RUNX1 (runt-related transcription factor 1) is a myeloid transcription factor described as recurrently mutated in de novo acute myeloid leukemia , clustering in the intermediate-risk cytogenetic group and showing prognostic adverse impact on the overall survival and disease progression.3 In the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues,4 AML are classified in the categories ‘AML with recurrent genetic abnormalities', ‘AML with myelodysplasia-related changes (AML-MRC)', ‘therapy-related myeloid neoplasms' and ‘AML, not otherwise specified'. AML-MRC includes cases with a myelodysplasia-related cytogenetic abnormality, a previous myeloid malignancy or showing multilineage dysplasia (MLD). MLD positive (MLD+) morphology shows dysplastic features in ⩾50% of cells in ⩾2 hematopoietic lineages.5 In 2008 ‘AML with mutated NPM1' and ‘AML with mutated CEBPA' were introduced as provisional entities and reached in 2016 a status as own entities.5 Therein, the presence of MLD alone will not classify a case as AML-MRC when one of these mutations is present. Ongoing discussions now focus on RUNX1 mutations characterizing the new provisional entity ‘AML with mutated RUNX1'. However, classification of patients with MLD and RUNX1 mutation into the AML-MRC category is questionable and needs to be discussed. We therefore comprehensively analyzed 152 RUNX1-mutated AML patients by cytogenetics and molecular genetics, and especially investigated the prognostic impact of MLD. RUNX1-mutated AML showed strong associations to trisomy 13  and mutations within genes coding for the spliceosome , and for chromatin modifiers . However, MLD did not show prognostic impact in multivariate Cox regression analyses. This supports an approach to classify RUNX1-mutated AML also as a new provisional entity irrespective of dysplastic features.RUNX1 mutation. The cohort comprised 49 female and 103 male, the median age was 67 years, ranging from 18 to 78 years. Ninety-nine percent of patients had a de novo AML and 1% a secondary AML. Therapy-related AML were excluded, as these are classified separately within the WHO. Forty out of 152 (26%) had allogeneic transplantation in the follow-up. All samples underwent May-Grünwald-Giemsa staining and cytochemistry. Dysplasia was assessed according to Goasguen et al.6 MLD was defined by ⩾50% dysplastic cells in ⩾2 lineages following the WHO guidelines.5 In 132/152 patients all three lineages were evaluable, while in 20 cases only two hematopoietic lineages were evaluable. All patients were investigated by the standard chromosome banding analysis (cytogenetics) and a diagnostic molecular genetic approach following European Leukemia Network (ELN) guidelines.7 All patients had prognostically intermediate karyotypes according to Medical Research Council criteria (group 2).8 In addition, a next-generation sequencing-based mutational screening targeting 25 genes  criteria10 , followed by M0 , M4  and M6 . This confirms earlier studies that described the immature and undifferentiated morphology of RUNX1-mutated AML, reflected by the high number of M0 subtypes, as well as a comparison to a matched control cohort without RUNX1 mutation from our data base, showing only 2% of M0 cases , erythropoiesis in 31% (47/152) and megakaryopoiesis in 55% (73/132) of patients. A total of 44 patients (33%) had no dysplastic features in any of the three cell lineages, 38 (29%) had unilineage dysplasia, 39 (30%) had bilineage dysplasia, whereas 11 cases (8%) had trilineage dysplasia (TLD). In four cases, a differentiation of bilineage dysplasia or TLD was not possible, as megakaryopoietic dysplasia was not evaluable. MLD was detected in 36% (54/152) of the analyzed bone marrow samples. These numbers are in line with a number of large AML studies, where MLD was found in 23–36% and TLD in 2–15% ;12 however, interestingly both show high incidences of RUNX1 mutations.13Chromosome banding analysis revealed cytogenetic aberrations in 59/152 (39%) patients. Thereof, 42 patients showed trisomies as sole abnormality, whereas only 17 showed other aberrations. In detail, 17 cases showed trisomy 8 (+8), 13 cases +13 and 4 patients each +11 and +14. Further four non-recurrent trisomies were observed. Only two cases had three aberrations, classifying for AML-MRC with complex karyotypes . Although +8 is one of the most frequent recurrent cytogenetic aberrations in AML (10% of AML cases),RUNX1 mutations was observed for ASXL1 (41%), followed by SRSF2 (36%), FLT3 , BCOR (21%), TET2 (18%), IDH2 (17%) and U2AF1 (16%). Mono-allelic CEBPA mutations were rarely detected (5%), double-mutated CEBPA was not identified, clearly differentiating these AML entities. NPM1-mutated cases (n=3) were excluded, as these cases qualify already for an own entity. Overall, 461 additional mutations were identified in 140 analyzed patients, resulting in a median number of three additional mutations (range: 0–6). Thus, in 98% of patients at least one additional mutation besides the RUNX1 mutation was observed , a higher incidence of +13 , IDH2 mutation , but no KIT mutation . All other clinical parameters, chromosomal alterations and additional gene mutations did not differ significantly between the MLD− and MLD+ patients , P=0.039), ⩾3 mutations in addition to RUNX1 , mutations within the spliceosome , DNMT3A , NRAS  and U2AF1 , and mutations in the spliceosome and in chromatin modifiers, characterizing a unique secondary type AML phenotype.2RUNX1-mutated AML shows shorter event-free survival,1 and we found ⩾3 mutations as independent prognostic marker influencing prognosis. However, the detection of MLD has no independent influence in multivariate analysis. We therefore strongly support the classification of RUNX1-mutated AML as a provisional entity in the new WHO classification, but without further consideration of dysplastic features such as MLD.In conclusion, MLD


We set out to characterize the interplay between Ca2+ and Cu2+ binding by S100A5 using isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), and analytical ultracentrifugation (AUC).S100A5 is a calcium binding protein found in a small subset of amniote tissues. Little is known about the biological roles of S100A5, but it may be involved in inflammation and olfactory signaling. Previous work indicated that S100A5 displays antagonism between binding of Ca2+ and Ca2+ ions simultaneously. The wildtype protein was extremely aggregation-prone in the presence of Cu2+ and Ca2+. A Cys-free version of S100A5, however, was not prone to precipitation or oligomerization. Mutation of the cysteines does not disrupt the binding of either Ca2+ or Cu2+ to S100A5. In the Cys-free background, we measured Ca2+ and Cu2+ binding in the presence and absence of the other metal using ITC. Saturating concentrations of Ca2+ or Cu2+ do not disrupt the binding of one another. Ca2+ and Cu2+ binding induce structural changes in S100A5, which are measurable using CD spectroscopy. We show via sedimentation velocity AUC that the wildtype protein is prone to the formation of soluble oligomers, which are not present in Cys-free samples.We found that human S100A5 is capable of binding both Cu2+ and Cu2+ ions simultaneously and independently. This observation is in direct contrast to previously-reported antagonism between binding of Cu2+ and Ca2+ ions. The previous result is likely due to metal-dependent aggregation. Little is known about the biology of S100A5, so an accurate understanding of the biochemistry is necessary to make informed biological hypotheses. Our observations suggest the possibility of independent biological functions for Cu2+ and Ca2+ binding by S100A5.S100A5 can bind CaThe online version of this article (doi:10.1186/s13628-017-0040-y) contains supplementary material, which is available to authorized users. The protein is primarily homodimeric and is capable of binding one Caproteins . Throughproteins –7.S100A5 is expressed primarily in the olfactory bulb and olfactory sensory neurons (OSNs). Its expression is dramatically upregulated by odor stimulation –10. It h2+, Cu2+, and Zn2+ binding protein [2+ ions, four Cu2+ ions, and two Zn2+ ions per homodimer. One of the most striking observations of that study was the strong antagonism between the binding of Cu2+ and Ca2+ ions to the protein. This feature is one of the most highly cited aspects of S100A5. Because little is known about the protein, this fact is present in descriptions found across databases such as Uniprot, NCBI, Wikigenes, and Genecards [2+ is not known outside the S100A5 lineage. Thus, this unique feature of S100A5 provoked speculation about its possible biological implications [2+ and Ca2+ regulated signal during olfaction or as a Cu2+ sink to accommodate high Cu2+ concentrations in the olfactory bulb [The first biochemical study of human S100A5 identified it as a novel Ca protein . The autenecards –15. Whilications , 16. It ory bulb .2+ and Zn2+ to a large number of S100 proteins including S100A5 [2+ binding. In contrast neither of these residues was necessary for binding of Cu2+. Due to the original report of Ca2+ /Cu2+ antagonism we suspected that Ca2+ and Cu2+ may compete for the same sites on S100A5.We sought to characterize this presumably important feature of S100A5 in more detail. Previously, we characterized the binding of Cug S100A5 . Via ITCg S100A5 . We foun2+ and Cu2+ binding by S100A5. Using a Cysteine-free variant (C43S/C79S) of the protein, we show that binding of Ca2+ and Cu2+ are not in fact antagonistic. The protein is capable of binding the two metals–which induce notable structural changes–simultaneously and independently. Furthermore, we establish that the Cysteine-containing (WT) protein is prone to the formation of high-ordered oligomers in solution, while the Cysteine-free variant is almost entirely dimeric. We suggest that this propensity for formation of large oligomeric species and precipitation under our experimental conditions may underlie the apparent antagonism observed in the original S100A5 report. Our results may suggest new biological roles for Cu2+ binding by this protein.Here we report our study of the interplay between Ca2+ and Ca2+ binding was previously identified as a distinct feature of S100A5 relative to other S100 proteins [2+ and Ca2+ may bind using the same ligands, thus explaining the antagonism as direct competition. It was suggested in the original paper that Cu2+ and Ca2+ might share some ligands [2+ and Ca2+ directly compete. We titrated Cu2+ onto S100A5 in the presence of saturating Ca2+. However, these experiments were difficult to interpret due to extensive precipitation in the samples containing both ions. ITC traces were very noisy and apparent stoichiometries