Intracerebral hemorrhage (ICH) remains a serious clinical problem lacking effective treatment. Urocortin (UCN), a novel anti-inflammatory neuropeptide, protects injured cardiomyocytes and dopaminergic neurons. Our preliminary studies indicate UCN alleviates ICH-induced brain injury when administered intracerebroventricularly (ICV). The present study examines the therapeutic effect of UCN on ICH-induced neurological deficits and neuroinflammation when administered by the more convenient intraperitoneal (i.p.) route.ICH was induced in male Sprague-Dawley rats by intrastriatal infusion of bacterial collagenase VII-S or autologous blood. UCN (2.5 or 25 μg/kg) was administered i.p. at 60 minutes post-ICH. Penetration of i.p. administered fluorescently labeled UCN into the striatum was examined by fluorescence microscopy. Neurological deficits were evaluated by modified neurological severity score (mNSS). Brain edema was assessed using the dry/wet method. Blood-brain barrier (BBB) disruption was assessed using the Evans blue assay. Hemorrhagic volume and lesion volume were assessed by Drabkin's method and morphometric assay, respectively. Pro-inflammatory cytokine  expression was evaluated by enzyme-linked immunosorbent assay (ELISA). Microglial activation and neuronal loss were evaluated by immunohistochemistry.Administration of UCN reduced neurological deficits from 1 to 7 days post-ICH. Surprisingly, although a higher dose  also reduced the functional deficits associated with ICH, it is significantly less effective than the lower dose . Beneficial results with the low dose of UCN included a reduction in neurological deficits from 1 to 7 days post-ICH, as well as a reduction in brain edema, BBB disruption, lesion volume, microglial activation and neuronal loss 3 days post-ICH, and suppression of TNF-α, IL-1β, and IL-6 production 1, 3 and 7 days post-ICH.Systemic post-ICH treatment with UCN reduces striatal injury and neurological deficits, likely via suppression of microglial activation and inflammatory cytokine production. The low dose of UCN necessary and the clinically amenable peripheral route make UCN a potential candidate for development into a clinical treatment regimen. Spontaneous intracerebral hemorrhage (ICH) accounts for approximately 15% of stroke incidents in Western populations and an even higher proportion, up to 20-30%, in Asian populations . Early sPathological changes in ICH can be divided into primary and secondary brain injury. Primary injury occurs rapidly as a result of physical destruction of tissues and mass expansion of the hematoma , and is UCN, a 40-amino-acid endogenous neuropeptide, belongs to the corticotrophin releasing hormone (CRH) family of peptides, which bind two G-protein coupled receptors, CRH-R1 and CRH-R2 ,10. Thesin vitro studies, we showed that UCN alleviates inflammation and neurotoxicity mediated by endotoxin-activated microglia  × 100, where df is the mean flow after administration and dF is the mean flow at baseline.Another 12 rats were randomly assigned for evaluation of rCBF. The rCBF was monitored with a laser probe  inserted during surgery and connected to a Laser Doppler Blood Flow Perfusion Monitor 403A . The probe was placed in the peri-hematomal region in the striatum . We collected the baseline rCBF data before and the rCBF at 0.5, 1 and 3 hours after collagenase administration. The rCBF at each time point was collected for 10 minutes and averaged. All data were normalized by the following formula: percentage change = [ method . The eva-1 × 100%.Brain edema formation peaks at days 3 post-ICH ,34. We t® 488 dye using a Microscale Protein Labeling Kit , according to the manufacturer's instructions. The Alexa Fluor® 488 dye-labeled UCN (2.5 μg), with fluorescence excitation and emission maxima of approximately 494 and 519 nm, was administered i.p. one hour after ICH. Three hours after injection of the fluorescently labeled UCN, the rats were re-anesthetized with chloral hydrate (0.4 g/kg i.p.), and their brains were removed immediately and sectioned to 20 μm thickness with a cryostat. To confirm the entrance of urocortin peptide into the brain, 20 μm thick brain slices were labeled with anti-UCN antibody. Sections were incubated overnight at 4°C with UCN primary antibody . The slices were then washed with PBS and incubated for 1 hour with secondary antibody  at room temperature. After rinsing with PBS buffer, the slices were examined under a fluorescence microscope. After counter-staining the nuclei with DAPI, the slides were washed and mounted on cover slips with anti-fading mounting medium . The presence of the labeled UCN in the striatum was evaluated under a fluorescence microscope.Urocortin was labeled with Alexa FluorHemoglobin content determined by spectrophotometric measurement is a good indication of the hemorrhagic volume (bleeding) on day 1 post-ICH. This volume, however, can be affected by breakdown of the hemoglobin, swelling (edema) of tissue, and production of many inflammatory mediators immediately following the bleeding. Therefore, we conducted both a spectrophotometric measurement for hemorrhagic volume and a morphometric measurement for lesion volume in the ICH + saline and ICH + L-UCN groups on days 1 and 3 post-ICH.Rats were randomly used in the assessment of hemorrhagic volume on day 1  post-ICH. The accumulated hemorrhagic volume was quantified by a spectrophotometric assay as reported by Park et al., with minor modifications . Briefly3) was computed by multiplying the blood clot area (for day 1) and lesion area (for day 3) in each section by the distance between sections [Rat brain sections were captured for morphometric measurement  prior to being used for the cytokine ELISA assay or the dry/wet method (brain edema assay). Briefly, rat brains on day 1  and day 3  post-ICH were cut coronally through the needle entry plane (identifiable on the brain surface), and then serially sliced (2-mm thickness) anterior and posterior to the needle entry site. Digital photographs of the serial slices were taken and lesion volume was computed using an image analysis program . The total lesion volume  and centrifugation at 12,000 g for 30 minutes, the supernatants were collected and stored frozen at -80°C. During quantification, the cytokines  were normalized to 100 μg of protein in the supernatant using a commercial ELISA kit from R & D Systems  according to the manufacturer's instructions.A total of 8 rats were used for immunohistochemistry on day 3 post-ICH. Rats were anesthetized as described above and transcardially perfused with cold 0.1 M phosphate buffer saline followed by cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Brains were removed and immersed in 4% paraformaldehyde for 24 hours and 30% sucrose for another 24 hours. Coronal brain slices (20 μm thickness) were cut. The slices were collected at +1.0, 0.0, and -1.0 mm (center of the hemorrhagic lesion) anterior and posterior to bregma using a cryostat (Leica CM 1900). Three serial slices were taken at each plane , and processed for the staining and counting of marker-specific cells . Antibod+ cells with dense immunoreactivity and showing a rod-like appearance were counted as activated microglia. Numbers of positive cells were counted in six squares (1 mm2) randomly located around the lesion in each slice. An average cell number in each plane  was calculated from 54 squares in 9 planes. All cell counting was done by an independent investigator.Negative control slices from each animal were prepared for immunohistochemical staining processed in an identical manner except the primary antibodies were omitted. OX-42t-test and are presented as mean ± standard deviation (SD). The statistical comparisons among multiple groups were made using one-way ANOVA, and multiple time points by two way ANOVA followed by Bonferroni correction. In all instances, n refers to the number of animals in a particular group. A p value of < 0.05 is considered statistically significant.Data were statistically analyzed using Prism software for Student's L/H-UCN improved neurological deficits in a dose- and time-dependent manner in collagenase-induced ICH injury Figure . In all In the autologous blood infusion model of ICH (n = 12), we selected the dosage of UCN shown to be most effective in the collagenase model  to investigate the drug's therapeutic effect Figure . The mNSOn day 1 post-ICH, UCN (ICH + L-UCN group) significantly reduced the water content of the ipsilateral hemisphere to 80.7 ± 0.3% compared to the ICH + saline group (81.4 ± 0.9%) Figure . The wat2, pCO2 and pH among the sham + saline, ICH + saline and ICH + L-UCN  groups , heart rate (HR), rectal temperature, pOs Figure .2, pCO2 and pH for the time points studied  were not significantly different between the groups  and H-UCN caused a significant decrease in MABP to a maximum of 25 mm Hg at 3.0 hours and 40 mm Hg at 0.5 hour post treatment Figure , respectBecause the low dose of UCN (2.5 μg/kg) showed better improvement in the neurological deficits in terms of reducing mNSS than the high dose (25 μg/kg), the dose of 2.5 μg/kg UCN was adopted for subsequent experiments.To clarify whether UCN affected the regional cerebral blood flow, we demonstrated that ICH alone and ICH + L-UCN did not affect perihematomal rCBF within 3 hours observation, while H-UCN significantly reduced rCBF by approximately 20% , the labeled UCN was localized in the striatum on both ipsilateral  and day 3   and declined on days 3 and 7 post-ICH Figure . The hem) Figure , suggestSince BBB disruption is very likely a contributory cause to the brain edema that peaks on day 3 post-ICH, changes in BBB disruption were determined by Evans blue dye assay on this day. Representative brain coronal sections Figure  show EvaPro-inflammatory cytokine  levels in the ipsilateral striatum in the ICH + saline group were significantly increased on day 1 post-ICH as compared with day 0 pre-ICH in the sham + saline group; levels of TNF-α Figure  and IL-1+ microglial cells  of UCN was less effective than the low dose (2.5 μg/kg) of UCN in attenuating neurological deficits. Brain edema leads to an increase in intracranial pressure (ICP), causing severe tissue damage. Our water content results indicate relief of edema by UCN. However, there were no significant differences between the two doses of UCN in effectively reducing brain edema, indicating the maximum effect on reduction of brain edema is reached at 2.5 μg/kg of UCN  (data not shown). The kinetics of UCN crossing the BBB into the brain were not appreciably different between the high dose and the low dose of UCN in ICH-induced injury brain. This phenomenon may be due to saturable entry of UCN through the BBB, inhibited by excess UCN ,46. DetaGlial cells (mainly astrocytes and microglia), apart from providing physical support and insulation, play important roles in maintenance and repair of neurons as well as neural transmission . The celSeveral drugs can produce anti-oxidative effects, which protect microglia from damage, preserving their phagocytotic function for faster hematoma clearance and promotion of neuroprotection in ICH -52. Howein vivo.Recently, microglial activation inhibitors such as MIF  and minocycline, have shown a promising reduction in brain edema and tissue damage, and attenuation of functional deficits in experimental ICH ,53. We iin vitro study also demonstrated that UCN (5 nM-5 μM) did not inhibit collagenase enzyme activity (data not shown); therefore, UCN may not reduce collagenase-induced bleeding. These results indicate that the neuroprotective effect of UCN may not be due to reducing bleeding volume, but most likely due to reducing brain edema and neuroinflammation.Whether UCN can attenuate bleeding (hemorrhagic) volume remains a question. We found that post-ICH treatment with UCN did not affect the accumulated hemorrhagic volume at 24 hours (day 1) after UCN treatment, but significantly reduced the lesion volume on day 1 and 3 after ICH. Besides, our in vitro studies, which demonstrated that femtomolar concentrations of UCN can inhibit TNF-α production in cultured microglia treated with endotoxin [in vivo anti-inflammation studies mentioned above [Clinically, a stroke patient is usually admitted to the emergency clinic within 1-3 hours. The patient should be treated as early as possible, so we chose to administer UCN at one hour post-ICH. The effectiveness of the low dose of UCN is consistent with our previous ndotoxin ,23. As ced above ,53, the However, the biological activities of UCN in inflammation remain controversial. Although most studies agree that UCN is a powerful anti-inflammatory agent -24,54-56in vivo [in vitro study [UCN is a cardioprotective agent in ischemia/reperfusion-induced injuries. Activation of the PI3K/Akt and ERK 1/2 signaling pathways is part of the underlying mechanism in cardiomyocytes -20. Furtin vivo . We alsoro study . This cuUCN is a potent anti-inflammatory agent that is a potential target for drug design and development for clinical use. Further investigation of UCN for clinical treatment of ICH is highly warranted.The authors declare that they have no competing interests.HKL performed all animal-related procedures, analyzed the data and generated the figures. CYP, MJW, JYW and CWH received funding for the project and provided input during the drafting of the manuscript. TYL assisted in evaluation of the behavioral tests and calculated cell density. HFP assessed the staining, and assisted in data collection. JSK assisted in preparation the manuscript. JYW contributed to the design and preparation of the manuscript. All authors read and approved the manuscript.


