texas Red intensities.Distinct regions of interest (ROI) along each line scan encompassing dendritic processes and somas were identified utilizing reference images and z stacks. Furthermore, an ROI devoid of processes was determined for each line scan to serve as a baseline intensity for normalizing observed Ca2+ entry was achieved through analysis of the change in Ca2+ Green-1 fluorescent intensity through MATLAB. Ca2+ transients were analyzed following the onset of stimulus application until the end of imaging (approximately 1 s). For each scanned process, three evoked Ca2+ transients were quantified from each of three protocols. For all line scans, each pixel along the length of the scan was quantified temporally in terms of Ca2+ Green and Texas Red intensity normalized by the mean green and red fluorescent intensity within the baseline ROI, respectively. This method was pioneered by Bloodgood and Sabatini (2+ Green fluorescence was subtracted from the Ca2+ Green intensity during the window from stimulus onset to imaging termination to determine the change in Ca2+ Green fluorescence (ΔG), an indicator of the change in internal Ca2+. ΔG was normalized by the pre-stimulus Texas Red fluorescence (R0) to obtain a ΔG/R0 curve. The area under the ΔG/R0 curve for each stimulus was then computed and the three values for each pixel of the line scan averaged together. As a result, a value for the area under ΔG/R0 was obtained for each pixel along the length of the line scan. For each line scan, the maximum value of area under ΔG/R0 within the dendritic or somal ROI was identified as a quantification of the Ca2+ transient, as shown in Figure 0, it was verified that the pixel was located within the process, then the path distance from that pixel to the soma was approximated in MATLAB based on neuronal reconstructions and line scan reference images.Measurement of action potential evoked CaSabatini . For eac2+ transients was performed using an arbitrarily assigned branch number as a variable in the analysis of covariance (ANCOVA) described below.For cells with scanned processes from at least three separate dendritic branches, a within-cell analyses of Ca2+ transients were analyzed using ANCOVAs . This aG93A and 13 wild type visually-identified motoneurons were recorded from the lumbar region as shown in Figure 2+ transients were measured with the calcium indicator Ca2+ green-1 dextran . Stimulation protocols were run simultaneously with line scans as shown in Figures 2+ green-1 at rest. But when action potentials were elicited, Ca2+ green showed a robust increase in fluorescence. Customized MATLAB software was used to measure the signal from 2.6 μm moving averages throughout the line scan and detect the pixel where the peak signal was centered. The anatomical characteristics of the dendrite at the location of the peak signal were used in later analysis. Three different protocols were applied to each neuron, each with three events: three depolarizing pulses of 50 ms (evoking one spike/stimulus), three pulses of 100 ms (evoking 3–5 spikes per stimulus), and three pulses of 200 ms (evoking about 10 spikes per stimulus). The three pulses of each protocol were averaged together (except where otherwise noted), and the area under the ΔGreen/Red curve , and to minimize changes in the peak and decay of the Ca2+ signal over time due to any potential increases in concentration of the Ca2+ sensitive dye over time  than WT motoneurons (solids lines), starting at 165 μm from the soma. Though axonal transport has been shown to be altered in mutant SOD1 motoneurons .After whole cell patch configuration was established, at least 20 min were allowed to pass for diffusion of the dyes. Since patch electrodes were large (2–4 MΩ resistance), diffusion took place rapidly and Texas red was clearly visible throughout the processes after this time. To further examine whether time may have had an effect on strength of the Ca2+ signals evoked with a single spike were quite robust  a significantly larger Ca2+ transient for the first (blue) of the three stimuli at many points in the dendritic processes, even while action potential amplitudes remained consistent across stimuli . This phenomenon was most pronounced with the spike trains evoked by the 200 ms pulses, and least pronounced in the 50 ms single-spike stimulus protocol .Each stimulus protocol was composed of 3 pulses of equal duration: three 50 ms pulses, . Stack sizes were 308 × 308 μm (512 × 512 pixels), and the depth ranged from 75 to 204 μm (step size = 1 μm). All of the morphological characteristics from the dendrites were compared to Ca2+ transients using ANCOVA. Ca2+ transients were found to correlate significantly with three factors: (1) number of spikes evoked/length of depolarizing current pulse, (2) distance from the soma, and (3) diameter of the dendrite at that location. More action potentials (from longer current steps), larger diameter dendrites and more proximal locations all contributed to larger Ca2+ signals (see Table G93A and 2 of 4 wild type) showed significant variation from one dendrite to the next. In other words, even when the contributions of branch diameter, distance from the soma and stimulus length were accounted for (using ANCOVAs) 3/7 motoneurons had dendrites with significantly different profiles of Ca2+ entry. For example, compare the Ca2+ transients from the P9 wild type motoneuron shown in Figure 2+ transients. These examples are taken at equal distances from the soma, are averages of three 200 ms stimuli delivered while performing 2PEF at each location of the dendritic field. The dendritic diameter does vary somewhat between these processes, but it cannot account for the magnitude of difference between signals. Only motoneurons in which ≥3 branches had been sampled at overlapping distances were included in the analysis (n = 7). The processes with lowest and highest signals were not consistently first or last in the recording order, and data was eliminated from analysis whenever there was any sign of photodamage  or physiological weakening of the cell . Rather, the different signals from a dendrite likely reflect Ca2+ microdomains as first suggested by Ladewig et al. .In order to capture anatomical characteristics, several z stacks were acquired after electrophysiological and Cag et al.  in hypogG93A and 13 wild type motoneurons were recorded. Comparing Ca2+ signals between these two groups, transients from SOD1G93A motoneurons were significantly smaller (p = 0.000) as shown in Figure 2+ channel activity in distal SOD1G93A dendrites . Table Overall, eight SOD12+ transients with (1) longer depolarizing steps/increasing numbers of evoked action potentials, (2) larger dendritic diameters, and (3) shorter distances from the soma, as has been documented in other neuron types , these channels probably are contributing to the Ca2+ signals here, along with the other high-voltage-activated Ca2+ channels Cav2.1 (P/Q) and Cav2.2 (N) type channels. Though classically the Cav2.1 channels mediate vesicle release at axon terminals, they have also been shown to contribute substantially to whole cell Ca2+ currents measured at motoneuron somata  channels. SK channels are present on motoneurons , metabotropic glutamate, and adenosine A1 receptor activation can all suppress Cav2 channel activity  in SOD1 motoneurons, while in contrast, the Cav1.3 channels responsible for the PIC appear to be more active . They also showed reduced amplitude dendritic Ca2+ transients evoked by spontaneous synaptic events (though the frequency of these spontaneous events increased). Since this study used the membrane permeable Ca2+ indicator Fura-2 AM, dye diffusion would not be a factor here  pathology are the largest, fast fatigable motoneurons which lack Cand Miles  describend Miles  reportednd Miles  observed2+ signals between regions that were differently filled with dye, or experienced differences in laser penetration (processes that were deeper in the slice than others). Specifically, use of 3000 MW Texas red to normalize transients detected by 3000 MW Ca2+ green-1 can control for