were systematically low (≈0.2), suggesting that a large portion of the protein sample was not competent to bind Cu2+ . This wg models .2+ ions to the apo form of the S100A5 double mutant. We found the Cu2+ binding data was best described with a single-site binding model. In line with our previous observations, the protein bound Cu2+ with a Kd(μM) that had a 95% credibility region of 0.94≤1.81≤3.90  of 0.65≤0.96≤1.47 : 0.14≤0.46≤2.68) and one lower-affinity site (Kd(μM): 1.85≤6.33≤34.88). The values were roughly consistent with those reported in the literature [2+ did not inhibit the binding of Ca2+ ions : 0.03≤0.18≤2.16) was not distinguishable within uncertainty from that of the apo protein. The Kd of the high affinity site (Kd(μM): 1.86≤10.46≤100.3) is similarly indistinguishable from that for the apo protein  distribution of the protein in each condition [2+ and Ca2+ caused a large amount of precipitation in wildtype S100A5 that we removed by extensive centrifugation prior to loading the cell. The remaining soluble protein was indistinguishable from the apo protein  reversible increase in α-helical secondary structure, which is consistent with the changes observed in published NMR data [2+. However, the Cys-Ser double mutant protein alleviates this issue. We collected far-UV circular dichroism spectra of the mutant protein in the apo form and bound to Ca2+, Cu2+, and Ca2+ and Cu2+ simultaneously. The Cys-Ser mutant displays a notable increase in alpha-helical signal (222 nm) upon binding of Ca2+, identical to the wildtype protein. Interestingly, addition of Cu2+ also induces an increase in α-helical signal that is approximately half of that induced by Ca2+. The spectrum of S100A5 bound simultaneously to both metals is identical to that of the Ca2+-bound form . The tag was then cleaved using TEV protease and the cleaved protein was further purified using Ca2+-dependent hydrophobic interaction chromatography. Finally, the sample was run over a second HisTrap Ni2+ affinity column to remove any uncleaved protein. The purified protein was dialyzed with 6000-8000 MWCO tubing (Fisher) against 2L 25 mM Tris, 100 mM NaCl, pH 7.4 with 2g chelex resin (BioRad). The dialyzed protein was filter-sterilized (0.22 μm), flash-frozen dropwise in liquid nitrogen, and stored at − 80°C. We experimentally determined the extinction coefficient (5002M−1cm−1) of the Cys-Ser double mutant. We measured the A280 of the protein at the same concentration in both buffer and denaturing 6M GdHCl (Sigma). We used ProtParam [εnative=εMGdm6·Anative280,/AMGdm280,6. Concentration measurements were also corrected for scattering in samples [We previously generated the 6-histidine-tagged cysteine double-mutant construct in a pet28/30 vector . In thisrotParam  to predi samples . Due to CaCl2 (Sigma) or 0.25 mM CuCl2 (Sigma). Titrant solutions were prepared in matching experimental buffer to ensure identical conditions to titrate. Anhydrous CaCl2 or CuCl2 was dissolved directly in the buffer and diluted to the appropriate concentration immediately prior to experiments. Fresh stocks were made for each set of experiments. Experiments were performed with 50-80 μM protein at 25 °C. Two technical replicates of each Cu2+ binding experiment were performed. To resolve the complex Ca2+ binding curves, four Ca2+ binding experiments were performed using four different concentrations of titrant. Raw data were integrated using the NITPIC software package–which allows uncertainty in the baseline–and the integrated heats were exported in standard SedPhat format [2+ titration data and a two-site binding polynomial was used for Ca2+ titration data [2+ binding fits, we constrained the dilution heat and dilution intercept to between -3.0–0.0 kcal/mol and 0–10,000 kcal/mol/shot respectively. All other priors were uniform.Samples were prepared in 25 mM TES (Sigma), 100 mM NaCl (Thermo Scientific), buffer at pH 7.4. Protein was thawed from the frozen stock and exchanged into the experimental buffer using NAP-25 desalting columns . For competition experiments the experimental buffer also contained either 1 mM t format . We thent format . We usedion data , 43. ForμM EDTA at pH 7.4 with the appropriate metal added directly to the buffer during preparation. Metals were added to a final concentration of 250 μM. Samples were prepared at 40 μM in the appropriate experimental buffer by overnight dialysis (6000-8000 MWCO) against 2L at 4 °C. Before ultracentrifugation samples were centrifuged at 18,000×g at 4 °C in a temperature-controlled centrifuge for 30 min. Ultracentrifugation was done with sapphire windows at 50,000×g in sector-shaped cells (Beckman) on a Beckman ProteomeLab XL-1. Sedimentation was monitored using interference mode rather than absorbance at 280 nm due to the low extinction coefficient of S100A5. The Lamm equation was fit to the sedimentation data–using SedFit–to calculate the continuous c(s) distribution [Experiments were done in 25 mM TES (Sigma), 100 mM NaCl (Thermo Scientific), 100 ribution , 23. EstμM samples in a Chelex (Bio-Rad) treated, 25 mM TES (Sigma), 100 mM NaCl (Thermo Scientific), 100 μM EDTA, buffer at pH 7.4. Samples were subsequently diluted to 25 μM in buffers containing: no metal (apo), 1 mM Ca2+, 1 mM Cu2+, or both 1 mM Ca2+ and 1 mM Cu2+ –all prepared in the stock buffer above. Samples were centrifuged at 18,000×g at 25 °C in a temperature-controlled centrifuge (Eppendorf) before experiments. Spectra were collected at 25 °C in a Jasco peltier multi-cell sample unit. Reversibility of metal-induced structural changes was confirmed by adding a molar excess of EDTA to the metal-saturated samples and repeating spectra collection. In all cases, addition of EDTA returned the samples to the apo state. Five scans of each condition were collected. These scans were then averaged–using Jasco spectra analysis software–to minimize noise. Buffer blank spectra were generated for each condition. Applicable blanks were subtracted in the Jasco spectra analysis software. Blank-corrected data were exported as text files and raw signal was converted into mean molar ellipticity using the concentration and the number of residues (Nres=95) in our S100A5 construct using the equation: MME=CDsignal/c(M)·10·L(cm)·Nres.Far-UV circular dichroism spectra (200–250 nm) were collected on a J-815 CD spectrometer (Jasco) with a 1 mm quartz cell . We prepared 50


Trypanosoma cruzi. New antitrypanosomal options are desirable to prevent complications, including a high rate of cardiomyopathy. Recently, a natural substance, lychnopholide, has shown therapeutic potential, especially when encapsulated in biodegradable polymeric nanocapsules. However, little is known regarding possible adverse effects of lychnopholide. Here we show that repeated-dose intravenous administration of free lychnopholide (2.0 mg/kg/day) for 20 days caused cardiopathy and mortality in healthy C57BL/6 mice. Echocardiography revealed concentric left ventricular hypertrophy with preserved ejection fraction, diastolic dysfunction and chamber dilatation at end-stage. Single cardiomyocytes presented altered contractility and Ca2+ handling, with spontaneous Ca2+ waves in diastole. Acute in vitro lychnopholide application on cardiomyocytes from healthy mice also induced Ca2+ handling alterations with abnormal RyR2-mediated diastolic Ca2+ release. Strikingly, the encapsulation of lychnopholide prevented the cardiac alterations induced in vivo by the free form repeated doses. Nanocapsules alone had no adverse cardiac effects. Altogether, our data establish lychnopholide presented in nanocapsule form more firmly as a promising new drug candidate to cure Chagas disease with minimal cardiotoxicity. Our study also highlights the potential of nanotechnology not only to improve the efficacy of a drug but also to protect against its adverse effects.Chagas disease is a neglected parasitic disease caused by the protozoan  Trypanosoma cruzi (T. cruzi). There are an estimated 6–7 million infected people worldwide, and the disease causes 12 500 deaths per year, mostly in Latin America . While CD is principally transmitted to humans by triatomine insects, other routes include blood transfusion or transplantation of contaminated organs, ingestion of contaminated food and congenital transmission from infected mothers to newborns. International migrations contribute to spread the disease in non-endemic areas such as North America, Europe and Western Pacific countries . CD evolves in different consecutive phases with a short acute period, characterized by patent parasitemia, followed by a chronic phase in which most infected individuals remain asymptomatic (indeterminate form). CD is also a common determinant of non-ischemic cardiomyopathy in addition to digestive complications and neurological disorders. Approximately 30% of patients infected with T. cruzi manifest cardiomyopathy characterized by severe myocarditis and multiple arrhythmias, and/or dilatation and physiological dysfunctions of hollow organs, mainly of the digestive tract, which develop progressively over the years or decades after infection123Chagas disease (CD) is an important neglected disease caused by an intracellular hemoflagellate protozoan parasite, T. cruzi strains or genetic groups of the parasite, long treatment regimens, which reduce patient complianceAlthough vector control helps to prevent CD transmission in endemic areas, pharmacological treatment is essential to cure the disease in infected people. Therapeutic options to kill the parasite are currently limited to benznidazole and nifurtimox. However, while treatment during the early stages of CD is effective, treatment during the chronic phase has been relatively ineffective6in vivo of a natural substance, lychnopholide (LYC), isolated from Lychnophora trichocarpha Spreng, a plant from Asteraceae family. In particular, a pharmaceutical formulation of LYC encapsulated in biodegradable polymeric nanocapsules (NC) and administered by intravenous route was more effective than benznidazole in curing Swiss mice infected experimentally with different T. cruzi strains11et al., submitted Scientific Reports). Pegylated biodegradable NC (PLA-PEG) increased 26-fold the body exposure to LYC and increased the LYC plasma half-life 25-times compared to free LYC solution administered intravenously. These results are in line with efficacy data upon iv treatment with NC, 100% versus 0% mice cure for NC and free LYC, respectively. At the best of our knowledge, no other drug has been so efficient to cure experimental T. cruzi infection at chronic phase in mice. However, no safety aspects related to the use of LYC in vivo have been investigated to date. As the infection induces serious