Here, in vitro and in vivo, we determined that tripeptidyl peptidase II antagonists strongly activated the cAMP/PKA signaling pathway that drives sperm capacitation-associated protein tyrosine phosphorylation. We demonstrated that in the absence of Ca2+, TPIII antagonists elevated the intracellular Ca2+ levels in sperm, resulting in a marked improvement in sperm movement, capacitation, acrosome reaction, and the in vitro fertilizing ability. This antagonist-induced release of intracellular Ca2+ could be blocked by the inhibitors of ryanodine receptors (RyRs) which are the main intracellular Ca2+ channels responsible for releasing stored Ca2+. Consistent with these results, indirect immunofluorescence assay using anti-RyR antibodies further validated the presence of RyR3 in the acrosomal region of mature sperm. Thus, TPPII can regulate sperm maturation by modulating intracellular Ca2+ stores via the type 3 RyR.Recent studies have identified Ca Mammalian sperm must undergo functional alterations after maturation in the epididymis before they can competently interact with oocytes. This process is referred to as capacitation. Cauda epididymal and ejaculated sperm can be capacitated both in the female reproductive tract and in chemically defined media. Nevertheless, caput epididymal sperm do not possess the ability to undergo capacitation and fertilize eggs 2+, and HCO3–; and protein tyrosine phosphorylation Sperm capacitation comprises a series of processes, including modifications in the distribution of surface protein; alterations in the plasma membrane characteristics; changes in enzymatic activities; modulation of intracellular constituents such as cyclic adenosine monophosphate (cAMP), Ca2+ signaling in sperm is critical for fertilization, and it plays a pivotal role in sperm maturation, including motility, capacitation, and acrosome reaction 2+ signaling in sperm is associated with male subfertility 2+ can directly stimulate adenylyl cyclase, leading to the activation of the sAC/cAMP/PKA signaling pathway in sperm 2+ stores in mature sperm is not well understood, and the mechanism by which intracellular Ca2+ stores are mobilized remains to be elucidated.Ca2+2+ levels in human spermatozoa induced by follicular fluid 2+ channels during the acrosome reaction in sperm was found to be repressed by inhibitors of chymotrypsin-like proteases Several studies have indicated that some proteolytic enzymes are closely associated with sperm maturation and calcium signaling. Calpain, a cysteine protease, has been shown to modulate sperm capacitation and acrosome reaction in association with extracellular CaTpp2 locus could not be obtained due to early embryonic lethality. However, Their Tpp2 heterozygotes were leaner than their wild-type littermates, while their food intake was normal Tpp2 mice with >90% reduced expression of TPPII compared to the wild-type mice were viable, fertile, and normal in appearance and behavior Tpp2−/− were viable and grossly indistinguishable from wide type littermates, but in these mice, TPPII deficiency activated cell type-specific death programs Recently, it was found that tripeptidyl peptidase II (TPPII) could operate mostly downstream of proteasomes in cytosolic proteolysis Our present work by using a pharmacological approach aimed to investigate the role of TPPII in sperm maturation and to identify the mechanisms by which it regulates sperm function.TPPII was widely discovered in a variety of tissues, including the testes, brain, spleen, lungs, liver, heart, and kidneys Since TPPII shows enzymatic activity, we considered that it may play an important role in sperm maturation, and investigated its function using two TPPII antagonists–butabindide and AAF-CMK. Butabindide is a reversible and the most specific TPPII antagonist 2+, and HCO3– in the medium is essential for the activation of this pathway 3–, and Ca2+. When the sperm were incubated in the absence of BSA or HCO3– for one hour, the acceleration of tyrosine phosphorylation induced by TPPII antagonists disappeared  function as another major intracellular Ca2+ channel. In mammalian sperm, IP3Rs have been detected at the acrosome of several species, including mice, and in some cases also in the RNE 3R pathway inhibitors could not block the increase of tyrosine phosphorylation induced by TPPII antagonists  and capacitation-associated protein tyrosine phosphorylation . The inhibitors of RyRs could block the elevation of TPPII antagonist-induced protein tyrosine phosphorylation and the increase of intracellular Ca2+. Thus, TPPII can modulate the intracellular Ca2+ store via ryanodine receptor type 3. These data showed that the release of Ca2+ from intracellular Ca2+ stores alone is sufficient to initiate sperm capacitation and that the initiation and completion of the capacitated state may be regulated by sperm cell itself. Similar to our findings, it was previously reported that thapsigargin-induced release of intracellular Ca2+ is sufficient to initiate hyperactivation of bull sperm that were incubated in a Ca2+-free medium 2+.Activation of sperm capacitation-associated tyrosine phosphorylation is involved in the sAC/cAMP/PKA signaling pathway. Carylation . Our stu pathway . Howeverfunction . Whereasrylation . Therefo3Rs) are another major intracellular Ca2+ release channel in sperm, and IP3R-gated intercellular Ca2+ stores are related to the regulation of sperm hyperactivated motility 3R pathway inhibitors–U-73122 (inhibitor of the hydrolysis of PIP2 to IP3) and 2-APB (inhibitor of IP3R)–were unable to block the TPPII antagonist-induced protein tyrosine phosphorylation  were purchased from the Animal Center of the Chinese Academy of Sciences . They were housed in the animal housing at our institute before manipulation. Food and water were freely available throughout the experiments. All protocols were conducted according to the approval of the Institute Animal Care Committee of Shanghai Institute of Biochemistry and Cell Biology (Permit Number: SYXK2007-0017).Western blot analysis of the TPPII protein in spermatozoa was conducted according to a previously described protocol Immunofluorescence was indirectly detected as described previously 2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM glucose, 21 mM sodium lactate, 0.25 mM sodium pyruvate, 25 mM NaHCO3, and 3 mg/ml bovine serum albumin (BSA). All the chemicals were purchased from Sigma and were of the highest purity available. A tenfold concentrated solution of all the ingredients was first prepared without CaCl2, BSA, and NaHCO3, sterilized by passage through a 0.22-µm filter, and stored at –20°C in single-use aliquots. Working media were prepared by adding CaCl2, NaHCO3 and BSA and gassing the medium with a mixture of 5% CO2 and 95% air overnight at pH 7.2–7.4. As described in a previous report 3 was derived by adding 25 mM NaCl instead of 25 mM NaHCO3. In some experiments, Ca2+- and BSA-free media were used and the Ca2+ and BSA were added back to above final concentrations if necessary.Enriched Krebs-Ringer bicarbonate (EKRB) medium was used throughout the study for mouse sperm preparation and capacitation, and the preparation was adopted from previously published reports 6 cells/ml in appropriate medium and assessed using a computer-assisted semen analysis (CASA) machine. After incubation for various time periods and following treatment with different antagonists or inhibitors, the sperm were concentrated by centrifugation at 6,000 g for 2 min at room temperature, washed in phosphate-buffered saline (PBS) three times, resuspended in Laemmli’s sample buffer without mercaptoethanol, and boiled for 5 minutes. After centrifuging at 6,000 g for 2 min, the supernatant was collected, and 2-mercaptoethanol was added to attain a final concentration of 5%. The sperm extract was either used immediately or stored at –70°C until analysis.The cauda epididymis was excised and freed from the fat pad, blood vessels, and connective tissue. The tissue was then transferred to a dish containing 1 ml EKRB medium prewarmed to 37°C, and cut in several places with iridectomy scissors to release the spermatozoa into the medium. After 5 minutes, the sperm suspension was transferred to a 5 ml centrifuge tube. The final concentration of sperm was adjusted to 3–4×10SDS-PAGE was carried out in 12% gel. The sperm extracts were electrophoretically transferred to PVDF membranes  in all experiments. The blots were blocked with blocking buffer  Triton X-100, 0.25% (m/v) gelatin; pH 7.5) and probed with a monoclonal antibody against phosphotyrosine  and enhanced chemiluminescence detection using an ECL kit (Amersham). To confirm equal protein loading, the blots were stripped and reprobed with anti-α-tubulin monoclonal antibody (Sigma).The analysis procedure was a modification of our previously published method 2+ for 0, 30, 60, and 90 minutes. The total protein was extracted from these sperm and the changes in protein tyrosine phosphorylation were determined according to the method described above.We injected 10 mg/kg butabindide and 10 µg/kg Ala-Ala-Phe-chloromethylketone (AAF-CMK) into the tail veins of mice according to their body weights. After one hour, cauda sperm from the treated mice were collected and incubated in the absence or presence of Ca2+ for 5 minutes, after which the sperm suspensions were loaded with the acetoxy-methyl ester of fura-2  and incubated for 30 min at 37°C and 5% CO2. The suspensions were then centrifuged in EKRB medium without Ca2+ three times at 300 g for 5 min each time to remove extracellular free Fura-2/AM. The spermatozoa were finally resuspended in fresh medium without Ca2+, and sperm aliquots