these variables.Our results show for the first time that high expressor SOD1 motoneurons may have an impedance to dye filling. It would be tempting to assume dendritic transport is altered similarly to axonal transport (Zhang et al., 2+ has broad impact on neurons, affecting the immediate electrophysiology, the short term cell signaling pathways, and in the long term, neurodegenerative pathways. In the present study we show that dendritic Ca2+ entry is highly variable even in wild type motoneurons. This likely allows for the fine tuning of synaptic inputs and intrinsic cell currents through neuromodulatory processes and variable expression of dendritic Ca2+ channels. In the SOD1G93A motoneurons, the complexity of Ca2+ entry is certainly not diminished. Dendritic Ca2+ entry can be significantly different from one branch to the next, and the overall Ca2+ signal in SOD1 motoneurons is smaller.Intracellular CaKQ designed, performed and analyzed the experiments, performed statistical analysis, interpreted the data and wrote the manuscript. JL designed the customized software to match transients to their anatomical parameters, performed the data analysis and statistical analysis. JS assisted with data analysis. CH developed the initial conception of these experiments, interpreted the data, and contributed to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mentoring is a key predictor of empowerment and prospectively a game changer in the quest to improve health inequities. This systematic review reports on the state of evidence on mentoring for Indigenous Australians by identifying the quantity, nature, quality and characteristics of mentoring publications.Thirteen databases were searched using specific search strings from 1983 - 2012. Grey literature was also canvassed. The resultant publications were mined to identify their outputs, nature, and quality. These were then conceptually mined for their characteristics to develop a model of mentoring that included the initiating environments, facilitating environments, operational strategies and outcomes.771 citations were identified; 37 full text publications met inclusion criteria and were assessed. Fifteen were eligible for review. Four of five original research publications used strong qualitative research designs. No publications were found before 1999; the largest proportion concentrated in 2011 (n = 4). Facilitating environments included: mapping participants’ socio-cultural and economic context; formal mentoring practices with internal flexibility; voluntary participation; integrated models with wrap-around services; mentor/staff competencies; and sustained funding. Mentoring strategies comprised: holistic scaffolding approaches; respectful, trusting, one-on-one mentoring relationships; knowledgeable mentors; regular contact; longer-term relationships and exit strategies; culturally-tailored programs; personal and social development opportunities; and specialised skills and learning opportunities. Outcomes varied in accordance to program aims and included improvements in aspects of education and employment, offending behaviours, relationships, and personal, social and professional development.Little research explored the effectiveness of mentoring, captured its impact qualitatively or quantitatively, developed appropriate measures or assessed its cost-effectiveness. There is a real need to evaluate programs particularly in terms of outcomes and, given there were no economic evaluations, costs. Commitments to improving Indigenous Australian mentoring rely on changes to funding structures and attitudes toward research. There was insufficient evidence to confidently prescribe a best practice model. Sufficient frequency of qualitative reporting between publications concluded that mentoring is a valuable empowerment strategy in the areas of health and wellbeing, education and employment and as a remedial and preventative measure in reducing offending behaviours. An evidence-informed mentoring model would take into account the key findings of the review. Nine (60%) publications were concentrated on youth mentoring.Most publications were centred on mentoring programs that aimed to improve education and employment outcomes for Indigenous Australians (11/15 or 80%). This focus was in sharp contrast to publications (3/15 or 20%) described as program innovations to address specific risk areas or problem behaviours, for example, changing offending behaviours for ‘at-risk’ youth ; young oThe facilitating environments, in which mentoring relationships developed, incorporated the operational elements of mentoring practice. Varied participant numbers were found across publications reporting figures in mentoring programs. They ranged from smaller programs with 10 mentees , 13 mentAll publications reported planned and systematic organisation of mentoring practices; nevertheless, in 9/15 or 60% publications voluntarily participation of mentees in programs were reported; the nature of participation for mentees was not mentioned in all other cases. The skills, qualities and competencies required by mentors to fulfil their commitments to Indigenous Australian mentees were taken into account. Eight/15 (53.3%) publications described having mentor training , 33, 37;The matching of mentors to mentees either in terms of mutual connections , gender Five/15 33.3%) publications explicitly demonstrated a reconciliatory approach other than partaking in cultural awareness training. For example, in the case of implementing a health promotion program, Paase and Adams  clearly .3% publiResourcing for mentoring programs reported (13/15 or 86.6%) was weighted in favour of government-derived funding with 7/13 or 53.8% , 33, 37 Mentoring strategies are the mechanisms that underlie mentoring relationships. While publications contributing to the review described diverse and sometimes poorly delineated processes in mentoring programs, four common program elements were evident in the mentoring models. These strategies were: 1) developing respectful, trusting, one-on-one mentoring relationships; 2) promoting personal and social development; 3) integrating social services, organisations and other support systems including communities and families; and 4) increasing specialised skills.Developing respectful, trusting, one-on-one mentoring relationships was reported in 13/15 or 86.6% of cases [of cases , 31–37. of cases  used groof cases  used peeof cases –36 and Nof cases  a minimuof cases  one day of cases  reportedPromoting personal and social development featured as a common and necessarily important strategy occurring in programs and mentor-mentee interactions. This saturation occurred with the exception of Nasir [of Nasir . Nasir cintegrated mentoring models - implementing a circle of care and support around mentees to support personal and social development. The three cases in which outside support was not integrated were all workplace mentoring programs [Most publications, 12/15 (80%) described programs , 30, 32:programs  demonstrincreasing specialised skills for mentees, and to a lesser extent building mentor skills. Strategies varied according to the nature of the mentoring program  and the existing skill sets within that cohort. Matching appropriate skills and development to the program aims were crucial for the overall success of programs and was resourced as much as possible through the mentors. For example, the AIME program [All publications focussed to varying degrees on  program –36 had aIncreasing specialised skills for mentees and mentors also involved accessing the cadre of organisations, services and/or other human resources that were integrated into the programs to support skill development. Targeted skills sets for mentees focussed on learning opportunities including literacy and numeracy; career development skills such as resume writing and work ethic and work experience; areas in the achievement of mentee goals such as education in the political system; and vocationally-oriented curriculum . For mentors, increased skills most often took the form of cultural competency