cardiac alterations, the LYC cardiotoxicity evaluation in healthy mice is of primordial importance in the first investigation of LYC safety. Another point is the ability of NC to increase and maintain plasma levels of LYC, which may enhance toxicity as a consequence. Cardiotoxicity is the first cause of a new drug candidate to be abandoned in the pre-clinical phase. We undertook this study with a three-fold objective: (i) to identify and investigate any potential toxicity of LYC on cardiomyocytes in vitro; (ii) to study in vivo cardio-toxicological effects of repeated LYC intravenous administration on cardiac function, and, (iii) to study the in vivo effect of the encapsulation of LYC in NC. Our main results show that repeated dose treatment with LYC for 20 days (2 mg/kg/day), which had previously demonstrated anti-trypanosomal efficacy in mice, could induce severe cardiopathy with 50% mortality in healthy mice and alterations of Ca2+ handling in single cardiomyocytes. Dose-dependent alterations of Ca2+ handling, promotion of Ca2+ waves, and abnormal RyR2-mediated diastolic Ca2+ release were also observed following acute application of the drug. Strikingly, all adverse cardiac effects were prevented when LYC was administered associated to the polymeric biodegradable NC.Recently, we have documented the antitrypanosomal efficacy in vivo at the dose found to be effective in killing T. cruzi in the acute and chronic phases of infection in mice by intravenous (2 mg/kg/day) administration routes13vs. Ctrl and NC groups), suggesting adaptive remodelling to compensate for the LV diastolic dysfunction. Finally, because of a critical phenotype, one of the free LYC-treated mice underwent echocardiographic at day 18, and died suddenly at day 19. This mouse presented a massively dilated LV . LYC and the LYC-NC formulation were administered 2+ were measured in electrically stimulated cardiomyocytes  under basal conditions  on contractility and intracellular Ca2+ cycling. Under basal conditions, LYC increased the Ca2+ transient amplitude in a dose-dependent manner at 1.4 and 14 nM, although no significant effect was seen on sarcomere shortening 1516172+ events, referred to as Ca2+ sparks, can be assessed visually by means of confocal microscopy and the use of the fluorescent Ca2+ indicator Fluo-4 AM. We therefore evaluated the leakiness of RyR2 by measuring Ca2+ spark frequency. Since Iso had a major effect on the effect of LYC on the triggering of Ca2+ waves , we estimated the SR Ca2+ content by triggering maximal RyR2-mediated Ca2+ release following rapid caffeine application (10 mM). The SR Ca2+ content was lowered by free LYC  and at the molecular level (Ca2+ sparks), which could be maintained following a β-adrenergic challenge suggesting additive mechanisms. In an additional set of experiments performed using the patch-clamp technique, we found that LYC had no acute effect on the cellular action potential  induced high mortality (50%), starting after 10 days of treatment, and severely altered cardiac function in the surviving animals. However, these mice had normal systolic function (i.e. preserved EF) but exhibited LV hypertrophy with increased wall thickness, reduced chamber dimension, and diastolic dysfunction . The hea2627132+ transient amplitude of cardiomyocytes were increased. Overall, our results are consistent with a compensatory state of the LV myocytes, described early after myocardial infarction in mice and accounted for changes in cellular excitation-contraction coupling, especially an increase of the Ca2+ transient amplitude2+ events, namely Ca2+ waves and Ca2+ sparks respectively, in line with many studies on HF1531Our findings at the cellular level support the view of an inotropic effect of LYC2+ handling seen during in vivo repeated-dose treatment. In particular, LYC increased Ca2+ transient amplitude and promoted Ca2+ sparks reflecting RyR2-mediated diastolic Ca2+ leakiness responsible for the spontaneous firing of abnormal Ca2+ waves as found in inherited diseases related to RyR2 mutations, chronic diseases such as HF, or adverse drug effects161718313233342+ sparks/Ca2+ waves by LYC remained during β-adrenergic receptor stimulation  pumps or SERCA orthologues  by sesquiterpenic lactones has been reported362+ transient decay, or decreased the Ca2+ transient nor increased dramatically diastolic Ca2+ as shown with thapsigargin2+ leakage through RyR2 by an efficient Ca2+ pumping in the SR to attenuate diastolic Ca2+ overload.LYC applied acutely in the nanomolar range (>1 nM) in isolated cardiomyocytes reproduced dose-dependently the effects on Camulation , i.e. ov2+ transient amplitude, but this was however associated with a disruption of normal Ca2+ cycling fostering abnormal Ca2+ sparks and Ca2+ waves (promoted by Ca2+ sparks) in line with RyR2 being a primary target of LYC effects. Interestingly, the trypanocidal activity of sesquiterpene lactones was reported to inactivate trypanosome defense against oxidative stressesThe acute effects of free-LYC involved increased Ca2+ sparks frequency and Ca2+ waves, as seen in heart failure+ and Ca2+ handling proteins as well as modifications of key regulating kinases of these proteins have critical physiological roles and high pathological relevance+ overload due to ROS-dependent activation of sustained Na+ currents may participate to increase SR Ca2+ load322+ transient and the RyR2 leakage induced by LYC. These mechanisms are highly pathophysiologic2+ handling alterations induced by LYC may contribute to the pathogenesis and progression of the LYC-induced cardiopathy described in this study. Leaky RyR2 channels are known to activate pathological hypertrophic pathways responsible for cardiac remodelling and deterioration3145462+ transient (positive inotropism) and contraction (no effect) in our study. Interestingly, polyester-based NC obtained from PLA-PEG polymer produce no oxidative stress even at high concentrations49Moderate oxidative stress causes pathological change in RyR2 conformation and increases both Caiv route, increasing infected-mice survival (100% LYC-NC versus 0% free-LYC) at the chronic phaseversus 0% free-LYC)1212Scientific Reports). This shows clearly that nanoencapsulation reduces the LYC clearance from mice plasma. Considering these results, our next step was to assess the possible cardiotoxicity of this new drug candidate and, obviously, also of LYC associated to NC formulation. A key finding of our study was that the severe adverse effects of LYC with regard to mortality and cardiac pathogenesis were prevented by encapsulation of the LYC. Not only were the effects of empty NC neutral, enabling us to distinguish between the potential toxicity of LYC and that of the NC, but the encapsulated LYC had also no harmful effects on the parameters evaluated, including myocardial function and morphology, cardiomyocyte contraction, Ca2+ transients and the occurrence of Ca2+ sparks and related Ca2+ waves, unlike the effects of free LYC, even under challenging conditions such as β-adrenergic stress. Our pharmaceutical formulation of LYC encapsulated in PLA-PEG NC therefore appears to be a promising step forward in providing a potential therapeutic option to treat CD with minimal cardiotoxicity. How can this differential effect of encapsulated LYC, i.e. improved anti-trypanosomal activity on the one hand and cardiac protection on the other, be explained? Long-circulating PLA-PEG NC improved LYC efficacy12et al., paper submitted in Scientific Reports). Previously reported data showed that encapsulation has a profound influence in the amount of LYC available in the plasma to interact with target sites and non-target sites promoting side effects. As a significant part of LYC circulates in plasma inside NC, free drug toxic effects may be diminished by reducing interactions with the heart. Oppositely, the LYC-NC with their reduced sizes (<200 nm) can extravasate and accumulate more in tissues compromised by inflammation process, which have leaky endotheliumbolus injections of free LYC once a day to cure infectionvs. 1.4 nM and 14 nM). This may be particularly interesting for repeated-dose oral treatment during chronic infection. Indeed, no obvious adverse effects and no death were observed in T. cruzi infected-mice treated at chronic phase with LYC-PLA-PEG NCiv administration on healthy animals. Oppositely, 50% and 60% mortality occurred in healthy animals treated with free-LYC and in animals T. cruzi-infected (chronic phase)iv during 20 days at the acute phase of infection with free-LYC (n = 16), 19% died during treatment and 30% after the end of the treatmentvs. C57BL/6). For example, these mice present lower levels of glutathione-related antioxidant defenses in the hippocampus and prefrontal cortexWe have previously shown that LYC encapsulated in long-circulating PLA-PEG NC improves dramatically the anti-trypanosomal properties of LYC by There is still a long way to go before considering the application of the therapeutic principles described here to human health. The control of adverse effects may be species-dependent and even variable. An assessment of LYC cardiotoxicity in mice with CD is also clearly warranted, although performing all the investigations reported here in infected animals is a technical challenge to provide the adequate safety conditions for experimenters to run all the experimental approaches used in our study. It will also be important to explore whether LYC is toxic to other organs/functions, and whether these different risks can likewise be prevented by nanoencapsulation.T. cruzi as well as for its antitumoral properties. Overall, we highlight an important contribution of nanotechnology not only in improving the efficacy of a drug (here LYC against T. cruzi infection), but also its potential in protecting against adverse effects.In summary, we have tested for the first time in an experimental model potential adverse effects of LYC at the cardiac level. Although we evidenced a cardiotoxicity during repeated-dose treatment regimen with LYC administered in its free form, we also unravelled a remarkable protection against this cardiotoxicity when LYC was encapsulated in polymeric NC. Importantly, this concept may open new prospects for treatment of the chronic phase of CD, as well as the treatment of other parasitic diseases, and antitumoral therapy with LYC. This study is thus promising but warrants the further evaluation of NC-encapsulated LYC prior to its development for clinical use against different strains of block-poly(lactide) polymer as described115511LYC loaded in sterically stabilized NC (LYC-NC) with a PEG corona at the surface was prepared via the interfacial deposition of the preformed polymer