were treated by different antagonists or inhibitors under different experimental conditions and the fluorescence was monitored. Spectrofluorometry was performed in a methylacrylate cuvette with magnetic stirring, and warmed to 37°C in a heated cuvette holder. The fluorescence intensity was measured after equilibration for 2 min on a Varian Cary Eclipse (USA) spectrofluorophotometer following excitation at 340 nm and emission at 510 nm. The [Ca2+]i was calculated using the equation [Ca2+]i = Kd (F – Fmin)/(Fmax – F), where Kd = 224 nM. Fmax and Fmin were recorded at the end of the incubation period. Fmax was determined after the addition of 20 µM digitonin, and Fmin was determined after addition of 10 mM Tris-EGTA to the cuvette.Measurements were performed as described elsewhere 2+ at 37°C in an atmosphere containing 5% CO2. At the end of the incubation period, they were collected for staining with chlortetracyclin (CTC) and assessing for sperm capacitation and acrosome reaction as described elsewhere Sperm aliquots were treated by TPPII antagonists in the medium for 60 min with or without Ca2, the eggs were washed free of unbound sperm, transferred to droplets of the same medium and returned to the incubator. Fertilization was assessed by recording the number of two-cell embryos 24 h after insemination.In vitro fertilization was performed as previously described with light modifications Figure S1Localization of TPPII protein in mouse sperm. Cauda sperm before (0 h) and after (2 h) capacitation were probed with anti-TPPII polyclonal antibodies. Control sperm (Co) were examined by anti-TPPII polyclonal antibodies which were pre-incubated with the corresponding antigen peptide. Sperm DNA was stained with propidium iodide (PI) and can be seen in red (bars 10 µm). A representative of three independent experiments is shown.(TIF)Click here for additional data file.Figure S23 protein in spermatozoa.Localization of RyR Spermatozoa were probed with (Exp) anti-RyR3 and (Co) anti-RyR3 pre-incubated with the corresponding antigen peptide. Sperm DNA was stained with propidium iodide (PI) and can be seen in red (bars 10 µm). A representative experiment of three experiments is shown.(TIF)Click here for additional data file.Figure S32+ stores via ryanodine receptor 3.Proposed mechanisms by which TPPII antagonists regulated sperm function by modulating intracellular Ca The inhibition of TPPII by AAF-CMK and butabindide resulted in the activation of cAMP/PKA-mediated protein tyrosine phosphorylation. This action of TPPII antagonists was dependent on extracellular HCO3– and BSA. Ryanodine receptor inhibitors but not IP3R inhibitors could block this TPPII antagonist-induced sperm protein tyrosine phosphorylation.(TIF)Click here for additional data file.Figure S4Effect of EGTA on TPPII antagonist-induced changes of sperm function. (A–C) Spermatozoa were treated with AAF-CMK  and butabindide  for 60 min in the absence of 1 mM CaCl2 and in the presence of EGTA at the dose of 25 and 50 µM. The percentage (A), VAP (B), and VCL (C) of sperm motility were examined using CASA. Results are expressed as the mean ± SEM (n = 4). *P<0.05 as compared with the corresponding control (Co) (unpaired t test). (D) Sperm were treated with AAF-CMK  and butabindide  for 60 min in the absence of 1 mM CaCl2 and in the presence of EGTA at the dose of 25 and 50 µM. Protein tyrosine phosphorylation was assessed by Western blot analysis. α-Tubulin was used as the loading control. The Western blot is a representative of four independent experiments.(TIF)Click here for additional data file.Figure S5Effect of BAPAT-AM on TPPII antagonist-induced changes of sperm function. (A–C) Spermatozoa were treated with AAF-CMK  and butabindide  for 60 min in the absence of 1 mM CaCl2 and in the presence of BAPAT-AM at the dose of 2.5, 10 and 25 µM. The percentage (A), VAP (B), and VCL (C) of sperm motility were examined using CASA. Results are expressed as the mean ± SEM (n = 4). *P<0.05 as compared with the corresponding control (Co) (unpaired t test). (D) Sperm were treated with AAF-CMK  and butabindide  for 60 min in the absence of 1 mM CaCl2 and in the presence of BAPAT-AM at the dose of 2.5, 10 and 25 µM. Protein tyrosine phosphorylation was assessed by Western blot analysis. α-Tubulin was used as the loading control. The Western blot is a representative of four independent experiments.(TIF)Click here for additional data file.


We aimed to investigate whether rats with low inborn maximal oxygen uptake  Ca2+ ATPase function in LCR (39%). Na+/Ca2+ exchanger activity was comparable between the groups. Diastolic SR Ca2+ leak was increased by 109%. This could be partly explained by increased ryanodine receptors phosphorylation at the Ca2+-calmodulin-dependent protein kinase-II specific Ser-2814 site in LCR rats. T-tubules were present in 68% of HCR cells whereas only 33% LCR cells had these structures. In HCR, the significantly higher numbers of cells with T-tubules were combined with greater numbers of myocytes where Ca2+ release in the cell occurred simultaneously in central and peripheral regions, giving rise to faster and more spatial homogenous Ca2+-signal onset.Atrial myocyte function was depressed in Low Capacity Runners (LCR) relative to High Capacity Runners (HCR) which was associated with impaired CaThis data demonstrates that contrasting for low or high aerobic capacity leads to diverse functional and structural remodelling of atrial myocytes, with impaired contractile function in LCR compared to HCR rats. Breeding started from N:NIH stock obtained from the National Institute of Health (USA), as described previously The Norwegian Council for Animal Research approved the study, which was in accordance with the Guide for the Care and Use of Laboratory Animals by the European Commission Directive 86/609/EEC.2 max was measured by uphill (25°) treadmill running in a metabolic chamber until exhaustion as previously described VO2, 0.33 mM NaH2PO4, 22 mM glucose, 50 µU/ml bovine insulin , 25 mM HEPESNaOH (pH = 7.4)) with 0.4 mM EGTA and immediately canulated through aorta and retrogradely perfused  with isolation buffer containing 0.4 mM EGTA for 2–4 min. Then the hearts were perfused with the enzyme solution containing isolation buffer supplemented with 0.048 mM CaCl2 and 1 mg/ml collagenase . From the digested hearts (10–15 minutes perfusion) left atria were removed, cut into 3–5 pieces, and further digested by gentle stirring for 5–10 min in fresh enzyme solution until myocytes appeared. Tissue chunks were then transferred to isolation buffer containing 0.096 mM CaCl2 and 10 mg/ml 0.1% bovine serum albumin (Sigma), cut into as small pieces as possible and mechanically agitated with a pipette. The cell suspension was centrifuged at 40×g for 2 minutes in a 15 mL plastic centrifuge tube, the supernatant was gently removed and the cell pellet was resuspended in 2 ml of isolation buffer with 0.026 mM CaCl2.Left atria from rats were isolated using a modified mouse model protocol 2+ recordings, Ca2+ concentration in the perfusion buffer was increased to 1.8 mM. Fura-2/AM-loaded  cardiomyocytes were field stimulated by bipolar electrical pulses at 2 Hz and then 5 Hz on an inverted epifluorescence microscope . Cell shortening was measured by video-based sarcomere spacing  and intracellular Ca2+ concentration was measured by counting 510 nm emissions with a photomultiplier tube  after exciting with alternating 340 and 380 nm wavelengths (340/380F ratio) . Quantification of the Sarcoplasmic reticulum (SR) Ca2+ content and rate constant for fractional contribution of Ca2+ removal by SR Ca2+ ATPase (SERCA2a) and Na+/Ca2+ exchanger (NCX) are previously described in Seidler et al. For intracellular Caet al. 2+- leak from the SR. To bring the cellular Ca2+-content to a steady state, cardiomyocytes were electrically stimulated at 1 Hz in normal HEPES based 1.8 mM Ca2+-solution for 30–60 seconds. After the last electric stimulus, perfusion was switched to a 0 Na+/0 Ca2+ containing solution and diastolic Ca2+ concentration was measured in quiescent non-stimulated cardiomyocytes (40 seconds) ± Tetracaine (1 mmol/L). The 0 Na+/0 Ca2+ solution prevents the Na+ - Ca2+ exchange, which is the primary Ca2+-influx and efflux mechanism at rest. Tetracaine blocks the Ca2+-leak over the RyR. The quantitative difference between diastolic Ca2+-concentration with and without tetracaine determines leak. After the 40 second period in 0 Na+/0 Ca2+ ± Tetracaine solution, caffeine was added (10 mM) to assess the SR Ca2+-content. Diastolic Ca2+-leak is presented as diastolic [Ca2+]i in relation to total SR Ca2+-content.A method similar to that established by Shannon 2+ transients were studied using a laser scanning microscope  and a Zeiss 63×1.23NA oil-immersion objective. To visualize T-tubular network, quiescent, non-perfused cardiomyocytes loaded with the membrane specific Di-8-ANEPPS dye  were confocal Z-stack scanned (488 nm excitation and detection at >514 nm). This was performed with pinhole of 1 airy unit and 0.38 micron thick stacks. T-tubule density was analyzed with custom-made applications in IDL 6.0 , by counting pixels stained with the dye relative to the total number of pixels after removing pixels associated with the external cell membrane. To study spatiotemporal characteristics of Ca2+ transients, Fluo-3/AM  loaded cardiomyocytes were confocal line-scan recorded (488 nm excitation and detection at >514 nm) during steady state stimulation at 1 Hz. Repetitive scanning of a line parallel to the transversal axis of the cell were used to visualize Ca2+ signal. For the Ca2+ synchrony analysis, the transients were divided