training and professional development through mentoring courses.Not all publications recounted the extent to which programs were effective. Of the nine that did, the rationales for program development intimated that a range of outcomes was being targeted. To a large extent, outcomes were matched to program aims. At times however, consequences of programs were unexpected. Most outcomes were qualitatively investigated and described, with only three reports quantitatively measuring their achievements –36. AIMELikewise the Tribal Warriors program  for postIn school environments, MacCallum, Beltman & Palmer (2005)  found thb however, these can provide valuable evidence and lessons for developing the field further. The slow progression of Indigenous Australian mentoring research is consistent with the findings of others in the broader field of mentoring. For instance, Crisp & Cruz [The findings show that literature around Indigenous Australian mentoring has an emergent lifespan of 14 years. They revealed that while outputs have increased, the volume of original research publications has not increased across time. All original research outputs were qualitatively-oriented. The methodological adequacy of the identified original research publications was strong; however, only one was peer-reviewed. There was no pattern in the volume of original research publications; these were randomly dispersed in number and nature across a 10 year lifespan from 1999 – 2009. This is not surprising given that publication outputs were heavily weighted in favour of descriptive documents: fourteen/15 publications were labelled descriptive and only one as intervention research. Specifically, such an output record means that Indigenous Australian mentoring research is still in its early exploratory phase and little research has explored the effectiveness of mentoring in its various forms, captured its impact qualitatively or quantitatively, developed appropriate measures or assessed it cost-effectiveness. This pattern is consistent with the results of several other reviews across a number of Indigenous-specific areas, including health, program transfer and implementation, sexual assault, cultural competency, child and maternal health and suicide prevention . While qp & Cruz  noted thThe main characteristics of Indigenous Australian mentoring were summarised in Figure This review included a range of mentoring initiatives aimed at increasing Indigenous Australian empowerment, engagement and social inclusion across a number of different developmental areas; particularly in education (school-based initiatives), employment (workplace initiatives) and broader health and wellbeing. Initiatives were embedded in ecological models of resilience models that worked from the premise of providing opportunities for building on existing strengths for mentees, mentors, families and communities, and service providers. In individual programs, mentee numbers and mentor-mentee ratios varied and appeared irrelevant; that is, outcomes were equally recorded against programs regardless of the participant intake or mentees allocated to each mentor. All publications predominately described a clear orientation toward formal mentoring processes, meaning that while participation for mentors and mentees was voluntary and there was internal flexibility, there were certain non-negotiable aspects and formal organisation of programs. Generally however, there were a number of flexible elements that contributed to their success. Thus developed were innovative integrated models of mentoring programs with wrap-around social services, communities and families and other support systems. These models had a variety of educational and life skill-related supports and in some instances clinical support e.g. psychologists. Consequently, these models link mentees and mentors, and also connect many individuals and resources in the mentors’ lives to the networks in the lives of the mentees. Since both mentees and mentors are connected to the program staff and service providers this approach has important implications for providing circles of holistic care needed to help programs deliver more targeted mentoring. To engage in this type of social mapping, it was evidenced that mentoring programs need to have a clear understanding of the environment and socio-cultural and economic milieu in which potential participants operate as well as clearly defined aims and outcomes to make a good match with strategies. How these criteria manifest, informs the type of program and its operational strategies.In order to enhance the benefits of mentoring, a focus on mentees’ support and development was matched with training and development of mentors. Mentor competency, particularly training in mentorship and cultural awareness were evidenced as critical prerequisites for successful program implementation. Given the long-term nature of successful mentoring practices, sustained funding was critically noted as vital to maximising the benefits of mentoring.Mentoring strategies were manifest in many different forms. As previously noted, mentoring was often reported as one strategy of a more extensive support/development program. Programs were culturally-tailored to meet mentee needs, both through implementing the integrated model approach  and acknowledging Indigeneity by ensuring cross-cultural training was provided to mentors. But support for Indigenous Australians through mentoring, like most other Indigenous programs, has often used short-term approaches. This approach is contrary to the evidence found in this review that longer-term investment is needed. The call for longer-term mentoring is made in international mentoring literature. Indeed, this evidence that suggests inadequate implementation and short-term programs has potentially harmful effects on mentees , 45. ThiPersonal and social development of the mentee was supported by a holistic scaffolding approach in which specialised skills and learning opportunities were provided. These development opportunities also extended to mentors. Mentors were provided with training in mentorship to ensure they had knowledge of and exhibited the most engaging mentor characteristics.The ways mentoring was used to support Indigenous Australians looked very different across program aims, and therefore a diverse number of outcomes were cited. Significant outcomes were realised in the largest proportion of mentoring publications, with clear benefits to both mentees and mentors, and others: with the exception of AIME reports –36, thesA rigorous search strategy was used and care taken to canvass a broad spectrum of multi-disciplinary peer-reviewed and grey literature. However, there are always possibilities of oversight when searching publications and thus potential that all relevant studies were not located. For instance, the omission of the analogous search terms might have meant that we missed relevant mentoring studies subsumed under different terminology; and search facilities in individual databases differed. Relevant publications might have been misclassified or excluded. Nevertheless, elevated levels of agreement between blinded coders suggest relevant publications have been classified or excluded with high level accuracy. Timeframes canvassed began from the emergence of the earliest mentoring literature and thus allowed for the optimal extraction of patterns of publication quantities and methodological changes. A careful hand-searching of included publications was implemented to mine for relevant data on mentoring characteristics. However, the human component of implementation means it is possible some elements were not located.The primary outcome of the review indicates that there is currently insufficient evidence from published documents to confidently prescribe a best practice model for Indigenous Australian mentoring. Nevertheless, there was sufficient frequency of qualitative reporting between the authors of publications to conclude that mentoring is potentially a valuable empowerment strategy, particularly in the areas of health and wellbeing, education