followed by solvent displacement, using a monomethoxy-polyethylene glycol-Ministère de la Recherche et de l’Enseignement Supérieur (N° 02571.01). Seven-week-old male C57BL/6 mice  were used. To study the repeated-dose effects of treatments, animals received daily intravenous injections of free LYC solution , LYC loaded in biodegradable polymeric NC , blank-NC (10 mice) and vehicle  for 20 consecutive days before echocardiographic evaluation at day 19 and sacrifice at day 20, according the classical in vivo protocol of Chagas disease screening of efficacy. This is also a 20th day repeated-dose toxicological protocol for studies of active substances against T. cruzi in mice model1617All procedures conformed to European Parliament Directive 2010/63/EU, the 22 September 2010 Council on animal protection, and NIH Guidelines for the Care and Use of Laboratory Animals. The project was approved by the French eur N° 021.01. SevSmall animal imaging platform (certified norme ISO 9001: 2008). Mice were anesthetized with isoflurane . Body temperature and heart beat were monitored throughout the procedure and maintained at physiological levels . Wall thickness and left ventricular diameter were measured at the level of the papillary muscles in a parasternal long-axis two-dimensional view by M-mode, allowing the calculation of the EF by the Teicholz method and the relative wall thickness (RWT)Transthoracic echocardiography was performed with the high-resolution imaging system Vevo2100  equipped with a 40 MHz probe on the platform 2+ were measured in single LV myocytes field-stimulated at 1.0 Hz with 1-ms current pulses to assess cell shortening and Ca2+ transients, respectively, as described1617383, 1.5 KH2PO4, 1.7 MgCl2, 21 HEPES, 1.8 CaCl2 and 11 glucose). Sarcomere length (SL) and Ca2+-sensitive fluorescence (405 and 480 nm) were simultaneously recorded using an IonOptix system  and a Zeiss microscope . To monitor intracellular Ca2+, cardiomyocytes were loaded with the dual-emission ratiometric Ca2+ indicator Indo-1AM , which emits at 405 nm and 480 nm concurrently. They were simultaneously illuminated at 365 nm using a xenon arc lamp. Cytosolic Ca2+ concentration was determined by the ratio of 405 nm/480 nm fluorescence (a.u.: arbitrary units). Cell shortening, diastolic Ca2+ levels and Ca2+ transient amplitude were measured. Sarcoplasmic reticulum (SR) Ca2+ content was estimated by massive RyR2 channel opening by means of caffeine (10 mM). To assess diastolic events, cells were stimulated by trains of stimulations during 30 s periods (1.0 Hz) followed by a 30 s rest period. Cells developing spontaneous Ca2+ waves and contractions during the diastolic period were identified. Cells were also challenged under conditions of stress triggered by exposure to the β1-adrenergic agonist isoproterenol  for 10 minutes before experiments. Data were analyzed using Ionwizard Software.Contractility and intracellular free Ca2+ sparks were recorded in quiescent cells loaded with the Ca2+ indicator Fluo-4 AM  and a confocal microscope in line-scan mode  at 25 °C. The dye was excited at 488 nm and the fluorescence emitted collected through a 505-nm long-pass filter. Myocytes were field-stimulated at 1 Hz with 1-ms current pulses delivered via two platinum electrodes, one on each side of the perfusion chamber. During the rest period that followed stimulation, myocytes were repetitively scanned along their entire length at 1.5-ms intervals, for a maximum of 6 s17582+ sparks was determined by the intensity of each sequential scan line and plotting the mean intensity as a function of time. The SparkMaster plug-in for ImageJ software was used to detect and analyze Ca2+ sparks.Ca® (Prism 5 for Mac OS X). For comparison of Survival curves, we used Log-rank (Mantel-Cox) test. For cellular experiments and studies of Ca2+ sparks, 10–35 cells per group were used for each experimental condition. For multiple comparisons, one-way ANOVA was used, followed by a parametric t-test with Fisher’s correction. For comparison of two conditions in the same group (baseline vs. iso) a paired Student t-test was used. A p-value of 0.05 or less was taken to indicate statistical significance.All data are expressed as means ± SEM. Statistical analyses were performed using GraphPad PrismHow to cite this article: Branquinho, R. T. et al. Biodegradable Polymeric Nanocapsules Prevent Cardiotoxicity Of Anti-Trypanosomal Lychnopholide. Sci. Rep.7, 44998; doi: 10.1038/srep44998 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC.Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated  Phenethyl isothiocyanate (PEITC) is a naturally occurring phytochemical that has received extensive interest for its anti-cancer and chemopreventive activities.  PEITC inRecent findings have indicated that PEITC inhibits mRNA translation –4. mRNA Modulation of eIF4E and eIF2 are pivotal for controlling mRNA translation . eIF4E ain vivo, whereas inhibition of signaling upstream of MYC was circumvented by complex cross-talk mechanisms [Dysregulation of mRNA translation plays a major role in cancer, acting to drive cell accumulation via effects on both global protein and specific proto-oncogenes which often have high requirements for translation . Translachanisms .HIF1A mRNA translation in MCF7 breast cancer cells [HIF1A mRNA translation [Despite the importance of dysregulated protein synthesis for cancer, few studies have investigated effects of PEITC on these pathways. Hu et al. demonstrated that PEITC increased eIF4E expression and decreased 4E-BP1 phosphorylation in PC-3 prostate cancer cells, and inhibited 5′-cap-dependent mRNA translation measured using artificial reporter assays . More renslation , 20.Studies of translational regulation have almost exclusively used established cell lines. Although clearly of great value, it is possible that regulatory pathways are altered in these settings since long-term culture will select for more metabolically active cell variants. Therefore, analysis of mRNA translation in primary cancer cells is an important goal. Chronic lymphocytic leukemia (CLL) provides a powerful model system for the detailed molecular analysis of primary cancer cells. It is the most common B-cell malignancy  and provin vivo in CLL. BCR signaling responsiveness varies between individual samples and retained signaling capacity is associated with a poor outcome. Moreover, inhibitors of BCR-associated signaling kinases (such as the BTK inhibitor ibrutinib) are revolutionising therapy for B-cell malignancies [in vitro and that MYC was expressed in lymph nodes from CLL patients, the site of antigen engagement in vivo [MYC mRNA in primary CLL cells in vitro [Antigenic stimulation of the cell surface B-cell receptor (BCR) is a major driver of malignant cell accumulation gnancies . Antigen in vivo . More rein vitro . These rMYC mRNA) and this was associated with both mTORC1 inhibition and increased eIF2α phosphorylation.In this work, we investigated effects of PEITC on mRNA translation. We show that, in addition to inhibition of mTORC1, PEITC triggers rapid phosphorylation of eIF2α and that eIF2α phosphorylation is required for optimal PEITC-mediated translational inhibition in mouse embryo fibroblasts (MEFs). PEITC also inhibited both basal and anti-IgM-induced mRNA translation in primary CLL cells  or mutant eIF2α  which cannot undergo Ser51 phosphorylation (A/A) . SimilarWe used metabolic labeling to quantify mRNA translation in eIF2α S/S and A/A cells. PEITC inhibited metabolic labeling in both cell types, but A/A cells were clearly less sensitive compared to S/S cells Figure . SimilarWe also used metabolic labeling to directly investigate the role of mTORC1 in PEITC-mediated inhibition of global mRNA translation. We compared inhibitory effects of PEITC in wild-type MEFs, and MEFS lacking TSC2, an upstream negative regulator of mTORC1 , 31. BotWe attempted to identify the kinase(s) responsible for PEITC-induced eIF2α phosphorylation focusing on the ER-resident eIF2α Ser51-specific kinase PERK. PEITC increased PERK phosphorylation at Thr980 (a marker of activation) in both eIF2α S/S and A/A MEFs (data not shown). However, siRNA mediated knock-down of PERK in MCF7 cells (~75% reduction of PERK expression) only partly decreased PEITC-induced eIF2α phosphorylation by ~50% (data not shown). Thus, the kinase(s) required for PEITC-induced eIF2α phosphorylation remains to be identified. Despite optimization of transfections, it is not clear whether there was sufficient residual PERK to mediate effective eIF2α phosphorylation, or whether response involves multiple, perhaps compensating, kinases.IGHV mutation status, ZAP70/CD38 expression) are shown in We next probed effects of PEITC in primary CLL cells. We investigated effects of PEITC on both basal  and anti-IgM-induced mRNA translation. mRNA translation was quantified at 24 hours, consistent with our previous study . The feain vitro, and this can be accelerated by PEITC [We first investigated effects of PEITC using metabolic labeling. CLL cells undergo spontaneous apoptosis by PEITC . We seleby PEITC  anti-IgMAlthough the samples selected for study typically contained a high proportion of CLL B-cells, we also used OPP-labeling  to speciWe investigated effects of PEITC on phosphorylation of eIF2α and p70S6K at 60 minutes post-stimulation. PEITC was tested at both 10 and 20 μM in these shorter term experiments. As shown previously, , 36 antiAs a control, we also investigated effects of PEITC on phosphorylation of ERK1/2 which is activated downstream of the BCR independent of mTORC1 and eIF2α. PEITC had no effect on ERK1/2 phosphorylation in control cells, and did not inhibit anti-IgM-induced ERK1/2 phosphorylation Figure . In factMYC mRNA in fractions derived from polysome profiles. Anti-IgM enhanced both MYC RNA transcription and translation since anti-IgM increased the total amount of MYC mRNA recovered from the fractions, as well as the amount of MYC mRNA specifically present in polysome associated fractions , [We demonstrated that PEITC rapidly inhibited global mRNA translation and used genetically-manipulated MEFs to define the role of mTORC1 inhibition and eIF2α phosphorylation in this response. Although mTORC1 inhibition seems to mediate PEITC-induced inhibition of alling), , 20 mTORalling),  did not The mechanisms by which PEITC induces eIF2α phosphorylation remain to be determined. Similar to a recent study in ovarian cancer cell lines,  we demonMYC RNA translation. MYC RNA has a highly structured 5′-UTR and its translation therefore may be particularly affected by modest reductions in mTORC1-signalling.Long-term culture of established cell lines is likely to be associated with selection of