into 5 equal strips. Time from stimulation to 50% peak Ca2+ release was measured for each strip by the programme LabTalk Origin  to determine spatial differences in systolic rise time of the Ca2+ transient from the edges to the center of the cardiomyocytes.Imaging of T-tubular network and spatiotemporal characteristics of CaProteins  from left atrium were heated in LDS loading buffer (Invitrogen) and subjected to electrophoresis on pre-cast 3–8% Tris-acetate denaturing NuPAGE gels (Invitrogen). After separation for 3 hours at 150 V/220 mA and 4°C, gels were incubated in 2× NuPAGE transfer buffer (Invitrogen) contatining 0.02% SDS for 10 minutes. And proteins were electro-transferred onto PVDF membranes  at 20 V overnight and 4°C . The membranes were blocked with Odyssey blocking buffer (LiCOR) prior to incubation with monoclonal anti-ryanodine receptor (RyR2) , polyclonal anti-pS2809-RyR2 , and monoclonal anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , Temecula, CA) antibodies overnight at 4°C. After incubation with secondary goat anti-mouse IRDye800LT and goat anti-rabbit IRDye680LT secondary antibodies (Li-COR) bands were detected using an Odyssey infrared imaging system . Quantitative analyses were performed with Odyssey v.3.0 software and ImageJ Data Acquisition Software .Data are presented as mean ± SD. Student T-test was used to identify statistical differences between the groups. Man-whitney Rank Sum test was used if normality test (Shapiro-Wilk) failed. The Fisher’s Exact test was applied to the categorical data. P<0.05 was considered statistical significant.2 max was 24% lower in LCR rats compared to HCR rats  with and without tetracaine provides a quantitative assessment of SR (RyR2) Ca2+ leak  specific Ser-2814 site is apparently induced in LCR rats  and no differences were observed when comparing U- vs. U –shaped transients and W- vs. W shaped transients between groups. This suggests that the slower time to peak in LCR was partly due to high proportion slow U-shaped transients. Further spatiotemporal analysis of U-shaped Ca2+ transient revealed that the central Ca2+ release within the myocytes was significantly slower than the edges .Two types of Ca p<0.05, . Analysi2+ handling in atrial myocytes.This is the first study to demonstrate that low inborn aerobic capacity is directly associated with reduced contractile function and impaired Ca2+ handling in heart failure 2+ handling in LCR compared to HCR rats, which confirms that there is an association between aerobic capacity and development of atrial myocyte function. Ca2+ amplitude together with duration of Ca2+ transient are main determinants of cardiac contraction 2+ amplitude was preserved at 2 Hz in LCR compared to HCR rats, still fractional shortening was depressed in LCR rats, indicating reduced Ca2+ sensitivity. At 5 Hz stimulation there was a significant decrease in Ca2+ amplitude in LCR rats. The observed negative frequency dependent alteration in systolic Ca2+ amplitude in the LCR (illustrated in 2+ to the SR by SERCA2a and 2) less developed T-tubule structures and reduced initiation sites for Ca2+ activated Ca2+ release.We have previously reported that left ventricular myocytes from LCR rats have impaired systolic and diastolic function relative to HCR rats 2+ removal 2+ and thereby not able to reload the SR with Ca2+ available between stimulation. Despite this obvious explanation we were unable to detect any significant difference SR Ca2+ content after caffeine-stimulated depletion. The stimulation frequency before caffeine stimulation in our experiments was, however, performed after 1 Hz electrical stimulation, which probably is too low to tax the capacity of SERCA2a. Therefore, despite that the SERCA2a capacity is reduced in LCR already at low frequencies compared to HCR, the capacity may still be adequate to maintain a preserved end-diastolic Ca2+ and SR Ca2+content at this frequency. Our finding of a significantly increased end-diastolic Ca2+ level at 5 Hz stimulation supports a failure of SERCA2a for reuptake of Ca2+ during increased Ca2+ cycling rates which potentially also mediated a reduced SR Ca2+ available for release.Earlier studies have shown that reduced SERCA2a function is related to a negative frequency dependent acceleration of Ca2+ transients in the myocytes from LCR compared to HCR rats, together with relative low number of atrial myocytes with T-tubules in LCR rats, suggests a lack of central initiation sites for Ca2+ response. The transients showing this spatial profile rises rapidly at the edges of the myocytes and more slowly in the interior, which is in agreement with association between lack of T-tubules and spatiotemporal characteristics of Ca2+ transients demonstrated in atrial cells previously 2+ channels (LTCCs) and RyRs that is necessary for Ca2+ induced Ca2+ release, occurs only at the cells periphery leading to dyssynchronous Ca2+ release 2+ dynamics has been reported in ventricular myocytes of HF models because of a loss of or reorganization of T-tubules leaving some orphaned RyRs that become physically separated from LTCCs 2+ release across the entire spatial dimension of the line scan was faster in HCR rats compared to LCR rats. This may be explained by the relative higher number of W-shaped Ca2+ transients due to more developed T-tubular network in HCR myocytes, which provide central initiation sites for Ca2+ release with faster and more spatial homogenous onset of Ca2+-signal. This is supported by Smyrnias et al. 2+ transients to have significantly faster recovery of systolic Ca2+ amplitude after complete depletion of Ca2+ by caffeine application. At increasing frequencies the functional consequences of delayed central Ca2+ rise in LCR rats will be even more pronounced because of the increased demand of rapid initiation of Ca2+ induced Ca2+ release. Therefore, we suggest an association between the observed differences in spatio-temporal characteristics of Ca2+-signal and the observed differences in atrial myocyte systolic performance due to the fact that slow rise in Ca2+ release may limit synchronous contractile activation, especially at high cardiac frequencies T-tubule system of variable extent has been reported in rat atrial cells 2+ leak in atrial myocytes is interesting since this is the first report showing that low aerobic capacity leads to a cellular substrate that may be more prone to triggering of atrial arrhythmias. Several studies on ventricle cardiomyocytes 2+ leak from the SR during diastole is a potent trigger for uncontrolled electrical activity that may cause spontaneous contractions and arrhythmias. On this basis several novel Ca2+ release RyR2-stabilizing drugs have been proposed 2+ leak 2+ leak, our data indicates that RyR2 serine-2814 phosphorylation is apparently increased in the LCR rats. Importantly, this suggests a deleterious signaling induced by contrasting for low aerobic capacity.The observation of increased diastolic SR Ca2+-handling. Reduced systolic Ca2+ amplitude in LCR rats was associated with reduced ability to initiate Ca2+ release from the SR that probably is caused by a less developed T-tubule network. Furthermore, low aerobic capacity in LCR rats led to an increased diastolic SR Ca2+ leak over the RyR2, which has been linked to cardiac arrhythmias in several studies on left ventricular myocytes. Our study therefore suggests that low aerobic capacity may lead to negative signaling in atrial myocytes with defective properties of Ca2+ handling that is not only negative for atrial function but also may cause a cellular substrate that is more prone for triggering of atrial arrhythmias. It is likely that the improved cardiomyocyte function and Ca2+ handling associated with high aerobic capacity has a positive effect during increased workload of the atria. It is furthermore tempting to speculate that the positive adaptations in the atrial cellular mechanisms may protect against atrial dysfunction such as atrial fibrillation.This study report for the first time that contrasting for low or high aerobic capacity leads to diverse functional and structural remodeling of atrial myocytes. Compared to rats with high aerobic capacity we found that low aerobic capacity in LCR rats was associated with reduced atrial myocyte contractility and diastolic relaxation that were associated with impaired Ca