and employment and as a remedial and preventative measure in reducing offending behaviours.There is a lack of evidence from peer-reviewed publications or in the grey literature on the most effective strategies for mentoring Indigenous Australians. The only measurable outcomes for effective mentoring practices lies in mentoring programs for Indigenous Australian secondary students –36. ImpoMethodologically, the review showed a predominance of descriptive publications in the field of Indigenous Australian mentoring across time. Descriptive research does not provide quality improvement in programs, nor does it provide evidence of how to improve programs or whether those programs were effective. There must be change in the patterns of research if the field is to advance. There is a real need to evaluate programs particularly in terms of outcomes and, given there were no economic evaluations, costs. Commitments to improving funding structures and attitudes toward research must change to improve mentoring outcomes across a range of social determinants of health for Indigenous Australians. While this review undertook to study mentoring for Indigenous Australians only, it is possible for the findings to be extrapolated to other similar structures, settings and populations.aEmpowerment is defined in this paper as: “a social action process by which individuals, communities, and organizations gain mastery over their lives in the context of changing their social and political environment to improve equity and quality of life” [of life” .bIt should be noted that the three AIME [ree AIME –36 repor


PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca2+]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA . Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca Uremia is characterized by the retention of several metabolites (collectively termed here as uremic solutes or uremic toxins). Based on the ability of these solutes to undergo clearance with dialysis, they are classified into three groups: small water-soluble molecules, middle molecules, and protein-bound solutes . IndoxylRecent studies have revealed that indolic solutes (IS and IAA) may enhance thrombosis through induce tissue factor (TF) in both endothelial cells (ECs) and vascular smooth muscle cells (vSMCs) ,4. Howevet al., RBC may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with PS exposure at the RBC surface ) using flow cytometry and results are represented by Ca2+ dependent fluorescence intensity. As illustrated in 2+] after 24 h of incubation. Furthermore, there was a significant increase of [Ca2+] in maximal levels of IS (1 mM) and IAA (50 μM) than that in median uremic concentration, respectively (p < 0.001).To explore the mechanism responsible for the PS exposure of RBCs in indolic uremic solutes, further experiments were performed to test cytosolic Ca2+], erythrocytes were exposed to IS (0.1 mM) and IAA (20 μM) for 24 h either in the presence of extracellular Ca2+ (1 mM) or in the nominal absence of Ca2+ and presence of the Ca2+ chelator EGTA (1 mM). As shown in 2+.In order to verify whether the PS exposure on erythrocyte by indolic uremic solutes was secondary to an increase of ) [2+]. Our results showed that cytosolic [Ca2+] was increased significantly in indolic uremic solutes and associated with increased PS exposure and RMPs release. Upon the increase of cytosolic [Ca2+], scramblase presumably mediate PS translocation to the erythrocyte surface with membrane blebbing and MPs shedding [2+ significantly blunted the effect of IS and IAA on lactadherin binding. Our study shows that the increased cytosolic [Ca2+] might contribute to IS and IAA-induced PS exposure and MPs generation in erythrocyte. The underlying molecular mechanism will be required to define our future studies.As eryptosis is triggered by the increase of cytosolic Ca([Ca2+]) ,26,27, fshedding ,29. RemoLactadherin, an effective PS-stained reagent was used as a novel probe to quantify and localize PS exposure on RBC. Moreover, lactadherin acts as an effective anticoagulant for inhibiting PCA of IS- and IAA-cultured RBC by blocked PS, which may prevent indolic uremic solutes induced thrombophilia.2+]. Alternatively, it is necessary to further study the possibility of applying lactadherin in thrombotic complications of uremia.In conclusion, we provide new insights into the roles of RBCs in the development of IS and IAA-related hypercoagulable states. We demonstrated that indolic uremic solutes increased PCA of RBC by inducing PS exposure and RMPs release, and the effect is at least partially due to increased cytosolic ), each group of RBCs were washed in Ringer solution and then loaded with 2 μM Fluo 3/AM for 1 h at 37 °C in the dark. Subsequently, RBCs were washed twice and then resuspended in 200 μL Ringer solution to a final concentration of 1×106/mL. Ca2+ dependent fluorescence intensity was measured and analyzed with BD FACS Diva Software.For detection of cytosolic Ca8) or MPs-containing suspension (prepared from 10 mL of the RBCs supernatants) was incubated with 100 μL of MDP at 37 °C. After 180 seconds, 100 μL of warmed 25 mM CaCl2 was added to start the reaction and the clotting time was recorded. All clotting assays were performed in triplicate. For the inhibition assay of coagulation time, 100 μL of RBCs or MPs suspensions were preincubated with 50 μL lactadherin  for 10 min at 37 °C. Clotting time was then recorded as above after addition of 100 μL MDP and 50 μL of warmed 50 mM CaCl2.Procoagulant activity (PCA) of RBCs and MPs was evaluated by one-stage recalcification time assay in a KC4A-coagulometer  . 100 μL 5 RBCs or 100 μL of RMPs-enriched suspension was incubated with 1 nM FIXa, 130 nM FX, 5 nM FVIII, 0.2 nM thrombin and 5 mM Ca2+ in FXa buffer  for 5 min at 25 °C. The reaction was stopped by addition of EDTA at a final concentration of 7 mM. After the addition of 10 μL S-2765 (0.8 mM) to each reaction, the quantity of FXa formed was determined immediately at 405 nm in kinetic mode on a Universal Microplate Spectrophotometer . Results were evaluated against the rate of substrate cleavage of a standard dilution of FXa. For the prothrombinase assay, RBCs were incubated with 1 nM FVa, 0.05 nM FXa, 1 μM prothrombin and 5 mM Ca2+ in prothrombinase buffer  for 5 min at 25 °C. EDTA and S-2238 were added to each microplate, and thrombin production was assessed as previously described [For intrinsic factor Xa assay, a total of 10escribed . Inhibitp < 0.05 was considered statistically significant.Data are expressed as mean ± standard deviation (SD), and statistical analysis was made by Wilcoxon or Kruskal-Wallis test as appropriate.


At low to mid sound frequencies cell depolarization follows the sound sinusoid and pulses of transmitter release from the hair cell generate excitatory postsynaptic currents (EPSCs) in the afferent fiber that translate into a phase-locked pattern of action potential activity. The present article summarizes our current understanding on the elementary properties of single IHC Ca2+ channels, and how these could have functional implications for certain, poorly understood, features of synaptic transmission at auditory hair cell ribbon synapses.Voltage-gated calcium (Ca The mammalian auditory system has evolved specialized structures and cellular mechanisms that allow sound information to be relayed to the brain with unparalleled temporal precision. Our ability to localize sound sources in the environment depends on the preservation of timing accuracy along the auditory pathway. Low-frequency sounds are localized by specialized cells in the brainstem that compare the temporal delay between the phase-locked activity originating from the two ears, which can be as small as ten microseconds Köppl, . The loc+ ions into the cell. The interplay of the transducer current with the cells basolateral membrane currents generates the characteristic IHC receptor potential  activates a proportion of voltage-gated Ca2+ channels that is thought to drive the tonic release of glutamate onto afferent terminals , the primary sensory receptors of the