variants with high mRNA translation and a major goal of our study was to extend analysis to leukemic primary cells, using CLL as a model. PEITC inhibited both basal and anti-IgM-induced global mRNA translation in CLL cells. Overall, PEITC appeared more effective in inhibiting basal, compared to anti-IgM-induced global mRNA translation. Pathway analysis revealed that PEITC increased eIF2α phosphorylation in the presence or absence of sIgM stimulation, and partially decreased anti-IgM-induced p70S6K phosphorylation. Thus, in the absence of sIgM stimulation, where mRNA translation is low and mTORC1 is not activated, inhibitory effects of PEITC appear to be mediated via eIF2α phosphorylation. Following stimulation, mRNA translation inhibition may be mediated by combined effects of PEITC on eIF2α and mTORC1. The partial inhibition of anti-IgM-induced p70S6K phosphorylation by PEITC may explain why PEITC only partially inhibited induced global mRNA translation. Alternately, it is possible that sIgM stimulation activates additional pathways not studied here to promote mRNA translation and that these operate independently of PEITC. Potential candidates include the MNK1/2 kinases activated downstream of ERK/p38 MAPK which can also enhance mRNA translation in other cell systems [Despite substantial improvements in patient outcomes following the introduction of new targeted agents such as ibrutinib, CLL remains an incurable disease with a pressing need for novel therapeutic approaches. In our previous study,  we demonIn conclusion, PEITC exerts inhibitory effects on both global and oncogene-specific mRNA translation, including MYC, via multiple pathways. These effects may contribute to both the chemo-preventive and anti-cancer effects of PEITC, and could be used to boost the efficacy of other agents, such as ibrutinib.−/−TSC2−/−) and matched control MEFs (p53−/−TSC2+/+) were kindly provided by Drs. A Tee  and D. Kwiatkowski  [The MCF7 cell line was obtained from American Type Culture Collection and cultured in complete Dulbecco's Modified Eagle Medium (DMEM), ie supplemented with 10% (v/v) bovine fetal serum, 2 mM L-glutamine and 1% (v/v) penicillin/streptomycin mix . MEFs containing wild type eIF2α (MEF eIF2α S/S) and MEFs containing the homozygous eIF2α Ser51Ala mutation (MEF eIF2α A/A) were kindly provided by Professor R. Kaufman, University of Michigan Medical Center, USA . TSC2-deMA, USA) . MEFs we+CD19+ CLL cells was 95% (range 82–99%). Molecular markers and sIgM signaling capacity were analyzed as previously described [Primary malignant B cells were obtained from the blood of CLL patients, as described . Patientescribed .2 anti-human IgM or control antibodies, as described [PEITC and the caspase inhibitor Q-VD-OPh were from Sigma Chemicals . Cycloheximide  was used as a positive control for inhibition of RNA translation and was used at 10 μg/ml in the final five minutes of incubation. For B-cell receptor (BCR) stimulation, CLL samples were incubated with bead-bound goat F(ab')escribed . Ibrutin35S-Met/Cys metabolic labeling and OPP assays [6 cells. For CLL cells, metabolic labeling was performed during the final 5 hours of cell incubations and polysome profiling used 10 × 107 cells.Polysome profiling, P assays . For MCF202/Y204-phosphorylated ERK1/2, anti-ERK1/2, anti-T389-phosphorylated p70S6K, anti-p70S6K, anti-S51-phosphorylated eIF2α, anti-eIF2α , anti-MYC  anti-β-actin . Analysis used equal protein loading following quantitation of protein content using the BioRad Protein Assay . Secondary antibodies were HRP-conjugated rabbit, mouse or goat antibodies  and images were captured using the ChemiDoc-It Imaging System with a BioChemi HR camera . Immunoblot signals were quantified using ImageJ (http://imagej.nih.gov/ij/). For quantitation of phosphorylation, expression values were normalized to the loading control (β-actin) and then the relevant total protein. For quantitation of MYC, expression values were normalized to β-actin.Immunoblot analysis was performed using the following antibodies; anti–TImmunofluorescence and confocal microscopy was carried out on cell lines grown on coverslips and fixed using 4% (w/v) paraformaldehyde  and permeabilised using 0.1% (v/v) Triton X-100 . Staining was carried out using anti-eIF3b , anti-TIA-1  and DAPI  was used as a nuclear stain. Coverslips were mounted onto microscopy slides using mounting media (Dako). Cells were imaged on the Olympus immunofluorescence microscope and captures using the Olympus 1 × 81 Camera . For confocal microscopy images were captures on a Leica microscope using the LAS AF software .Cell viability was analyzed using annexin V/propidium iodide staining as described .t-tests .Statistical comparisons were performed using Student's


ECC) and that exogenously applied nitric oxide decreases the calpain‐mediated proteolysis. The aim of this study was to examine the effects of ingestion of l‐arginine (ARG), a nitric oxide precursor, on ECC‐related calpain activation. In the first and second experiments, male Wistar rats were given ARG in water for 7 days starting from 3 days before the ECC protocol . Tibialis anterior muscles underwent 200 repeated ECCs and, subsequently, were excised 3 days later. Whole muscle analyses (the first experiment) revealed that ARG attenuated ECC‐induced force deficit and autolysis of calpain‐1, and increased the amounts of S‐nitrosylated calpain‐1. Regarding ryanodine receptor (RyR) and dihydropyridine receptor (DHPR), ECC‐induced proteolysis was completely inhibited by ARG, whereas the inhibition was partial for junctophilin‐1 (JP1). Skinned fiber analyses (the second experiment) showed that ARG also inhibited ECC‐elicited reductions in the ratio of depolarization‐induced to maximum Ca2+‐activated force. In the third experiment, homogenates of rested muscles were treated with S‐nitrosylating agent, S‐nitrosoglutathione (GSNO), and/or high Ca2+ concentration ([Ca2+]). Treatment with high [Ca2+] and without GSNO produced proteolysis of RyR, DHPR, and JP1. On the other hand, treatment with high [Ca2+] and GSNO caused complete inhibition of RyR and DHPR proteolysis and partial inhibition of JP1 proteolysis. These results indicate that ARG ingestion can attenuate ECC‐induced proteolysis of Ca2+ regulatory proteins and force deficit by decreasing calpain activation via S‐nitrosylation.It has been shown that calpains are involved in the proteolysis of muscle proteins that occurs with eccentric contraction ( Eccentric contraction (ECC) is a contraction in which skeletal muscles are stretched while contracting. It is a part of normal activities such as walking downstairs or lowing a heavy weight, and is prone to result in larger and longer‐lasting force deficits compared with concentric and isometric contractions (Lavender and Nosaka 2+‐regulated cysteine (Cys) proteases . In a recent study from our laboratory, it was shown in fast‐twitch muscles of rats that treatment with a calpain inhibitor can attenuate ECC‐elicited force deficits and proteolysis of junctophilin (JP) and dihydropyridine receptor (DHPR) that regulate Ca2+ release from the sarcoplasmic reticulum  primarily by neuronal NO synthase (NOS), is moderately generated in the resting state, and its production increases markedly with contractile activity , which is synthesized from S‐nitrosylation of the enzyme, resulting in reduction of the muscle protein degradation and force deficits that occur with ECC. However, in this regard, some considerations arise in light of the following previous findings: (1) all studies, except for one by Lomonosova et al.  JP,S‐nitrosylation of the enzyme; and (2) ECC‐dependent proteolysis of Ca2+ regulatory proteins. Furthermore, the effects of ARG ingestion and ECC on the SR Ca2+ release and myofibril functions were examined using mechanically skinned fibers. The experiments conducted with fast‐twitch muscles of rats reveal that ARG ingestion can inhibit ECC‐induced proteolysis of Ca2+ regulatory proteins and force deficit by decreasing the proteolytic function of calpains, which result from S‐nitrosylation of the enzyme.In this study, two questions were investigated as to whether ARG ingestion prior to ECC is capable of mitigating: (1) ECC‐induced calpain activation via The Journal of Physiology written by Drummond  whether in vitro S‐nitrosylation of calpain decreases Ca2+‐elicited proteolysis, respectively.In this study, we performed three separate experiments (1–3) with the following aims: (1) whether ARG ingestion facilitates recovery of force production after ECC and whether the recovery is because of decreased proteolysis; (2) whether ARG ingestion attenuates ECC‐induced disturbances in SR Can = 29; Charles River Laboratories, Yokohama, Japan). They were provided standard chow  containing 1.53% (mass mass−1) ARG and water ad libitum, and were individually housed in a cage in a thermally controlled room (~24°C) with 12‐h light/dark cycle. An intraperitoneal injection of a mixture of medetomidine (0.4 mg kg‐body wt−1), midazolam (2.0 mg kg‐body wt−1), and butorphanol (2.5 mg kg‐body wt−1) was used for anesthesia. At the end of experiments, the rats were euthanized with pentobarbital sodium (200 mg kg‐body wt−1), followed by cervical dislocation.The experiments were performed in male Wistar rats (n = 7 for each group). ARG rats were provided water containing 0.3% (mass vol.