The precise role of CaMKIIδB in the transcriptional cross-talk of Ca2+-handling proteins during heart failure remains unclear. In this work, we aim to determine the mechanism of CaMKIIδB in modulating the expression of sarcolemmal Na+–Ca2+ exchange (NCX1). We also aim to address the potential effects of calmodulin antagonism on the imbalance of NCX1 and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) during heart failure. Eight weeks after transverse aortic constriction (TAC)-induced heart failure in mice, we found that the heart weight/tibia length (HW/TL) ratio and the lung weight/body weight (LW/BW) ratio increased by 59% and 133%, respectively. We further found that the left ventricle-shortening fraction decreased by 40% compared with the sham-operated controls. Immunoblotting revealed that the phosphorylation of CaMKIIδB significantly increased 8 weeks after TAC-induced heart failure. NCX1 protein levels were also elevated, whereas SERCA2 protein levels decreased in the same animal model. Moreover, transfection of active CaMKIIδB significantly increased NCX1 protein levels in adult mouse cardiomyocytes via class IIa histone deacetylase (HDAC)/myocyte enhancer factor-2 (MEF2)-dependent signaling. In addition, pharmacological inhibition of calmodulin/CaMKIIδB activity improved cardiac function in TAC mice, which partially normalized the imbalance between NCX1 and SERCA2. These data identify NCX1 as a cellular target for CaMKIIδB. We also suggest that the CaMKIIδB-induced imbalance between NCX1 and SERCA2 is partially responsible for the disturbance of intracellular Ca2+ homeostasis and the pathological process of heart failure.Ca Predominant expression of the CaMKIIδ isoform contributes to cardiac decompensation by enhancing the ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca2+ leak, and attenuating CaMKIIδ activation has been shown to limit the progression to heart failure 2+ homeostasis 2+-mediated transcriptional gene regulation. Transient expression of CaMKIIδB induces hypertrophy and ANF production in neonatal rat ventricular myocytes 2+-handling proteins during heart failure.Ca2+ channels and increases Ca2+ influx in the heart. This triggers the release of more Ca2+ from the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA2) via RyRs, an event typically referred to as Ca2+-induced Ca2+ release. During relaxation, Ca2+ is immediately transported into the SR via SERCA2 and extruded by sarcolemmal Na+–Ca2+ exchange (NCX1) in cardiomyocytes 2+ overload has substantial, adverse effects in failing hearts. Several investigators have shown that during heart failure, the expression and activity of the Ca2+-sequestering SERCA2 is decreased, RyR is hyperphosphorylated, and/or activity and protein levels of NCX1 are increased et al. showed that inducible cardiomyocyte-specific excision of the Serca2 gene leads to a substantial reduction in diastolic function in mice 2+ homeostasis, NCX1 is reported to be up-regulated at the transcriptional level during cardiac hypertrophy, ischemia, and failure et al. demonstrated that the feline 1831Ncx1 H1 promoter is sufficient for the up-regulation of Ncx1 in response to pressure overload in transgenic mice 2+-handling proteins in a failing heart. Accordingly, we tested the hypothesis that heart failure induced by transverse aortic constriction (TAC) is associated with an increase in CaMKIIδB activity, an imbalance of Ca2+-handling proteins and altered Ca2+ homeostasis.In the physiological context, the depolarization of an action potential activates L-type Ca2+ handling during heart failure, we sought to determine the potential role of CaMKIIδB in modulating both the expression of NCX1 and NCX1/SERCA2 balance during the pathological process of heart failure. Here, we demonstrate a correlation between the activation of CaMKIIδB and the elevation of NCX1 protein levels during TAC-induced heart failure in mice. The over-expression of CaMKIIδB led to an increase in NCX1 expression and a disturbance of the NCX1/SERCA2 balance via class IIa histone deacetylase (HDACs)/myocyte enhancer factor-2 (MEF2)-dependent signaling. Moreover, treatment with a calmodulin antagonist improved cardiac function and normalized the NCX1/SERCA2 balance away from heart failure. These findings not only define a novel function for CaMKIIδB in Ca2+-handling proteins but also highlight the potential therapeutic benefits of a novel calmodulin antagonist against heart failure.To better understand the molecular basis of aberrant Caad libitum. All the animal experiments were carried out in accordance with the National Institute of Health guidance for the care and use of laboratory animals, were approved by The Committees for Animal Experiments in Tohoku University in Japan (21–252) and Zhejiang University in China (SCXK 2007–0029).Adult male DDY mice weighing 35–40 g were obtained from Nippon SLC . Eight-week-old males were acclimated to the local environment for 1 week, which included housing in polypropylene cages at 23±1°C in a humidity-controlled environment maintained on a 12-h light–dark schedule (lights on 8:00 a.m. – 8:00 p.m.). Mice were provided food and water Transverse aortic constriction (TAC) was performed on mice as described previously Mouse heart samples were cut into transverse sections and stained with hematoxylin and eosin (H&E) as described previously After 6 weeks of drug administration , the mice were sacrificed by cervical dislocation; then, the hearts and lungs were removed and weighed as previously described Non-invasive echocardiographic measurements were performed using ultrasonic diagnostic equipment  as described previously All animals were sacrificed after 6 weeks of drug administration, and their hearts were immediately harvested and stored at −80°C until immunoblotting analyses were performed, as described previously Adult mice cardiomyocytes were isolated as described previously Confocal immunocytochemical imaging was performed as previously described NCX1 promoter region using a ChIP kit  according to manufacturers' protocol. Briefly, cardiac cells were treated with 1% formaldehyde and collected by brief centrifugation, lysed in lysis buffer, and micrococcal nuclease digestion (MNase digestion). Protein-DNA complexes were immunoprecipitated using MEF2 antibody  or IgG as a control. MEF2/DNA formaldehyde cross-linkages were reversed by heating at 65°C for 2 h, DNA purified by DNA Clean-Up Column and resuspended in 50 µl of DNA Column Elution Solution. PCR was performed using 1∶10 dilution of input chromatin (input DNA) or immunoprecipitated chromatin (IP DNA). NCX1 primers sequences were forward 5′- TTCGGCTTTCGCTTCC ATACA-3′ and reverse 5′- CTCTGGCCTTCTGCTTTTCCT-3′. PCR conditions included: an initial 2 min denaturation at 94°C followed by 40 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s; a final 5 min elongation at 72°C.ChIP assay was performed on MEF2 in the Small interfering RNAs (siRNAs) against MEF2 and control siRNA were obtained from Santa Cruz Biotechnology. siRNAs were introduced into cardiomyocytes with transfection medium according to the manufacturer's instructions. After 6 h incubation, the fresh serum-free DMEM was changed, and then cells were transiently transfected with 0.3 µg/well of pGL3-NCX1 reporter construct using Lipofectamine 2000 (Invitrogen). For co-transfections, pRL-TK (Promega) encoding Renilla luciferase (Rluc) served as an internal control. 0.3 µg/well of pGL3-NCX1 reporter construct was transfected along with activated FLAG-tagged CaMKIIδB (CaMKIIδB-T287D). After transfection for 24 h, ET (100 nM) was added to the serum-free DMEM and cultures were incubated for another 24 h. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) with a luminometer .2+ transient was used as an index of SR Ca2+ content. Changes in the caffeine-induced Ca2+ release from the SR were determined using a ratio of the fluorescence emission at 510 nm in response to excitation at 340 and 380 nm.Adult mice cardiomyocytes were isolated as described previously In situ DNA fragmentation was assessed using a TUNEL assay as previously described P<0.05 was considered statistically significant.Differences between groups were assessed with Student's t-test and one-way ANOVA. Survival was expressed by the Kaplan-Meier method, and survival curves were compared by the log-rank test. All values are expressed as mean ± S.E.M. A value of 2+-handling proteins by Western blot ]. In cardiomyocytes, transfection with active CaMKIIδB led to enriched NCX1 in the membrane, as shown in the immunochemistry assay . In addi2+/calmodulin signaling has been shown to regulate MEF2/HDAC function, we explored the possibility that CaMKIIδB also plays a role in regulating NCX1 expression via the MEF2-mediated pathway. First, we tested the efficacy of MEF2c siRNA to knockdown MEF2C in cultured adult cardiomyocytes in the presence of ET (100 nM). Transfection withMEF2c siRNA (300 ng/ml) almost completely inhibited MEF2 protein expression, as measured by Western blot analysis  and MEF2 siRNA vectors. The ET treatment produced a more than a 5-fold increase in reporter activity, whereas the MEF2 siRNA resulted in significant decreasing . Co-tranin vitro and in vivo through inhibition of Ca2+/CaM-dependent enzymes via downstream targets during the pathological process of cardiac hypertrophy 2+-handling proteins. As shown in We previously reported on the cardioprotective effects of calmodulin antagonists, DY-9760e and DY-9836, To gain further insight into the mechanisms of the NCX1/SERCA imbalance in a TAC-induced failing heart, the effects of a calmodulin antagonist on alterations of SERCA2 and NCX1 were examined 8 weeks following TAC treatment. SERCA2 protein levels were decreased in the TAC-treated animals , whereas2+-handling proteins after TAC treatment, we measured the Ca2+ content in the SR by assessing caffeine-induced Ca2+ release in cultured adult cardiomyocytes. The Fura-2 ratio at 340/380 nm was not significantly different between the three groups at the baseline. When Ca2+ release from the SR was triggered by the application of 10 mM caffeine, transient Ca2+ elevation was significantly decreased in the TAC group  did not affect basal Ca2+ levels or caffeine-induced Ca2+ release (data not shown).To verify the pathological relevance of CaMKIIδB over-expression and CaAC group . As showCardiac apoptosis contributes to the phenotypic changes associated with heart failure. Changes in apoptosis after 8 week of TAC was detected by TUNEL assay. Images of TUNEL were recorded as described in P<0.05), as observed at 64 days. The reduced mortality in response to DY-9836 treatment suggests that the maintenance of Ca2+ homeostasis protects the heart from cardiac injury under TAC pathological conditions.Eight weeks after TAC, the treated mice had developed cardiac hypertrophy, as measured by an increased HW/TL ratio compared with the sham group . TAC als2+ content and an imbalance of NCX1/SERCA2 in