cochlea, whereby sound-induced deflection of the hair bundle gates mechano-electrical transducer (MET) channels allowing the depolarizing flow of K2+ influx and increasingly faster vesicular fusion  of the low-to-mid-frequency sinusoidal sound wave independent of its intensity  using non-stationary fluctuation analysis of macroscopic (whole-cell) Ca2+ tail currents  to estimate the channel number, single channel current and open probability (Po). The latter is a direct measure of elementary Ca2+ current (iCa) size and Po that is used to work out the number of channels from the size of the macroscopic Ca2+ current (ICa). The number of Ca2+ channels per IHC estimated from fluctuation analysis is about 1, 800  depolarizing voltage steps, in order to collect a large number of openings for reliable analysis. However, Ca2+ channels clearly underwent inactivation during these long pulses, as indicated by the shape of the ensemble-average currents  increased from 0.024 to 0.06 in middle-turn IHCs  is approximately 23 nm from the Ca2+ channel. Because of the high affinity of the Ca2+ sensor  amplitude until the onset of hearing at around post-natal day 12, whereupon the release of synaptic vesicles becomes linearly dependent on ICa amplitude, as described above , or that an even looser spatial relation exists between them and voltage-gated Ca2+ channels. Until this is not demonstrated, such interpretation can be regarded as an interesting theoretical possibility. Morphologically, maturation-dependent linearization of vesicular release on ICa amplitude is accompanied by a change in the IHC active zone/postsynaptic density complexes that are initially multiple, small and spot-like and then become large single structures. There is also a topographical re-arrangement of Ca2+ channels, which matches that of the active zones: Ca2+ channels are initially located in several smaller, round clusters and then form a larger stripe-like cluster , the fraction of channel openings long enough to saturate the Ca2+ sensor will be dominant over the fraction of shorter, non-saturating, openings. For instance, if a channel open duration equals to one tenth of τ  to large multi- or monophasic events seemingly resulting from the fusion of up to 20 vesicles  the ribbon itself, or proteins associated with the ribbon, facilitate the coordinated fusion of multiple vesicles docked at the active zone  negligible. Early studies on CaV1.2 channels also showed different modes of gating , with the few channels opening in bursts (mode 2) underlying the majority of the macroscopic current recorded in whole-cell.2+ channels enter mode 2, accounting for the increase in the macroscopic current.We also found that, with depolarization, single Ca2+ entry occurs during the burst opening of Ca2+ channels would have important implications as far as signal transmission at IHC afferent synapses is concerned. As discussed above, various lines of evidence suggest that IHC Ca2+ channels and release sites are within the spatial range of nanodomains, such that the opening of one channel might be sufficient to saturate the Ca2+ sensor and trigger vesicle release. One could hypothesize that, under these conditions, the most effective modality for Ca2+ channel opening in terms of vesicle release is burst opening. However, it is unlikely that the prolonged entry of Ca2+ during a burst is the necessary factor for vesicle release, since depolarizations of only a few hundred microseconds must be sufficient to trigger a release event during mid-frequency sound stimulation (see above).The likelihood that the majority of Ca2+ channels are associated with a release-ready vesicle , with and average Po across all 5 channel of 0.2  4·(1–0.8) = 0.16. In this example, a single bursting channel, even when the group of channels shows the same average Po as the non-bursting channels, would increase the probability of at least one Ca2+ channel being open at each active zone, from 0.67 to 0.84, which would increase the overall reliability of vesicle release.It is more likely that a predominance of burst openings would improve the reliability of vesicle release. Let us assume, for example, that five Ca2 Figure . This av) Figure . In the 2+ microdomain or nanodomain control of IHC exocytosis. In a microdomain, Ca2+ influx through several Ca2+ channels at a presynaptic site would resemble the macroscopic ICa. Since ICa amplitude and activation speed increase with depolarization (Figure 2+ channel opening decreases significantly with depolarization (Figure 2+-dependent exocytosis and modify the phase of the response, interfering with phase-locking to that particular sound frequency.Afferent fibers innervating apical- and middle-turn cochlear IHCs show phase-locked spiking activity to sound frequencies up to a few kHz, independent of stimulus intensity Figure . The indn Figure , it is en Figure . Therefo2+ channel facilitation that occurs at voltages close to the resting membrane potential is compensated by synaptic vesicle depletion during prolonged trains of activity, producing a constant synaptic delay despite varying stimulus intensity (Goutman, A mechanism by which the accuracy of afferent phase-locking could be preserved independent of sound intensity has been proposed, involving the balance between short-term facilitation and depression of transmitter release at the hair cell ribbon synapse (Cho et al., Goutman, . HoweverGoutman, . This mo2+ channel currents could underlie the preservation of phase-locked transmission in a nanodomain in two possible ways. In the first scenario, as the IHC depolarizes, the amplitude of iCa decreases due to the reduction in driving force for Ca2+ entry (Figure 2+ sensor saturation for exocytosis may be comparable at different stimulus levels. This mechanism would allow the preservation of a constant phase relationship to changing sound intensity (Figure iCa. During the repolarizing phase of a rapid cyclic stimulus, the large increase in driving force for Ca2+ would increase the amplitude of the current flowing through an already open Ca2+ channel. If repolarization is sufficiently fast, the amplitude of the elementary Ca2+ current would quickly rise to levels high enough to promptly saturate the Ca2+ sensor. These “saturation events” will cluster in a limited time window during the repolarizing phase, regardless of the amplitude of the foregoing depolarization, as long as it has opened the Ca2+ channel. During the macroscopic Ca2+ “tail” currents elicited by fast repolarization, the amplitude of elementary currents would be maximized with minimal jitter (Figure 2+ current development occurs during the early repolarizing phase of the cycle (Figure 5 in Li et al., 2+ currents have been shown to elicit time-locked M-EPSCs (Goutman, 2+ tail current upon repolarization rather than to the peak ICa. In the amphibian papilla, in which each afferent fiber receives input from several synaptic ribbons, the faster, less variable tail Ca2+ currents would seem better suited for triggering synchronous vesicle fusion than the stochastic opening of Ca2+ channels at different presynaptic sites. Therefore, in this second scenario, although IHC depolarization is necessary to open Ca2+ channels, subsequent IHC repolarization appears optimally suited for synchronizing post-synaptic activity with sound frequencies around the phase-locking limit.The properties of single Cay Figure . This woy Figure . In a ser Figure . Their dr Figure . Recent r Figure , even whr Figure  is takenGoutman, . FinallyGoutman,  and frogGoutman,  appear b2+ channels underlie several as yet unexplained features of the afferent response. The sub-ms first latency would ensure that at least some Ca2+ channels open with very short delay, allowing even relatively high frequency stimuli to be followed. The bursting behavior of Ca2+ channels, on the other hand, would increase the reliability of signal transmission. Finally, in a nanodomain where vesicle fusion is controlled by a single Ca2+ channel, the elementary current amplitude and opening latency could balance each other to produce constant phase-locking of the afferent response despite variations in sound intensity. The elementary tail currents would provide a rapid and large Ca2+ influx at the highest sound frequencies possible.In conclusion, it is possible that the elementary properties of IHC CaJM, PS, SLJ, and SM substantially contributed to the conception or design of the work, analysis and/or interpretation of data, drafting of the text and /or figures, revision, final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