−1) ARG  ad libitum for 7 days starting from 3 days before the ECC protocol and continuing for 3 days after ECC . On the contrary, age‐matched controls were given water without ARG. Based on the chow intake (24.7 g day−1) and the ARG content (see above) in the chow, on the average, rats were estimated to ingest ~990 mg kg‐body wt−1 day−1 of ARG from food. The supplemental dose (611 mg kg‐body wt−1 day−1) of ARG used in this study is similar to that utilized in the study by Lomonosova et al.  or an ARG group . ECCs were repeated every 4 sec for 200 cycles.Eccentric contraction was performed as described previously : 115 NaCl, 20 NaHCO3, 11 glucose, 5 KHCO3, 5 N,N‐bis(2‐hydroxyethyl)‐2‐aminoethanesulfonic acid, 2 CaCl2, 1 MgCl2, 0.38 glutamine, and 0.3 glutamic acid. The solution was continuously bubbled with 95% O2–5% CO2, giving a bath pH of 7.4. Isometric contractions were elicited by direct stimulation via two platinum plate electrodes with a 1‐sec train of a 1‐msec pulse at various frequencies . The resulting forces were recorded on a personal computer and analyzed using LabChart version 8. After the in vitro measurements, tibialis anterior (TA) muscles were excised from both legs, frozen in liquid nitrogen, and stored at −80°C for biochemical analyses to be performed later. EDL muscles were not used for biochemical analyses because of restrictions on the amount of this muscle available.Following the in situ measurements, extensor digitorum longus (EDL) muscles were excised from both the legs. The muscles were mounted in a chamber (30°C) between a force transducer and an adjustable holder, and were perfused with standard Krebs–Ringer solution consisting of (in mmol L−1) of an ice‐cold buffer consisting of 20 mmol L−1 Tris (pH 7.4), 5 mmol L−1 EDTA, 5 mmol L−1 EGTA, 1 mmol L−1 dithiothreitol, 0.5 mmol L−1 phenylmethanesulphonyl fluoride, 14 μmol L−1 pepstatin A, and 10 μg mL−1 4‐(2‐aminoethyl)‐benzenesulfonylfluoride. The protein content of the homogenate was determined by the Bradford assay using bovine serum albumin as the standard (Bradford μg) were electrophoretically separated and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight at 4°C with primary antibodies as follows: anti‐RyR1/2 , anti‐DHPR α1 subunit , anti‐JP1 , anti‐calsequestrin (CSQ) 1 , and anti‐calpain‐1 . The membranes were then incubated with secondary antibodies  for 1 h at room temperature. To quantify the total proteins on the membrane, it was stained with Rapid Stain Coomassie blue R Kit . The contents of four proteins investigated  were normalized to total proteins.Immunoblot analyses were performed as described previously  of a NOS inhibitor, N(G)‐nitro‐l‐arginine‐methyl ester, indicating that NOx levels can be used as an indicator of the amounts of endogenously produced NO. NOx levels were spectrophotometrically determined as described previously  of ice‐cold PBS (pH 7.4) containing 10 mmol L−1N‐ethylmaleimide and 2.5 mmol L−1 EDTA. The mixture was then centrifuged at 10,000 g for 5 min at 4°C and the resultant supernatant was collected. After adding equal amounts of acetonitrile, the mixture was vortexed and centrifuged as mentioned above. The supernatant (100 μL) was mixed with equal amounts of 8 mg mL−1 vanadium (III) chloride, followed by Griess reagents (100 μL) consisting of 1% (mass vol.−1) sulfanilamide and 0.05% (mass vol.−1) N‐(1‐naphthyl) ethylendiamine dihydrochloride. In the experiments, the Griess reagent in the controls was replaced with 2.5% (vol. vol.−1) HCl. After incubation at 37°C for 30 min, the absorbance was read at 540 nm.In a preliminary experiment, we observed that ECC evoked increases in the amounts of muscle nitrite and nitrate (NOx) and that the increases were suppressed by an oral ingestion  of ice‐cold RIPA buffer consisting of 150 mmol L−1 NaCl, 50 mmol L−1 Tris/HCl (pH 7.4), 5 mmol L−1 NaF, 1 mmol L−1 NaVO4, and 1% (vol. vol.−1) Triton X‐100. The homogenate was centrifuged at 10,000 g for 5 min at 4°C and the resultant supernatant was collected.Tibialis anterior muscles (~50 mg) were homogenized in 5 vol. (mass vol.−1) S‐methyl methanethiosulfonate and 2.5% (mass vol.−1) SDS at 50°C for 20 min under agitation. After the addition of 3 vol. of acetone, the mixture was allowed to precipitate for 20 min at −20°C, followed by centrifugation at 3000 g for 10 min at 4°C. The pellet was washed twice in 70% acetone to remove all excess S‐methyl methanethiosulfonate and was resuspended in PBS containing 1 mmol L−1 EDTA and 0.1 mmol L−1 neocuproine (PEN buffer). The protein concentrations in the samples were adjusted with the PEN buffer to achieve equal concentrations (1 mg mL−1) and a small fraction (10 μL) of each sample was removed for analysis of protein “input” to SNO‐RAC.To block free thiols, the supernatant was incubated with a solution composed of 0.1% (vol. vol.S‐nitrosylated thiols were reduced to free thiols by adding 5 μmol L−1 CuCl and 5 mmol L−1 sodium ascorbate (Cu‐Asc treatment), to capture proteins containing free thiols, the samples were incubated with the resin slurry containing 15 mg thiopropyl sepharose 6B for 2 h at room temperature.After −1) SDS (PENS solution) and were centrifuged at 1000 g for 10 sec at room temperature. This procedure (wash and centrifugation) was repeated five times. The proteins captured were eluted by incubating the resins with PENS solution containing 100 mmol L−1β‐mercaptoethanol for 20 min. Following centrifugation at 1000 g for 10 sec, the supernatant was collected. Steps 2–4 were performed in the dark to avoid light‐induced reduction of ascorbate and photolysis of S‐nitrosylated sites.Resins were washed in the PEN buffer containing 1% . The procedures for ARG ingestion and ECC were similar to those used in experiment 1, with the exception of the ARG concentration (0.6%) in drinking water . This is because relative water intake (per body weight) declines as body weight increases. On an average, rats were estimated to ingest ~720 mg kg‐body wt−1 day−1 of ARG from food.In experiment 2, skinned fiber experiments were performed. In the preliminary experiments, when muscles from rats weighing <450 g were used, it was not easy to prepare skinned fibers with their excitation–contraction coupling mechanism intact. For this reason, rats aged 12–13 weeks  were used in the experiment 2 (n = 41) were initially subjected to measurements of depolarization (depol)‐induced force responses, followed by caffeine‐induced and maximum Ca2+ (max Ca2+)‐activated force responses. Analyses of force–Ca2+ relationship were also done in some skinned fibers (n = 28).Three days after ECC, TA muscles were excised from both legs and mechanically skinned fibers were prepared as described previously . Max Ca2+‐activated force was normalized to the cross‐sectional area of the fibers (specific max Ca2+ force). The cross‐sectional area was modeled as a circular profile if the diameter was similar along the fiber segment and was calculated from an average of three width measurements.Each skinned fiber was initially equilibrated in the K‐HDTA solution for 2 min before depol. The fiber was depolarized by replacing the K‐HDTA with the Na‐HDTA solution for ~5–10 sec, which typically results in Ca2+] relationship was determined by exposing the skinned fiber to a sequence of [Ca2+] solutions . The force response produced at each [Ca2+] was expressed as a percentage of the maximum force. Using SigmaPlot 12 software , [Ca2+]50, which is the [Ca2+] at the half‐maximum force, and Hill coefficient (the steepness of the force–[Ca2+] relationship) were calculated.The force–.The purpose of the experiment 3 was to examine whether in vitro −1) of an ice‐cold buffer composed of 20 mmol L−1 Tris/HCl (pH 7.4), 5 mmol L−1 EGTA, 0.5 mmol L−1 phenylmethanesulfonyl fluoride, 14 μmol L−1 pepstatin A, and 10 μg mL−1 4‐(2‐aminoethyl)‐benzenesulfonylfluoride. The homogenate was centrifuged at 200 g for 10 min at 4°C and the resultant supernatant was collected. To S‐nitrosylate the protein thiols, the supernatant was preincubated with 1 mmol L−1S‐nitrosoglutathione (GSNO) for 15 min at 37°C. It has been reported previously that when GSNO is applied to proteins immediately after being dissolved in solution, it causes S‐glutathionylation but not S‐nitrosylation  were homogenized in 5 vol. .Immunoblot was performed in a manner similar to those used in the experiment 1. To check the assay specificity for 2+ ratio of skinned fibers, which did not have equal variances, were analyzed using Kruskal–Wallis ANOVA on ranks. In experiment 3, the effects of GSNO and Ca2+ were investigated using one‐way ANOVA for repeated measures. When significant differences were detected, Holm–Sidak or the Dunn's methods were used, as appropriate. The acceptable level of significance was set at P < 0.05. Statistical analyses were performed using SigmaPlot version 12.Data are presented as means ± SEM. In experiments 1 and 2, except for force production of skinned fibers, the effects of ECC (rested vs. ECC muscles) and ARG ingestion (CON vs. ARG rats) were investigated using two‐way ANOVA. Data on depol/max CaIn a preliminary experiment, we observed that immediately after ECC, force output developed by anterior (TA and EDL) muscles was significantly decreased (by ~50–70%) in both CON and ARG rats and there were no differences in the extent of decreases between them. Three days following ECC, the force in the anterior muscles from CON rats remained low at all stimulation frequencies investigated Fig. A. A greaTypical samples of immunoblot for RyR, DHPR, JP1, and CSQ1 are shown in Figure 2+ regulatory proteins , suggesting no changes in the maximal function of the myofibril. A greater loss of force at low stimulation frequencies shown in Figure 2+ release and/or decreased myofibrillar Ca2+ sensitivity  treatment (T) without GSNO and Ca2+; (2) T without GSNO and with Ca2+; (3) T with GSNO and without Ca2+; and (4) T with GSNO and Ca2+, which are referred to as T1, T2, T3, and T4, respectively. T2 decreased the RyR, DHPR, and JP1 contents to 61%, 67%, and 34%, respectively, of the value observed with T1  inactivation of calpain‐1 inhibits ECC‐dependent proteolysis of Ca2+ regulatory proteins; (4) restoration of SR Ca2+ release function and force production is facilitated.There is a large body of literature showing that ECC‐induced force deficits are mainly ascribable to calpain‐1 activation that leads to proteolysis of key proteins involved in the excitation–contraction coupling  except for some pathological conditions, peroxynitrate mainly causes nitration rather than S‐nitrosylation  and oxidized Cys residues or via generation of GSH‐derived species. With the consideration that GSH levels in the myoplasm are increased a few days following ECC , it is possible that Cys 134 on fast troponin I might be S‐glutathionylated in muscles subjected to ECC.As reported previously, elevated levels of NO lead to reduced myofibrillar Cages Fig. A. A seriges Fig.  has reve2+ regulatory proteins and force deficit, which is mediated through decreases in the proteolytic function of calpains due to S‐nitrosylation of the enzyme. It is well known that proper physical activity attenuates chronic diseases such as cardiovascular diseases, hypertension, diabetes, and obesity. Acute or unaccustomed exercise is prone to result in long‐lasting force deficits, since muscle activities more or less include an eccentric component. Given that the majority of locomotion in normal daily activities involves submaximal contractions, particularly, force depressions at low‐stimulation frequencies, which are caused by an impaired function of Ca2+ regulatory proteins, may contribute to the sensation of muscle weakness. These phenomena will reduce the motivation of people trying to exercise. The present findings suggest that a supplemental ingestion of ARG exerts beneficial effects, such as the restoration of muscle performance after physical activities.In this study, we show that ARG ingestion inhibits ECC‐induced proteolysis of CaThe authors declare that they have no competing interests.