TAC-induced heart failure; 2) active CaMKIIδB up-regulates NCX1 protein expression in adult cardiomyocytes via HDAC/MEF2-dependent signaling; and 3) the antagonism of calmodulin restores SR Ca2+ levels and regulates the imbalance between NCX1 and SERCA2 by inhibiting CaMKIIδB activity, then rescues cardiomyocytes from apoptosis and improves cardiac function.We can draw several conclusions from the present study, as follows: 1) aberrant CaMKIIδB expression is associated with a disturbance in SR Ca2+ handling have been described in the cardiomyocytes of failing hearts. However, the contribution of CaMKIIδ isoforms to transcriptional regulation, Ca2+ handling and cardiomyocyte apoptosis, which are factors associated with heart failure, remains unclear. It has been shown that the over-expression of CaMKIIδC phosphorylates Ca2+-handling proteins, such as RyR, PLB and L-type Ca2+ channel complex, but has no effect on the SERCA and NCX1 protein levels in rabbit cardiomyocytes 2+-handling protein expression remain to be determined. Therefore, in our present study, we seek to address the role of CaMKIIδB in the TAC-induced imbalance of NCX1 and SERCA2.Numerous disturbances in the function and/or expression of proteins involved in Ca2+ in cardiomyocytes. Additionally, a critical role for HDACs has been proposed in the CaMKII-mediated induction of hypertrophy Ncx1 promoter contains several consensus sequences for a number of potential DNA-binding factors, including two MEF2 elements at positions −166 and −22 Ncx1 promoter to shape the magnitude of a NCX1-mediated signal. Thus, we postulate that the CaMKIIδB-induced NCX1 up-regulation is transcriptionally mediated in the TAC model, which may include the translocation of HDACs to the cytosol and MEF2 transcripts, as well as lead to increased expression of proteins directly involved in compensatory Ca2+ homeostasis.We first examined whether CaMKIIδB was involved in the regulation of NCX1 expression. Notably, transfection with active CaMKIIδB induced NCX1 protein up-regulation, as detected by Western blot and immunocytochemical assays. Our results show that an elevation of NCX1 occurs with activation of CaMKIIδB during TAC-induced heart failure, possibly implying a greater reliance on NCX1 to remove cytosolic Ca2+ from the cytosol to the SR of cardiomyocytes, thus maintaining the store of releasable Ca2+ that is necessary for contraction. We propose that reduced SERCA2 function would be expected to cause immediate, severe myocardial contractile dysfunction. In the present work, we further measured the Ca2+ content of the SR by assessing caffeine-induced Ca2+ release in TAC mice. Our data demonstrated that the Fura-2 ratio at 340/380 nm was significantly decreased by 40% in the TAC-treated adult cardiomyocytes. Likewise, the reduced SERCA2 function was associated with an increase in NCX1 expression, which could compensate for reduced SR Ca2+ cycling by enhancing transsarcolemmal Ca2+ cycling by means of NCX during heart failure. Thus, the present data imply that the over-expression of CaMKIIδB might induce an NCX1/SERCA2 imbalance that exacerbates the perturbation of Ca2+ homeostasis and causes cardiac dysfunction. Several recent studies have indicated that SERCA2 protein degradation is stimulated by peroxynitrite and prevented by calpain inhibitors 2+/calmodulin stimulates the degradation of spectrin by calpain in TAC mice In addition, our results demonstrate an increase of NCX1 levels in conjunction both with SERCA down-regulation in the failing heart of TAC animals. SERCA2 transports Ca2+ suppression in mice, concides with inhibitory effect on the apoptosis of cardiac myocytes in vivo and improved survival rate. Therefore, the prolonged calmodulin antagonist treatment might be prevents progression of cardiac hypertrophy to terminal event (heart failure). An enhanced SR-stored Ca2+ concentration contributes to the cytosolic Ca2+ homeostasis required to rescue cardiomyocytes from apoptosis and recover contractile function. Thus, we propose that the inhibitory effect of the calmodulin antagonist on CaMKIIδB activity contributes to rescue from CaMKII-mediated up-regulation of NCX1 during TAC-induced hypertrophy, which maintains the balance of NCX1/SERCA2 and strengthens the SR Ca2+ concentration. It has been reported that CaMKII can directly phosphorylate cardiac SERCA2, resulting in an increase in its enzymatic activity under physiological conditions Although hypertrophy is formed in response to hemodynamic overload, prolonged left ventricle hypertrophy may result in congestive heart failure. The abnormalities in calcium handling and apoptosis of cardiac myocytes have been reported to be involved in the transition from compensated cardiac hypertrophy to decompensated heart failure 2+ cycling towards more ICa/NCX cycling and a reduction in SR Ca2+ uptake and release. This result in a reduction of the SR stored Ca2+ concentration during TAC-induced heart failure. The inhibition of CaMKIIδB activity and restoration of the NCX1/SERCA2 balance by a calmodulin antagonist partially mediates its protective effects in cardiomyocytes during heart failure. The present study suggests that targeting calmodulin as well as specifically targeting CaMKIIδB may prove to be an effective approach for the treatment of cardiac hypertrophy and heart failure.In summary, our data revealed that the CaMKIIδB-mediated aberrant expression of NCX1 and the imbalance of NCX1/SERCA2 lead to a shift in CaFigure S1Effect of pharmacological inhibition of CaMKII and HDAC on NCX1 expression. (A) The NCX1 overexpression induced by ET is associated with CaMKIIδB phosphorylation. (B) Effect of HDAC inhibitor on NCX1 expression following ET treatment. β-tubulin was used as the loading control. Con, control; ET, endothelin-1; TSA, Trichostatin A.(TIF)Click here for additional data file.Figure S2Effect of calmodulin antagonist on phosphorylation of PLB in TAC mice. Representative immunoblots from heart tissue lysates of control and TAC mice with or without  treatment, assayed with phospho-PLB antibody (Thr17 or Ser16). ββ-tubulin was used as the loading control.(TIF)Click here for additional data file.Figure S3The calmodulin antagonist inhibited cleavage of caspase-3 following TAC. Representative immunoblots of cleaved caspase-3 in sham and TAC mice with or without  treatment. β-tubulin was used as the loading control.(TIF)Click here for additional data file.Table S1Echocardiographic measurements in mice with aortic banding. HR: heart rate; LV: left ventricular; LVIDd: LV end-diastolic internal diameter; LVIDs, LV end-systolic internal diameter; FS: LV fractional shortening. **P<0.01 vs Sham; #P<0.05, ##P<0.01 vs TAC.(DOC)Click here for additional data file.


NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1β (IL-1β) by activating caspase-1. Although several models for inflammasome activation, such as KTo investigate how extracellular DAMP signals activate the NLRP3 inflammasome and the molecular pathogenesis of CAPS.2+ and cAMP.Using a combination of genetic, pharmacological, and biochemical approaches, we provide evidence that the CaSR is essential for NLRP3 inflammasome activation, which is directly controlled by intracellular Ca2+ or other CaSR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CaSR reduces inflammasome activation in response to known NLRP3 activators. The CaSR activates the NLRP3 inflammasome through phospholipase C (PLC), which catalyzes inositol trisphosphate (IP3) production and thereby induces release of Ca2+ from endoplasmic reticulum (ER) stores. The increased cytoplasmic Ca2+ promotes the assembly of inflammasome components, and intracellular Ca2+ is required for spontaneous inflammasome activity in cells from CAPS patients. CaSR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1β production from CAPS patients’ peripheral blood mononuclear cells is attenuated by increasing cAMP.Ca2+ and cAMP are two key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS. In addition, our data suggest a broader spectrum of potential targets for therapy of CAPS as well as other inflammatory conditions involving the NLRP3 inflammasome, including gout, type 2 diabetes mellitus, atherosclerosis, and Alzheimer’s disease.Taken together, these findings suggest that CaNone declared.


This initiative is the first of this kind in Australia and supports the development of a trained volunteer mentoring network, offering opportunities for people in early recovery to develop supportive mutually beneficial mentoring relationships through regular contact with someone who has lived through and recovered from body image or eating issues for a minimum six month mentoring contract.Recovery Mentors at Isis- The Eating Issues Centre Inc. receive Peer Mentor Training that focusses on awareness of mentoring role, boundaries, skills, risk management and self- care, developing social networks and becoming a non-professional ‘buddy’. Mentoring is provided as an additional support alongside the mentees current contact with professional medical and mental health support.This poster will outline aims and components of the Isis Recovery Oriented Peer Mentor Program that has been trialled at Isis from late 2012 to present, and includes qualitative results and identified areas for further development. This project values lived experiences of recovery and seeks to build capacity to provide effective mentoring support, reflect on the mentoring journey and factors that contribute to recovery. Preliminary results show the program is effective in reducing social isolation, increases hope in the possibility of recovery and provides linkages back to community.