During malaria, accelerated death of infected erythrocytes may decrease parasitemia and thus favorably influence the clinical course of the disease. In the present study, phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. A 48 h treatment of human erythrocytes with nelfinavir significantly increased the percentage of annexin-V-binding cells (≥5µg/mL), significantly decreased forward scatter (≥2.5µg/mL), significantly increased ROS abundance (10 µg/mL), and significantly increased [Ca2+]i (≥5 µg/mL). The up-regulation of annexin-V-binding following nelfinavir treatment was significantly blunted, but not abolished by either addition of the antioxidant N-acetylcysteine (1 mM) or removal of extracellular Ca2+. In conclusion, exposure of erythrocytes to nelfinavir induces oxidative stress and Ca2+ entry, thus leading to suicidal erythrocyte death characterized by erythrocyte shrinkage and erythrocyte membrane scrambling.The HIV protease inhibitor, nelfinavir, primarily used for the treatment of HIV infections, has later been shown to be effective in various infectious diseases including malaria. Nelfinavir may trigger mitochondria-independent cell death. Erythrocytes may undergo eryptosis, a mitochondria-independent suicidal cell death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress and increase of cytosolic Ca Nelfinavir, a specific HIV protease inhibitor, has originally been developed for the treatment of HIV infections and subsequently been shown to be effective in further infectious diseases including SARS, tuberculosis, and malaria ,2,3,4,5.2+ activity ([Ca2+]i), ceramide i determined utilizing flow cytometry.The present study explored, whether and how nelfinavir stimulates eryptosis. To this end, erythrocytes from healthy volunteers were exposed to nelfinavir and phosphatidylserine abundance at the erythrocyte surface, cell volume, abundance of reactive oxidant species and i). [Ca2+]i was quantified utilizing Fluo3 fluorescence. As shown in 2+. To this end, erythrocytes were incubated for 48 h in the absence or presence of 10 µg/mL nelfinavir, both in the presence or nominal absence of extracellular Ca2+. As shown in 2+ significantly blunted the effect of nelfinavir on annexin-V-binding, an observation pointing to a role of Ca2+ entry from extracellular space in the stimulation of cell membrane scrambling by nelfinavir. However, even in the absence of extracellular Ca2+, nelfinavir significantly increased the percentage of annexin-V-binding erythrocytes. Thus, eryptosis was in part triggered by mechanisms other than entry of extracellular Ca2+.Oxidative stress is known to activate Cai.e., the triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and erythrocyte cell membrane scrambling with phosphatidylserine translocation from the cell interior to the erythrocyte surface. The nelfinavir concentration required for stimulation of erythrocyte cell membrane scrambling (5 µg/mL) was similar to plasma concentrations (10 µg/mL) reported in vivo i), which leads to cell shrinkage by activation of Ca2+ sensitive K+ channels, K+ exit, cell membrane hyperpolarization, Cl− exit and thus cellular loss of KCl with osmotically obliged water [The nelfinavir induced erythrocyte shrinkage was presumably secondary to increase of cytosolic Caed water .2+ entry from extracellular space leading to increase of [Ca2+]i, a powerful stimulator of cell membrane scrambling with phosphatidylserine translocation i was presumably in part the result of oxidative stress, which activates oxidant sensitive Ca2+ permeable erythrocytic cation channels [The effect of nelfinavir on [Cachannels . Beyond channels ,56,57,58Plasmodium imposes oxidative stress on the host erythrocyte leading to activation of several host cell ion channels including Ca2+-permeable erythrocyte cation channels [2+ entry triggers cell membrane scrambling, phosphatidylserine translocation, binding to phosphatidylserine receptors at phagocytes, phagocytosis and thus removal of the infected erythrocytes from circulating blood [Eryptosis serves to clear defective erythrocytes prior to hemolysis with release of hemoglobin, which may undergo glomerular filtration in the kidney and precipitate in the acidic lumen of renal tubules thus occluding affected nephrons . Eryptoschannels ,60. The ng blood . Eryptotng blood ,61,62,63ng blood , lead inng blood , treatmeng blood  or preseng blood . It is tng blood , they arAs phosphatidylserine exposing erythrocytes are engulfed by macrophages and thus rapidly cleared from circulating blood, excessive eryptosis may lead to anemia . Moreovein vitro for 48 h at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, and 1 CaCl2; the pH was adjusted to 7.4 and the temperature kept at 37 °C. Where indicated, erythrocytes were exposed to nelfinavir , which was dissolved in DMSO . For comparison, the effect of 1 µL DMSO/mL Ringer was tested.Fresh Lithium-Heparin-anticoagulated blood samples were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003 V). The blood was centrifuged at 120 g for 20 min at 23 °C and the platelets and leukocytes-containing supernatant was disposed. Erythrocytes were incubated 2 and then stained with Annexin-V-FITC  in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur . In some experiments erythrocytes were preincubated in Ca2+ free solution. For determination of annexin-V-binding, addition of Ca2+ was required during the 15 min incubation with FITC-annexin V. Immediately thereafter measurements were done so that the exposure to Ca2+ was too short to trigger significant phosphatidylserine translocation.After incubation under the respective experimental condition, a 150 µL cell suspension was washed in Ringer solution containing 5 mM CaClOxidative stress was determined utilizing 2',7'-dichlorodihydrofluorescein diacetate (DCFDA). After incubation, a 150 µL suspension of erythrocytes was washed in Ringer solution and then stained with DCFDA  in Ringer solution containing DCFDA at a final concentration of 10 µM. Erythrocytes were incubated at 37 °C for 30 min in the dark and then washed three times in Ringer solution. The DCFDA-loaded erythrocytes were resuspended in 200 µL Ringer solution, and ROS-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD).2 and 5 µM Fluo-3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µL Ringer. Then, Ca2+-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur.After incubation, a 150 µL cell suspension was washed in Ringer solution and then loaded with Fluo-3/AM  in Ringer solution containing 5 mM CaClFor the determination of hemolysis, the samples were centrifuged  after incubation under the respective experimental conditions and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis. Hemolysis is expressed in % of total hemolysis.t test as appropriate. n denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and 2+ activity.Nelfinavir stimulates eryptosis, the suicidal erythrocyte death characterized by erythrocyte cell membrane scrambling and cell shrinkage. The effect is paralleled by, and at least partially due to, oxidative stress and increase of cytosolic Ca