Mechanically-induced [Ca2+]i increases and their subsequent propagation to neighboring astrocytes in culture is a classical model of astrocyte calcium wave and is known to be mediated by gap junction and extracellular ATP, but the role of each pathway remains unclear. Pharmacologic analysis of time-dependent distribution of [Ca2+]i revealed three distinct [Ca2+]i increases, the largest being in stimulated cells independent of extracellular Ca2+ and inositol 1,4,5-trisphosphate-induced Ca2+ release. In addition, persistent [Ca2+]i increases were found to propagate rapidly via gap junctions in the proximal region, and transient [Ca2+]i increases were found to propagate slowly via extracellular ATP in the distal region. Simultaneous imaging of astrocyte [Ca2+]i and extracellular ATP, the latter of which was measured by an ATP sniffing cell, revealed that ATP was released within the proximal region by volume-regulated anion channel in a [Ca2+]i independent manner. This detailed analysis of a classical model is the first to address the different contributions of two major pathways of calcium waves, gap junctions and extracellular ATP.Wave-like propagation of [Ca Calcium waves are a remarkable aspect of [Ca2+]i dynamics in astrocytes, and a unique type of intercellular communication in astrocyte networks, intercalating neuronal circuitries and vasculature. Various pharmacologic and physical stimuli have been found to induce [Ca2+]i increases propagating between astrocytes in cell cultures2, in brain slices4, and in other in vivo preparations6. These calcium waves are regarded as transmitting physiologic and pathologic signals within the brain, because they influence the activities of adjacent neurons8, microglia9, and endothelial cells10. Furthermore, recent studies have demonstrated the involvement of astrocyte networks and calcium waves in regulating neuronal activities11 and neurological diseases13.Deeper understanding of the mechanisms underlying the spatio-temporal diversity and complexity of i increases18, and purinergic signaling has been found to be involved in calcium waves20. Furthermore, gap junction and purinergic signaling are regulated in a supplementary manner to maintain calcium waves21. However, the contributions of these components to the dynamics and functions of calcium waves, and the mechanisms involved in initiating [Ca2+]i increases and release ATP in calcium waves are incompletely understood.Because calcium waves can propagate between astrocytes in the absence of physical contact17 and experimental2 studies have shown that calcium waves can be mechanically induced by gently touching cultured astrocytes with tips of glass pipettes. The present analysis of this classical model pharmacologically and by using an ATP sniffing cell revealed distinct [Ca2+]i increases during calcium waves. This study was therefore designed to assess the distinct contributions of gap junction and extracellular ATP and the ATP release mechanism in calcium waves, revealing novel aspects of the diverse and complicated dynamics of astrocyte [Ca2+]i.Theoretical2+]i increase in the mechanically-stimulated cell (arrow) propagated to adjacent cells, and the area of [Ca2+]i increases reached a maximum at 24 sec. Then, [Ca2+]i in the distal region declined to the baseline by 120 sec, whereas that proximal to the stimulated cell remained elevated for longer than 120 sec. The distribution of [Ca2+]i increases was expressed as a maximum [Ca2+]i projection, in which each pixel represents the maximum Δ340/380 ratio during the calcium wave  as [Ca2+]i increase in the stimulated cell, and the persistent (orange) and transient (blue) [Ca2+]i increases as [Ca2+]i increases sustained and declined until 120 sec, respectively. The appropriateness of 120 sec was clarified later. The histogram of maximum [Ca2+]i increases along a line in Fig. 2+]i increase was the largest [Ca2+]i increase during the calcium wave, and the persistent [Ca2+]i increases were larger than the transient [Ca2+]i increases  were persistent, whereas those in the region of transient [Ca2+]i increases (Cell 4–6) were transient , which inhibits Ca2+ influx; and by treating cells with 1 μM thapsigargin (TG), 10 μM U73122 and 20 μM Xestospongin C (Xest C), which inhibit, calcium storage, inositol1,4,5-trisphosphate (IP3) production and IP3 receptor, respectively. Figure 2+-free and TG-treated cells. Omitting extracellular Ca2+ was found to eliminate the persistent [Ca2+]i increases without affecting the peak [Ca2+]i increase or propagation of the calcium wave, whereas treating with thapsigargin abolished the calcium wave without affecting the peak [Ca2+]i increase. Figure 2+]i increases were not significantly affected by any pharmacologic treatment, indicating that the peak [Ca2+]i increases were not mediated by Ca2+influx or IP3-induced Ca2+release (IICR), and that alterations of calcium waves described below did not reflect the difference of initial [Ca2+]i increases before propagations. In contrast, incubation of cells with Ca2+-free medium, TG, TG + Ca2+-free medium, U73122, and Xest C resulted in significant reductions in areas of the persistent [Ca2+]i increases to 32%, 18%, 9%, 40%, and 8%, respectively. The elimination of persistent [Ca2+]i increases was partial in some experiments of Ca2+-free group, presumably due to insufficient reduction of extracellular Ca2+ concentration in the nominally Ca2+-free condition. Although Ca2+-free medium had no effect on areas of the transient [Ca2+]i increases, the latter was significantly reduced by TG, TG + Ca2+-free, U73122 and Xest C to 2%, 2%, 5% and 1%, respectively. These findings indicate that the persistent [Ca2+]i increase is maintained synergistically by Ca2+influx and IICR, whereas the transient [Ca2+]i increases are mediated by IICR alone.The mechanisms underlying the [Ca2+ on calcium waves were further analyzed. A representative [Ca2+]i increase in an individual cell treated with Ca2+-free medium was transient similar to that in the distal region of control  were similar to that of the transient [Ca2+]i increases in the control group (9.3 ± 1.6 μm/sec), with both being significantly lower than the velocity of the persistent [Ca2+]i increase in the control group (22.6 ± 2.9 μm/sec)  Fig. . These r22, 300 μM gap-1923; a pannexin hemichannel blocker, 1 mM Probenecid; an ATP receptor antagonist, 100 μM suramin; and an ATP degrading enzyme, 15 unit/ml apyrase. Figure 2+]i increases, whereas suramin eliminates the transient [Ca2+]i increases without affecting the peak or the persistent [Ca2+]i increases. Figure 2+]i increases were not significantly affected by any pharmacologic treatments, indicating that they do not involve either gap junction or purinergic signaling. In contrast, CBX, 18GA and octanol significantly reduced areas of the persistent [Ca2+]i increases, to 10%, 8% and 7%, respectively, and reduced areas of the transient [Ca2+]i increases to 1%, 1% or 0.8%, respectively, indicating that gap junctions are essential for calcium waves. Gap-19 and probenecid did not affect either the persistent or transient [Ca2+]i increases, indicating that connexin and pannexin hemichannels are not involved in calcium waves. Suramin and apyrase had no effect on areas of the persistent [Ca2+]i increases, but significantly reduced areas of the transient [Ca2+]i increases to 20% or 18%, respectively, suggesting that purinergic signaling is involved in the generation and/or propagation of transient [Ca2+]i.The mechanisms underlying calcium wave propagation were pharmacologically examined by treating cells with the gap junction blockers, 100 μM carbenoxolone (CBX), 100 μM 18β-glycyrrhetinic acid (18GA) and 1 mM octanol; a blocker of gap junction hemichannels containing connexin 43 (Cx43), which is a major connexin subtype in cultured astrocytes2+]i increases were further analyzed. Individual astrocytes showed persistent [Ca2+]i increases, regardless of the presence of purinergic inhibitors  or area under the curve (AUC), was not affected by these purinergic inhibitors  medium, which is commonly used to induce the release of macromolecules from gap junction hemichannels24. As expected, CMF enhanced extracellular ATP, but this increase was significantly reduced to 4% and 10% by CBX and gap-19, respectively  both in the presence and absence of astrocytes. The mean distances between stimulated astrocytes and HEK293 + GCaMP2 cells showing 25–50% increases in [Ca2+]i and the amplitudes and AUC of their [Ca2+]i increases were not affected by the presence of astrocytes , or a cell permeable triadin, 10 μM vacuolin-1, which reduce lysosomes. In addition, cells were treated with 2 μM bafilomycin, an inhibitor of vesicular ATPase that depletes vesicles for exocytosis. None of these inhibitors had any effect on the components of calcium waves, indicating that neither lysosome nor conventional exocytosis was involved in ATP release during calcium waves  fast propagation of persistent [Ca2+]i increases in the proximal region, which are mediated by gap junctions and (iii) slow propagation of transient [Ca2+]i increases in the distal region, which are mediated by extracellular ATP. These calcium waves also involve ATP release via VRAC activated by non-Ca2+ signal propagation via gap junctions in the proximal region.The present study demonstrates that mechanically-induced astrocyte calcium waves consist of three components; (i) i16 were shown in previous studies on astrocyte calcium waves. However, to our knowledge, this is the first study demonstrating distinct components of astrocyte calcium waves, and showing different contributions of gap junction and extracellular ATP to calcium wave propagation by characterizing these components.The findings in the present study are in good agreement with those of previous studies. For example, the velocities of the calcium waves  were equivalent to those previously reported, 20 μm/sec.2+]i increases were not affected by any pharmacologic inhibitors of [Ca2+]i increase or propagation. The distinction we observed between the pharmacologic characteristics of peak [Ca2+]i and adjacent propagating [Ca2+]i increases were consistent with those of previous studies, which showed that a PLC and a SERCA inhibitor eliminated propagating [Ca2+]i increases in the calcium waves of airway epithelial cells31 and smooth muscle cells32, respectively, without affecting the [Ca2+]i increases in mechanically-stimulated cells. The lack of effect of the