Congenital amusia is defined by marked deficits in pitch perception and production. Though historically examined only in otherwise typically developing (TD) populations, amusia has recently been documented in Williams syndrome (WS), a genetic, neurodevelopmental disorder with a unique auditory phenotype including auditory sensitivities and increased emotional responsiveness to music but variable musical skill. The current study used structural T1-weighted magnetic resonance imaging and diffusion tensor imaging to examine neural correlates of amusia in 17 individuals with WS (4 of whom met criteria for amusia). Consistent with findings from TD amusics, amusia in WS was associated with decreased fractional anisotropy (FA) in the right superior longitudinal fasciculus (SLF). The relationship between amusia and FA in the inferior component of the SLF was particularly robust, withstanding corrections for cognitive functioning, auditory sensitivities, or musical training. Though the number of individuals with amusia in the study is small, results add to evidence for the role of fronto-temporal disconnectivity in congenital amusia and suggest that novel populations with developmental differences can provide a window into understanding gene-brain-behavior relationships that underlie musical behaviors. As a complex auditory stimulus, music engages multiple brain areas and networks and can be a window into understanding neural connectivity . RecentCongenital amusia and its neural correlates have historically only been studied in TD adult populations without cognitive impairments. While this work has been an important first step in understanding the neural architecture underlying amusia, expanding the lens to other populations such as children and those with developmental differences can enhance our knowledge about how and why amusia develops. In contrast to acquired amusia , whii.e., time spent listening to or playing music), or low-level auditory processing )rameters . The tecrameters .r = −0.008, p = 0.978), gender , handedness , IQ , sensitivity to specific  sounds , or sensitivity to sound characteristics . DTT was not significantly related to cumulative years of individual musical training  but was strongly related to the number of types of music lesson experiences .Raw data can be found in the To examine brain areas related to DTT, zero-order correlations were conducted between DTT scores and the gray and white matter volumes of the right and left transverse temporal gyri, superior temporal gyri, and pars orbitalis. To examine structural connectivity, correlations were conducted between DTT scores and FA of right and left inferior and superior segments of the SLF. Based on results in TD individuals, we hypothesized that lower DTT scores (consistent with amusia) would be associated with reduced white matter volume in the right pars orbitalis and reduced FA in the right inferior and superior SLF. We then conducted partial correlations to examine if correlations remained significant when controlling for potential confounding factors of age, IQ, handedness, sound sensitivities, and musical training. Results did not change when controlling for age or handedness.versus without, amusia via Mann-Whitney U tests. Given the small sample size, dichotomous analyses should be considered exploratory. The four individuals with amusia had fewer types of music lessons than those without amusia but did not differ in respect to age, handedness, IQ, gender, sound sensitivities, or other musical engagement variables .As many studies of amusia in TD individuals have utilized a between-group rather than continuous design, we similarly conducted dichotomous analyses comparing volume and FA of these same brain areas in individuals with WS with, r = 0.507, p = 0.038; see r = 0.504, p = 0.046) or sound characteristics , was marginal after controlling for cumulative years of training , and was not significant when controlling for IQ  or lesson exposure . DTT scores were also marginally associated with gray matter volume in the right pars orbitalis , it was marginal after controlling for sensitivity to specific sounds  or sound characteristics , and not significant when controlling for IQ  or lesson exposure . Additionally, though zero-order correlations did not reveal a significant relationship between DTT score and gray matter volume of left pars orbitalis, this relationship was significant when controlling for cumulative years of training  but not when controlling for other potential confounds. Similarly, there was a significant relationship between DTT score and left  and right  superior temporal gyrus gray matter volume only when controlling for sensitivity to specific  sounds but not when controlling for other potential confounds. No other significant relationships between DTT and gray or white matter volumetric data were found in the a priori regions of interest.The zero-order correlations of the volumetric data revealed that Distorted Tunes Test (DTT) score was significantly correlated with white matter volume in the right pars orbitalis , cumulative years of training , sensitivity to specific sounds , sensitivity to sound characteristics , or lesson exposure . The relationship between DTT score and FA in the right superior SLF remained significant when controlling for sensitivity to specific sounds  or sensitivity to sound characteristics  but did not remain significant when controlling for other variables in the model. Additionally, DTT score was significantly correlated with FA in the left superior SLF when controlling for cumulative years of training , sensitivity to specific sounds , or sensitivity to sound characteristics  but not when controlling for other variables.Zero-order correlations between DTT and the fractional anisotropy (FA) data revealed that DTT scores were significantly correlated with FA in the right inferior portion of the superior longitudinal fasciculus  and left  pars orbitalis, and there was a trend toward less white matter volume in the right pars orbitalis . Amusics also had reduced FA in the right inferior  and superior  SLF. Following Bonferroni correction, only the group difference in FA in the right inferior SLF remained.Preliminary dichotomous analyses comparing the 13 non-amusics to the 4 amusics revealed that the amusics had significantly less gray matter volume in the right  on a series of melodic and rhythmic musical production tests versus 14 participants with WS who demonstrated impairments on these tasks. In particular, left planum temporale volume was associated with participants’ abilities to sing the final note of a melodic phrase, a task which involves both perception and production abilities. However, lack of information about the participants’ musical training and the method for measuring performance on the musical tasks limit the generalizability of these findings. Wengenroth et al. [i.e., perceiving complex sounds based on the fundamental frequency rather than harmonic overtones). Again, the musical training of the WS participants was not well characterized, as the participants were described as having “only minor musical training” and being matched to the TD group. Findings from the present study suggest that previous volumetric studies may have been limited by a focus on regions associated with the primary auditory cortex rather than the entire network involved in auditory processing. Indeed, functional neuroimaging studies have reported more diffuse activation to music throughout the neocortex in WS versus TD controls, including the frontal lobes [Previous research in WS has focused on associations between auditory cortex volumes and specific auditory skills. Martens et al.  reportedh et al.  reportedal lobes ,69.Beyond adding to our knowledge about the variability of musical skills in individuals with WS, the present study adds to a growing body of literature on the neural basis of amusia in general. Our finding of reduced FA in the right inferior SLF in individuals with amusia in WS is highly consistent with converging evidence from structural and functional MRI, DTI, and ERP studies of amusia in TD. Specifically, this work suggests that amusia is due to disconnectivity in the right fronto-temporal pathway connecting the right auditory cortex and inferior frontal lobe rather than isolated dysfunction in the primary auditory cortex ,15,16,35versus superior SLF to values in the published literature. Nevertheless, we can make conjectures about how the inferior and superior portions of the SLF in the current paper compare with the inferior and superior components of the arcuate fasciculus examined in a previous study of connectivity in TD amusics.While the relationship between amusia and right fronto-temporal disconnectivity was present for both the inferior and superior components of the SLF in the present study, it was particularly strong in the inferior SLF and remained significant after controlling for cognitive abilities or musical training. A benefit of using ROQS comes from the fact that ROI selection and FA analysis are performed on an individual basis. This avoids potential confounds that can be introduced by non-linear spatial normalization techniques . The semet al. [Loui et al.  defined et al. . In broaIt is noteworthy that while the correlation between amusia and right SLF withstood corrections for variance due to IQ and musical training, the relationship between amusia and white matter volume of the right pars orbitalis did not. Amusia may reflect an impairment in auditory-motor feedback or integration, such that amusics are unable to use auditory feedback to guide musical motor behaviors . Preet al. [Though the current results in WS were predominantly right lateralized, reduced gray matter volume in the left pars orbitalis and reduced FA in the left superior SLF were found when controlling for cumulative years of training . Similarly, though the aggregated findings from studies in TD individuals suggest that the neural correlates of amusia are predominantly right lateralized, bilateral differences in cortical thickness have been reported in the IFG and auditory cortex of amusics , as wellet al.  suggest et al.  and leftet al.  auditoryet al. .Additionally, our results revealed an association between poorer pitch performance on the DTT and reduced gray matter volume in the superior temporal gyri bilaterally but only when controlling for variance due to general auditory sensitivities . Recent studies of TD individuals with amusia also find reduced gray matter volume  and incri.e., timbre, dynamics, articulation, etc.) may be more important than tonal expectancies for the emotional connection to music in WS [versus TD individuals [Additionally, these findings that superior temporal gyri volume only correlated with DTT performance when controlling for auditory sensitivities may further our understanding of the heightened emotional responsiveness to music in WS ,22. Previc in WS . Indeed,ividuals , which mThe consistency of the neural basis for amusia in WS with previous results from TD individuals suggests that WS may be a model system for further exploration of the roles of genes and environment in the development of musical skill. Studies of congenital amusia in TD individuals have hypothesized that amusia is due to anomalous neural development rather than a lack of exposure to musical experiences and/or training. Individuals with WS typically have as much, if not more, musical exposure than TD individuals with regard to their musical listening and playing habits . In a prStudying amusia in WS may also yield an increased understanding of the specificity of the neural correlates of amusia. For example, previous studies have suggested that amusia is associated with deficits in visual-spatial perception , althougResearchers have also suggested that there may be a shared neural basis for amusia and language difficulties such as dyslexia . Loui anThe current study uniquely extends the neural basis of amusia to a known genetic syndrome. Additional strengths of the study include the use of both volumetric and connectivity neuroimaging measures; the use of individual-specific (versus group-averaged) data via the use of ROQS; the well-characterized musical background of the sample; and the exploration of relationships while controlling for cognitive, auditory sensitivity, and music training factors. Furthermore, we quantified musical training in ways that are both comparable to the TD literature yet meaningfully reflective of the training experiences of individuals with developmental disabilities. Future studies can extend this work by using additional measures of amusia beyond the DTT, including tests of pitch discrimination thresholds and musical production abilities, as well as basic hearing assessments such as pure tone thresholds. Studies could also examine how amusia in WS relates to their short-term and working memory capacities across different auditory tasks given the pitch specific memory deficits in TD amusics . Future et al. [The challenges of conducting neuroimaging studies in a rare syndrome such as WS have been noted previously ,69. The et al. , however