However, the cellular mechanisms of the effects of chronic clenbuterol administration on skeletal muscle are not completely understood. As the intracellular Ca2+ concentration must be finely regulated in many cellular processes, the aim of this study was to investigate the effects of chronic clenbuterol treatment on force, fatigue, intracellular calcium (Ca2+) homeostasis and Ca2+-dependent proteolysis in fast-twitch skeletal muscles , as they are more sensitive to clenbuterol-induced hypertrophy. Male Wistar rats were chronically treated with 4 mg.kg−1 clenbuterol or saline vehicle (controls) for 21 days. Confocal microscopy was used to evaluate sarcoplasmic reticulum Ca2+ load, Ca2+ -transient amplitude and Ca2+ spark properties. EDL muscles from clenbuterol-treated animals displayed hypertrophy, a shift from slow to fast fiber type profile and increased absolute force, while the relative force remained unchanged and resistance to fatigue decreased compared to control muscles from rats treated with saline vehicle. Compared to control animals, clenbuterol treatment decreased Ca2+-transient amplitude, Ca2+ spark amplitude and frequency and the sarcoplasmic reticulum Ca2+ load was markedly reduced. Conversely, calpain activity was increased by clenbuterol chronic treatment. These results indicate that chronic treatment with clenbuterol impairs Ca2+ homeostasis and this could contribute to the remodeling and functional impairment of fast-twitch skeletal muscle.Clenbuterol, a β Like other β–adrenoceptor agonists, clenbuterol can induce skeletal muscle hypertrophy, by stimulating protein synthesis and inhibiting proteolysis Clenbuterol is a βClenbuterol effects on skeletal muscle seem to be fiber-type specific. Indeed, clenbuterol treatment causes hypertrophy only of fast skeletal muscle fibers 2+ concentration is implicated in many cellular processes, such as excitation-contraction coupling, cell survival, energetic metabolism and gene transcription signaling 2+ transient, which is mainly elicited by the Ca2+ release from Ryanodine receptors (RyR) and is strongly influenced by sarcoplasmic reticulum (SR) Ca2+ load, is a major determinant of skeletal muscle functional properties. Acute administration of clenbuterol on skinned skeletal muscle fibers has a direct effect on sarcoplasmic reticulum (SR) Ca2+ release 2+ content and the Ca2+-transient amplitude 2+) homeostasis in skeletal muscle are lacking. Therefore, in this study, we assessed the effect on muscle Ca2+ homeostasis of chronic treatment (21 days) with clenbuterol in male Wistar rats. Real time laser scanning confocal microscopy was used to investigate Ca2+-transient amplitude, Ca2+ spark spatio-temporal properties, which reflect RyR functional properties, and SR Ca2+ load in EDL muscles. In addition, we also evaluated the effects of clenbuterol treatment on Ca2+-activated proteolysis because it could be altered by Ca2+ homeostasis disturbances. EDL muscles have a fast-glycolytic phenotype and were chosen because they are less affected by myonecrosis and they are more sensitive to clenbuterol-induced hypertrophy 2+ transients in rat slow-twitch soleus muscle have been already described The cellular mechanisms explaining the effects of chronic β–adrenoceptor agonist administration on skeletal muscle are not completely understood. Regulation of intracellular Ca−1.day−1 diluted in saline solution) or of an identical volume of saline vehicle (CTR). Rats were sacrificed with a lethal dose of pentobarbital sodium 24 h after the last injection.This study was approved by the Ethics Committee on Animal Experimentation of the Languedoc Roussillon Region, France, in accordance with the guidelines from the French National Research Council for the Care and Use of Laboratory Animals (Permit Number: CEEA-LR-1069). Surgery was performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering. Twenty-two 6-weeks/old Wistar male rats (body mass: 236.3±3.8 g) were randomly allocated to the control  or clenbuterol-treated  group. They were housed in standard cages with a 12 h∶12 h light-dark cycle and provided with food and water ad libitum. Every day at 9 am for 21 days, rats were weighted and received a subcutaneous injection of clenbuterol  equilibrated with 95% O2–5% CO2 gas and maintained at 25°C. The pH was set to 7.4.These conditions are optimal for maintaining muscle viability in vitro for the duration of the experimental protocol.For muscle contraction studies and Cag for 10 min, 4°C). Supernatants were stored at −80°C. The protein content of supernatants was determined using the bicinchoninic acid (BCA) protein assay .For calpain activity measurements, EDL muscles were homogenized in ice-cold buffer  and then centrifuged . Supernatants were stored at −80°C.For western blotting, muscles were homogenized in ice-cold lysis buffer  and then centrifuged (12 0002.Muscle phenotype was determined on cryosections (6 µm) by myosin ATPase histochemical staining as previously described in vitro according to the methods described in detail previously 0), i.e. the muscle length producing maximal twitch tension, was determined. All subsequent measurements were made at L0. The tension-frequency response was then determined  using stimulation trains of 500-ms, with pulse duration of 0.5 ms at frequencies of 1–100 hz. Stimulus trains were separated by one-min interval. The maximum isometric tetanic tension (P0) was then determined. Three minutes after the tension-frequency determination, the resistance to fatigue was evaluated using a low-frequency fatigue protocol of 50-hz trains of 700 ms delivered every 2 s for 5 min lim) was defined as the time taken to produce a 50% reduction from the initial maximum power output. After all measurements, muscles were removed from the bath, trimmed of the connective tissue, blotted dry and weighed on an analytical scale. The specific force  was calculated by dividing the tension (P0) by the whole-muscle CSA. The whole-muscle CSA was determined by dividing the muscle volume  by the average fiber length (Lf). f was determined by multiplying L0 by 0.44, which is the previously determined fiber length to muscle length ratio for the EDL muscle EDL muscle fiber contractile properties were assessed 2+, in Krebs-Ringer solution for 1 h. Muscles were then mounted under stretch  to suppress movement artifacts 2+ transients were triggered by field stimulation with a single 2 ms pulse just above the stimulation threshold for visible fiber contraction under a microscope. Fluorescence images were acquired in line-scan mode  with a Zeiss  LSM 510 NLO confocal system , as previously reported 2+-transients were identified on raw fluorescence microscopy images. Image strips of the manually identified area of a single fiber were extracted. After subtracting the photomultiplier tube (PMT) offset current, strip images were converted to ΔF/F, where F is the fiber mean resting fluorescence calculated from the image area preceding the action-potential stimulation and the subsequent Ca2+-transient. The temporal fluorescence time courses of each fiber were generated by spatial averaging of the ΔF/F images, resulting in the mean spatial F value for each temporal coordinate.Intact EDL muscles were loaded with 50 µM Rhod-2 AM , a fluorescent probe for intracellular Ca2+-release events  were measured as previously reported 2ATP, 4.53 mM MgCl2, 1 mM EGTA, 0.29 mM CaCl2, and 2 mM malate, pH 7.0). Single fibers were manually dissected under a dissecting microscope , mounted in an experimental chamber containing the internal-like buffer, and permeabilized by adding 0.01% saponin at room temperature for 30 s. Fibers were slightly stretched to the sarcomere length (approximately 2.8–3.2 µm) to reduce movement artifacts. Fibers were bathed in internal-like buffer containing 50 µM of Fluo-3 , a fluorescent Ca2+ indicator. Fluorescence images were acquired with the confocal system operated in line-scan mode . Fluo-3 was excited with an argon/krypton laser at 488 nm and fluorescence was recorded at 525 nm. Image acquisition was performed along the fiber longitudinal axis. Potential spark areas were empirically identified using an auto-detection algorithm 2+-spark activity corresponding to brief transients (<100 ms) of localized fluorescence. Determinations of the spatio-temporal properties of individual Ca2+-sparks were made on spatial (x) and temporal (t) profiles of sparks centered at the peak amplitude. Parameters derived from the ΔF/F temporal profile include its amplitude, the rise time and the time constant for the decay rate given by the time-to-half decay (Tlim). The