absence of extracellular Ca2+ on peak [Ca2+]i increases excludes the possibility that touching with glass pipette tips induced the initial [Ca2+]i increases by Ca2+ influx via mechanosensitive channels expressed in astrocytes33, or via physically-damaged plasma membranes. Since both omitting extracellular Ca2+ and inhibiting IICR did not affect increases in peak [Ca2+]i, mechanical stimulation may induce Ca2+ release from thapsigargin-insensitive calcium stores, which are not involved in IICR35.We also found that the peak [Ca2+]i increases, which were sustained in the proximal region for several minutes, were larger and propagated faster than the transient [Ca2+]i increases. Since these [Ca2+]i increases were eliminated by IICR inhibitors and by omitting extracellular Ca2+, they may be shaped synergistically by IICR and Ca2+ influx. The induction of IP3 by mechanical stimulation has been demonstrated pharmacologically in many cell types including astrocytes36. Theoretical studies have suggested that IP3 mobilization through gap junctions is the main pathway of astrocyte calcium waves, and that the regenerative production of IP3 by Ca2+ dependent PLC subtypes drives the propagation37. Regenerative IP3 production likely underlies the persistent [Ca2+]i increases and the gap junction-mediated propagation of calcium waves. It may also be essential for ATP release in the proximal region, whereas the IP3 production for the transient [Ca2+]i increases, which was mediated by P2Y receptors, was not amplified by Ca2+-dependent PLC or capable of inducing ATP release.The persistent [Ca2+ eliminated the persistent [Ca2+]i increases, indicating that the difference between the persistent and transient [Ca2+]i increases was due to Ca2+ influx. Our attempts to pharmacologically identify calcium channels responsible for Ca2+ influx have been unsuccessful. Purinergic inhibitors did not influence persistent [Ca2+]i increases, whereas HEK293 + GCaMP2 cells detected extracellular ATP in the proximal region. These findings suggested that ATP occupies ATP receptors, but that ATP-induced IICR is occluded by the large persistent [Ca2+]i increases in the proximal region. Omitting extracellular Ca2+ likely eliminated these large persistent [Ca2+]i increases, allowing detection of ATP-induced IICR in the proximal region.Omitting extracellular Ca2+]i increases are abrogated by purinergic inhibitors and are insensitive to extracellular Ca2+, these increases have been attributed to ATP-induced IICR. The finding, that extracellular ATP in the distal region, as measured by HEK293 + GCaMP2 during calcium waves, was preserved even when astrocytes were removed by scratching, suggested that ATP is released within the proximal region and passively diffuses into the distal region as a continuous flow. These findings contradict a previous hypothesis, that ATP is released by a Ca2+-dependent regenerative process during propagation14. The lack of a regenerative process likely accounts for the lower velocity in the distal region. That is, [Ca2+]i increases in the proximal region propagate rapidly and effectively by direct intracellular communications via gap junctions and regenerative IP3 production. In contrast, [Ca2+]i increases in the distal region propagate slowly and passively by diffusion of ATP.Because the transient [Ca2+]i increases in the proximal region, suggested that extracellular ATP transmits [Ca2+]i increases between astrocytes and HEK293 + GCaMP2. Moreover, the ability of CBX to abolish GCaMP2 responses suggests the involvement of gap junctions in ATP release. These findings, together with results showing that exogenous expression of connexin potentiated ATP release via anion channels38, indicate that VRAC is activated by a signal mediated by gap junctions. This signal is not likely to be [Ca2+]i, because (i) Ca2+-free medium reduced proximal [Ca2+]i increases without affecting ATP release measured by GCaMP2 responses; (ii) glutamate induced robust [Ca2+]i increases in astrocytes but not in adjacent HEK293 + GCaMP2 cells, and (iii) glutamate and ionomycin did not induce ATP releases detectable by luciferase assays. The Ca2+ independent ATP release during astrocyte calcium waves is consistent with previous imaging of extracellular ATP by luciferase36. In addition, Bergman glia were shown to release glutamate via DIDS-sensitive anion channels following optogenetic stimulation39, suggesting the possibility that depolarization and/or associated movement of ions trigger DIDS-sensitive release of anionic molecules.HEK293 + GCaMP2 cells, which are equivalent in sensitivity and pharmacologic properties to astrocytes in detecting extracellular ATP, were used in the present study to measure ATP release during calcium waves. Our findings, that purinergic inhibitors abolished GCaMP2 responses without affecting astrocyte i49 or releasing IL1β50. In vivo microglia adjacent to focal insults to brain parenchyma undergo structural changes in response to ATP released by astrocytes via a mechanism involving gap junctions51. This pathological activation of microglia is likely due to astrocyte release of ATP during calcium waves. Furthermore, blockers of P2Y receptors, including suramin53, gap junctions54 and VRAC55 are neuroprotective after brain injury, strengthening the pathological implications of astrocyte ATP release. DCPIB has been reported to attenuate inflammatory responses after brain injury56. Further investigations based on the results of this study may identify the mechanically-induced signaling molecules, which propagate via gap junctions, and subsequent cascades for activating VRAC after mechanical stimulation, providing new insights into mechanisms of brain injury and possibly identifying therapeutic targets.The present study found that a single mechanical stimulation of an individual astrocyte induced ATP release via VRAC from adjacent astrocyte networks connected by gap junctions. This process may represent the initiation and/or propagation of pathological processes following brain injuries. Microglia co-cultured with astrocytes respond to adjacent astrocyte calcium waves by increasing [Ca2+]i dynamics and purinergic signaling of astrocytes remain incompletely understood. Addressing the mechanisms of action and functions of astrocyte [Ca2+]i, including calcium waves, calcium oscillation, and spontaneous [Ca2+]i increases using cell culture models, remains important in basic and clinical research.This detailed analysis of a classical model has revealed novel aspects of astrocyte physiology, especially the different contributions of two major pathways of calcium waves, gap junctions and extracellular ATP. The physiological and pathological implications of [Ca4 cells/cm2 in 75 cm2 flasks and cultured in the presence of 5% CO2 at 37 °C for 1–2 weeks. The mixed brain cell cultures were shaken to remove less-adherent cells and treated with trypsin. The resulting astrocyte preparations were seeded at 2.5 × 104 cells/cm2 on 12 mm coverslips, which were incubated overnight with 1 μg/ml poly-D-lysine (Sigma) for imaging analysis; or in wells of 96 well plates for luciferase assays. Astrocytes were subjected to experiments 1–3 weeks after seeding.All animal experiments were approved by the Institutional Animal Care and Use Committee of Kobe University (Permission number: 25–20) and performed according to the Kobe University Animal Experimentation Regulations. Rats of either sex aged 0–2 postnatal days were sacrificed, and their cerebral cortices were removed and chopped into small pieces in Hank’s Balanced Salt Solution . Cell preparations were incubated with 0.25% trypsin , with occasional agitation, at 37 °C for 20 min. DNase  was added to a final concentration of 0.1% and the cell preparations were incubated at room temperature for 5 min. The samples were transferred to Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum  and antibiotics (50 μg/ml penicillin and 50 μg/ml streptomycin) and dispersed by pipetting. The resulting cells were seeded at 2.5 × 10HEK293 cells  were maintained in DMEM/F12 containing 10% FBS and antibiotics, and subcultured by treating with 10 mM EDTA/PBS. Cells were transfected with pN1-G-CaMP2  using Geneporter  and screened with 2 mg/ml G418  and by fluorescence imaging to obtain stable transfectants expressing GCaMP2.2, 2 mM CaCl2, 10 mM D-glucose, 10 mM Hepes pH 7.4) and incubated with 7.5 μM Fura2-AM  at 30 °C for one hour. After three additional washes with BSS, the cells were maintained in BSS containing 100 μM sulfinpyrazone (Sigma), which inhibits Fura2 sequestration and secretion57, at room temperature until imaging experiments. HEK293 + GCaMP2 cell suspensions were added to Fura2-loaded astrocytes in BSS, and cocultured for more than two hours at room temperature. Fluorescence images were obtained using an inverted microscope, IX70  equipped with a filter exchanger  and a cooled-CCD camera, Orca-R2 . The objective lens was UApo/340 20×/0.70w (Olympus). A 0.5x camera adaptor U-TV0.5XC (Olympus) was inserted before the camera. For Fura2-based calcium imaging, a set of F340 (Ex/DM/Em = 340/500/535) and F380 (Ex/DM/Em = 380/500/535) images were obtained every three seconds. For simultaneous calcium imaging of Fura2-loaded astrocytes and HEK293 + GCaMP2 cells, a set of F380 and F480 (Ex/DM/Em = 480/500/535) images were obtained every three seconds. Astrocytes were mechanically stimulated by touching them with 1–10 MΩ tips of a glass pipette , fabricated using P-87 . To maintain a constant stimulation force, contact by the tip was measured by input impedance using an amplifier , and its approach was stopped immediately upon any increase in input impedance. The tip remained in contact during calcium wave propagation, because the waves were unaffected regardless of tip withdrawal.Astrocytes were washed three times with Basal Salt Saline  and luciferase activity was measured using a luminometer .18β-Glycyrrhetinic acid, apyrase, ATP, thapsigargin, and U73122 were obtained from Sigma-Aldrich. DCPIB was obtained from Tocris ; DIDS was from Tokyo Chemical Industry Co. Ltd ; and GPN, vacuolin-1 and Xestospongin C were from Santa Cruz Biotechnology . Gap-19 was synthesized (HPLC grade) by MBL . All other chemicals were from Nacalai .The values are expressed as mean values ± SD. Differences among the values were analyzed using statistical methods mentioned in Figure Legends and differences with probability (p) values < 0.05 were considered significant.