Binding abilities of current inhibitors to MDMX are weaker than to MDM2. Polarizable molecular dynamics simulations (MD) followed by Quantum mechanics/molecular mechanics generalized Born surface area (QM//MM-GBSA) calculations were performed to investigate the binding difference of inhibitors to MDM2 and MDMX. The predicted binding free energies not only agree well with the experimental results, but also show that the decrease in van der Walls interactions of inhibitors with MDMX relative to MDM2 is a main factor of weaker bindings of inhibitors to MDMX. The analyses of dihedral angles based on MD trajectories suggest that the closed conformation formed by the residues M53 and Y99 in MDMX leads to a potential steric clash with inhibitors and prevents inhibitors from arriving in the deep of MDMX binding cleft, which reduces the van der Waals contacts of inhibitors with M53, V92, P95 and L98. The calculated results using the residue-based free energy decomposition method further prove that the interaction strength of inhibitors with M53, V92, P95 and L98 from MDMX are obviously reduced compared to MDM2. We expect that this study can provide significant theoretical guidance for designs of potent dual inhibitors to block the p53-MDM2/MDMX interactions. The tumor suppressor protein p53 plays a key role in maintaining genetic integrity and preventing tumor development34578Recent studies showed that sequence identity in the N-terminal domain of MDM2 and MDMX reaches ~54%, and these two oncoproteins share highly similar overall structure 101112. B101141314152225et al. to calculate residue-specific interactions in the p53-MDM2 complex and the results proved that van der Waals interactions drive the p53-MDM2 binding4748et al. used MD simulations and binding free energy calculations to study the binding modes of p53 and nutlin to MDM2/MDMX and their results suggested that p53 and nutlin have higher affinity for MDM2 than MDMX5152et al. combined MD simulation with principal component (PC) analysis to explore the conformation changes of MDM2 and their results showed that the binding cleft of MDM2 undergoes a large conformational changeDue to great success of molecular simulations and predictions of binding free energies in insight into the inhibitor-protein interactions, conformation changes and structure-affinity relationship of proteins272829303132333435363738394041424344et al. successfully integrated AMOEBA polarizable force field with binding free energy calculation to investigate binding modes of inhibitors to trypsinet al. applied MD simulation involving CHARMM fluctuating charge force field to investigate the structures of six small proteins and the results suggested that these proteins maintained native structures during simulationCompared to classical molecular force field, polarizable force field plays more important role in stabilization of protein structure545556575960In this work, two peptide inhibitors pDI6W (LTFEHWWAQLTS) and pDIQ (ETFEHWWSQLLS) were selected to probe interaction mechanism of inhibitors with MDM2 and MDMX at the atomic level. The median inhibitory concentration (IC50) values of pDI6W to MDM2 and MDMX are 36 and 250 nM, respectively, and the corresponding values of pDIQ to MDM2 and MDMX are 8 and 110 nM6465676869To evaluate the effect of polarizable and non-polarizable MD simulation on the stability of structure, the RMSD values of all backbone atoms in the complexes were calculated relative to their corresponding crystal structures and plotted in The combination of the Cpptraj program65To measure the strength of interactions between inhibitors and MDM2/MDMX, the absolute binding free energies were calculated by using QM/MM-GBSA method implemented in Amber programbind) of the pDI6W-MDMX, pDI6W-MDM2, pDIQ-MDMX, pDIQ-MDM2 complexes are −8.94, −11.02, −9.18 and −11.82 kcal · mol−1, respectively, which agrees well with the experimentally determined values. This result shows that the binding abilities of two inhibitors to MDMX are weaker than to MDM2. Additionally, the entropy changes (−TΔS) induced by inhibitor bindings produce a good correlation with the interaction enthalpy (ΔHtot). This result should be rational because the stronger the interaction enthalpy is, the more the restrictions of motion freedoms are.According to vdW), non-polar solvation energy (ΔGsurf) and quantum mechanics energy (ΔGqm) provide favorable contribution to the inhibitor bindings. Although the electrostatic interaction (ΔEele) also favors in the inhibitor bindings, this favorable factor are completely screened by the stronger unfavorable polar solvation energy (ΔGgb). Compared to the pDI6W-MDM2 complex, the van der Waals interaction, non-polar solvation energy and quantum mechanics energy of the pDI6W-MDMX binding are decreased by 2.5, 0.42 and 0.97 kcal · mol−1, respectively. Similarly, the pDIQ-MDMX binding also leads to the decrease of 2.65, 0.83 and 0.44 kcal · mol−1 in the van der Waals interaction, non-polar solvation energy and quantum mechanics energy, respectively. Overall, the decrease in the van der Waals should be a main origin of weaker binding abilities of inhibitors to MDMX than to MDM2.As seen from vdW) and molecular surface-correlated energies (γ·ΔMSA) is favorable for the inhibitor bindings. Despite favorable contributions of the intermolecular Coulomb interactions (ΔEc), this favorable force was completely counteracted by the unfavorable reaction energies (ΔGR). Compared to the MDM2 binding complexes, the pDI6W-MDMX binding leads to the decrease of 3.72 and 0.45 kcal · mol−1 in ΔEvdW and γ·ΔMSA, respectively, and for the pDIQ-MDMX binding, ΔEvdW and γ·ΔMSA are reduced by 5.7 and 0.60 kcal · mol−1, respectively. Thus, the decrease in the van der Waals interactions is the main origin of less efficiency of inhibitors on MDMX than on MDM2. This result is in good agreement with the previous QM/MM-GBSA calculations. Studies of Joseph et al. proved that p53 and nutlin also produce weaker affinity to MDMX than to MDM2To further support the above conclusion, the binding free energies were also computed by using the SIE method and the results were given in To further explore the origin of weaker binding of inhibitors to MDMX than to MDM2, the inhibitor-residue interaction were calculated by using the residue-based free energy decomposition method and the results depicted in −1. These residues involve K51, L54, I61, M62, Y67, Q72, H53, V93, H96, I99 and Y100. One can observe that the residue L54 produce the strongest interaction  with pDI6W. This interaction mainly arises from two contributions, one from the CH-CH interactions between the alkyls of L54 and L26′, another from the hydrogen bonding interaction between the atom NE1 of W23′ and the carbonyl oxygen O of L54  dihedral angle of the sidechain in Y99 (MDMX) and Y100 (MDM2) and their frequency distribution were calculated through the polarizable MD simulation shown in . One canThe free energy landscapes were constructed by using the backbone ψ and φ angle to further probe the conformational change of Y99 (MDMX) relative to Y100 (MDM2), as shown in The residue L54 in MDM2 is substituted by M53 in MDMX and the length of the side chain of M53 is longer than the one of L54. To reveal how this change affect the inhibitor bindings, the time evolution of the Chi1 (χ1) dihedral angle of the side chain in M53 and L54 were calculated through the MD trajectory, as well as their frequency distribution were depicted in The above analyses suggest that the conformations of M53 and Y99 in MDMX are highly different from the one of L54 and Y99 in MDM2. The surface mode display of MDM2/MDMX  structuret al. also suggested that special conformation of M53 and Y99 leads to a steric clash with inhibitors, which agrees with our current results19The above dihedral analyses suggest that the Chi1 angle of Tyr100 in MDM2 is about 186°, while the one of Y99 in MDMX is about 286°. The longer sidechain of M53 in MDMX relative to L54 in MDM2 points to the outside of the binding groove. To clearly clarify this difference, the structural superimposition analyses of MDM2 and MDMX were performed by using the lowest energy structure extracted from MD trajectory and depicted in et al. also proved that the polarizable MD simulations can better maintain the hydrogen bonding interactions than the non-polarizable MD simulations64In this work, the polarizable MD simulations of a combination FF02.r1 and POL3 and the non-polarizable MD simulations of the combination FF03 and TIP3P were performed to evaluate the effect of force field on the stability of protein second structure. The results indicate that the polarizable MD simulations can produce more hydrogen bonds and to stabilize the second structure. Meher and Zuo et al. suggest that the reduction of van der Waals contacts induce weaker binding of inhibitors to MDMXQM/MM-GBSA and SIE method were applied to calculate the binding affinities of inhibitors to MDM2 and MDMX. The predicted binding free energies using QM/MM-GBSA method quantitatively agree well with the experimentally determined values. The calculated results of two methods show that inhibitors produce weaker binding to MDMX than MDM2, and the decrease in the van der Walls interaction of inhibitors with MDMX relative to MDM2 is the main factor of weaker bindings of inhibitors to MDMX. Binding free energy predictions of Joseph et al. the closed conformation constructed by the orientations of M53 and Y99 were also observed to affect the binding of inhibitors to MDMX19The time evolutions of dihedral angle Chi1 for M53 and Y99 in MDMX and L54 and Y100 in MDM2 were evaluated using polarizable MD trajectory. The results indicate that the conformations of M53 and Y199 are considerably different from L54 and Y100. The orientations of M53 and Y99 shape a closed conformation that prevents inhibitors from reaching the deep of the hydrophobic cleft in MDMX and reduce the van der Waals interactions of inhibitors with M53, V92, P95 and L98 in the cleft. Thus, the closed conformation of MDMX binding cleft caused by the orientations of M53 and Y99 is the main origin of weaker bindings of inhibitors to MDMX than to MDM2. In studies of Phan and Popowicz The crystal structures of MDM2/MDMX complexed with inhibitors pDI6W and pDIQ were obtained from the protein data bank (PDB): 3JZR for the pDI6W-MDM2 complex, 3JZP for the pDI6W-MDMX complex, 3JZS for the pDIQ-MDM2 complex and 3JZQ for the pDIQ-MDMX complex78−1. Periodic boundary condition was adopted to avoid edge effect throughout MD simulations and a time step of 1 fs was set in all simulations.Initial bad steric contacts with the solvent or within protein molecules were removed by energy minimization of three steps. Firstly, 6000-step energy minimization, involving the steepest decent minimization of 3000 steps and conjugate gradient one of 3000 steps, was carried out in which a straining force was applied to all protein atoms. Then another 6000-step energy minimization was performed by refreezing all backbone atoms using the same restraining force. Finally, another 6000-step energy minimization was carried out without any restriction. Then, the system was heated from 0 to 300 K in 1 ns and equilibrated at 300 K for another 1 ns. Next, a 30-ns simulation without restriction was conducted at constant pressure and 300 K, and the conformations were saved every 2 ps. During MD simulations, the particle mesh Ewald (PME)The binding free energies of two inhibitors to MDM2/MDMX were calculated using QM/MM-GBSA approach in Amber based on 200 conformations extracted from the last 10 ns of MD trajectories with an interval of 50 psG is the total binding free energy, Gcomplex, Greceptor and Ginhibitor are the free energies of the complex, receptor and inhibitor, respectively. H and TΔS represent the enthalpy and entropy contributions of system. The terms Evdw, Eele and Eint are van der Waals interaction, electrostatic interaction, and internal energy, respectively. The terms Ggb and Gsurf are the polar and non-polar solvation free energy, respectively. Ggb is calculated using the modified GB model developed by Onufriev et al.Gsurf is approximated by the experiential equation γ and β were set to 0.0072 kcal. mol−1. Å−2 and 0.0 kcal · mol−1, respectivelywhere ΔIn the case of QM/MM-GBSA calculations, the enthalpy term was computed by applying the QM/MM approach. The residues involving the hydrogen binding interactions are described at the QM level using the semi-empirical Hamiltonian PM6 method to treat the hydrogen binding interactions. Our previous studies proved that this method can accurately evaluate the hydrogen bonding interactionset al. to predict the binding affinities of inhibitors to proteins91The SIE method was developed by Naim Ec and EvdW are the intermolecular Coulomb and van der Waals interaction energies in the bound state, separately. These two terms were computed with the Amber ff02 force fields. The term ΔGR represents the change of the reaction energy induced by inhibitor bindings and was calculated by solving the Poisson equation with the boundary element method (BRI BEM)MSA represents the change of the molecular surface area upon bindings. The Amber van der Waals radii linear scaling coefficient (ρ), the solute interior dielectric constant (C) are the parameters optimized by fitting to the absolute binding free energy for a set of 99 protein-ligand complex. The optimized values of these parameters are α = 0.1048, Din=2.25, γ = 0.0129 kcal/(mol · Å2) and C = −2.89 kcal · mol−1, respectively, which was used to successfully evaluate the binding modes of the other inhibitor-protein binding complexes71in which How to cite this article: Chen, J. et al. Probing Origin of Binding Difference of inhibitors to MDM2 and MDMX by Polarizable Molecular Dynamics Simulation and QM/MM-GBSA Calculation. Sci. Rep. 5, 17421; doi: 10.1038/srep17421 (2015).