width of the Ca2+-spark  was determined from the spatial profile. The time course and spatial width of the fluorescence were then assessed and the following criteria were then used to classify a perturbation of ΔF as a Ca2+-spark: ΔF/F amplitude >0.2, half duration >6 ms and <50 ms, and half width >0.7 µm and <4 µm.Local Ca2+ content was evaluated as previously described 2+-spark measurement, including permeabilization with saponin. Ca2+ release was elicited by adding to the 250 µl of internal-like buffer (containing the fluorescent Ca2+ indicator Fluo-3) in which the fibers were bathed another 250 µl of the same solution with 100 µM of 4-chloro-m-cresol (4-CmC). Ca2+-transient was imaged using fast time series of x–y confocal images . Fields of one or two fibers were imaged at the acquisition rate of one image every 400 ms. Fluo-3 was excited with an argon/krypton laser at 488 nm, and fluorescence was recorded at 525 nm. The mean fluorescence was calculated in regions of interest and reported as a function of time. The peak of the ΔF/F signal in response to 4-CmC exposure was used to assess SR Ca2+ content, as previously described The SR CaN-succinyl-Leu-Tyr-7-amido-4-methylcoumarin, SLY-AMC), as previously described 2, 1 mM Dithiothreitol (DTT) 1, 0.15 nM Pefabloc and 5.8 nM Pepstatin A) for 10 min. After addition of 5 µl of 25 mM SLY-AMC in dimethyl sulfoxide, buffer was added to adjust the volume to 1 ml to obtain a SLY-AMC final concentration of 125 µM. The fluorescence signal of the released AMC was monitored using a Perkin Elmer spectrofluorimeter at 37°C for 15 min . Control assays were performed without CaCl2 and in the presence of 10 mM EDTA and 10 mM EGTA. Calpain activity was determined as the Ca2+-dependent cleavage of SLY-AMC and was expressed as arbitrary units per minute of incubation per milligram of muscle protein.Calpain activity was determined in whole EDL muscle homogenates  using a calpain-specific fluorogenic substrate  and polyclonal anti-calpain 2  or saline vehicle (CTL) for 21 days and then the EDL muscles were harvested for morphological and functional investigations. As previously described To investigate the effect on muscle Ca0 was markedly increased after 21 days of CBL treatment (+87.5±2.3%) compared to CTL, whereas the sP0 values , were not different between groups  after chronic CBL treatment compared to CTL . Global calpain activity, assessed by using spectrofluorimetric techniques, was drastically increased by 115% following CBL treatment  and a phenotypic shift to a faster muscle profile. These effects are consistent with what is usually reported in the literature on fast skeletal muscle 0) of EDL muscle was increased following clenbuterol treatment, while the specific force (sP0) was not different compared to controls, suggesting that the strength improvement was only due to the increased skeletal muscle mass. Analysis of representative recording of the decrease in EDL tension during the fatigue protocol highlighted that the initial tension was higher and appeared earlier in clenbuterol-treated EDL muscles than in untreated controls, in accordance with the increase in muscle mass and the phenotypic shift to a faster contractile profile. Fatigability of EDL muscles from clenbuterol-treated animals was nevertheless increased, as the peak tetanic force fell more quickly in clenbuterol-treated EDL muscles than in controls, and Tlim, used as an index of resistance to fatigue, was significantly reduced. This last result confirmed that clenbuterol treatment negatively affects muscle endurance, in agreement with the phenotypic remodeling.Surprisingly, while the effects of β2-agonists on skeletal muscle mass and phenotype have been extensively studied, few experiments have evaluated the impact of chronic treatment with clenbuterol on skeletal muscle function. In humans, 4-week clenbuterol treatment in orthopedic patients did not improve muscle absolute strength 2+ signaling plays a critical role in regulating skeletal muscle functional properties 2+ homeostasis, resulting in an increased Ca2+ leak from the SR. To date, no studies have evaluated the impact of chronic treatment with clenbuterol on skeletal muscle Ca2+ signaling. It is generally accepted that Ca2+ spark spatio-temporal properties and frequency reflect RyR1 function, as indicated by previous publications by our group. For instance, we previously used Ca2+ spark analysis to demonstrate the contribution of RyR in skeletal muscle pathophysiology 2+-release events (Ca2+-sparks) are reduced in EDL muscle fibers from clenbuterol-treated animals compared to control, suggesting changes in the SR Ca2+ release properties. The decreased amplitude of Ca2+ sparks could be explained by a decreased SR Ca2+ load as suggested by results in cardiac myocytes 2+ regulatory sites on the luminal side and on the myoplasmic side of the SR could explain how a reduction in SR Ca2+ content might affect Ca2+ release 2+ load reduces Ca2+-spark frequency and amplitude in smooth muscle from rat 2+ load was markedly reduced compared to untreated controls. The decrease in SR Ca2+ load could be a consequence of increased passive Ca2+ leak from the SR as already reported following acute clenbuterol treatment in mechanically skinned skeletal muscle fibers from rats 2+-transients in EDL muscles from clenbuterol-treated animals. This result is in accordance with the reduced amplitude of Ca2+ sparks and may also be a consequence of the reduced SR Ca2+ load. A reduction in Ca2+-transient amplitude could induce a decrease in the maximum capacity of force production by skeletal muscle, as already demonstrated following acute clenbuterol treatment 2+ sensitivity. Indeed, it has already been reported that chronic clenbuterol administration can improve Ca2+ sensitivity in rat slow-twitch skeletal muscle fibers Ca2+ is a second messenger that regulates many signaling pathways. Therefore, Ca2+ homeostasis disturbances may also potentially disturb skeletal muscle metabolic and catabolic processes. For example, Ca2+ participates in the regulation of calpain activity. In particular, calpain 1  binds Ca2+ rapidly when the calcium concentration increases within a physiological range in vivo is more ambiguous, due to its apparent requirement of supraphysiological Ca2+ concentrations 2+ leak from the SR could mediate a decreased SR Ca2+ load and finally may reduce Ca2+-sparks and global Ca2+-transient amplitude. Another consequence of the increased Ca2+ leak from the SR could be the activation of calpains. Such association between SR Ca2+ release disturbances and calpain activation has already been reported in a mouse model of Duchenne muscular dystrophy (mdx mouse), a pathological condition that is associated with increased SR Ca2+ leak 2+-transients 2+ regulation disturbances and calpain activation. Recent studies have confirmed that chronic clenbuterol treatment in rats leads to increased calpain activities in fast-twitch skeletal muscles 2+ homeostasis. In particular, it would be interesting to evaluate the potential role of calpain activation in SR Ca2+ release changes. Indeed, Ca2+-activated proteases can induce proteolysis of SR Ca2+ release channels 2+ exposure results in disruption of excitation-contraction coupling (E-C coupling) and titin, an effect possibly mediated, at least in part, by Ca2+-activated proteases 2+-induced disruption of E-C coupling 2+-release, leading to a functional impairment of skeletal muscle.Ca2+ signals associated with excitation-contraction coupling in fast-twitch skeletal muscles. This effect occurs in association with increased calpain activities. Together, these alterations might contribute to the structural and functional adaptations of skeletal muscle to chronic clenbuterol treatment. As clenbuterol is considered a good candidate for the treatment of muscle disuse, our study underlines that more experiments are necessary to further elucidate the impact of chronic clenbuterol treatment on muscle metabolism and function.In this study we demonstrate that chronic clenbuterol treatment induces significant changes in various Ca