However, no structural data has been reported on how the enzymes bind the γ-lactams and achieve their enantioselectivities. We previously identified an isochorismatase-like hydrolase  with (+)-γ-lactamase activity, which constitutes a novel family of γ-lactamase. Here, we first discovered that this enzyme actually hydrolyzed both (+)- and (−)-γ-lactam, but with apparently different specificities. We determined the crystal structures of the apo-form, (+)-γ-lactam bound, and (−)-γ-lactam bound forms of the enzyme. The structures showed that the binding sites of both (+) and (−)-γ-lactam resemble those of IHLs, but the “cover” loop conserved in IHLs is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrated that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 would reduce the binding affinity of (−)-γ-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of γ-lactam. Our results would facilitate the directed evolution and application of Mh33H4-5540 in antiviral drug synthesis. All the (+)-γ-lactamases also show low sequence identities to (−)-γ-lactamases. A (−)-γ-lactamase which was isolated from an Aureobacterium species has been characterized and expressed in Escherichia coli (E. coli)E. coli cells. Based on the complex structure, a mechanism of (−)-γ-lactam hydrolysis was suggested, which agreed with that of α/β hydrolase enzymesComamonas acidovorans was also solved, which showed high sequence identity to formamidases and acetamidasesCompared with chemical synthesis, enzymatic resolution of racemic γ-lactam with (+)-γ-lactamase has proven to be more efficient and environmentally friendly to give optically pure (−)-γ-lactam67891011121314Microbacterium hydrocarbonoxydans with a high (+)-γ-lactamase activityComamonas acidovorans, Bradyrhizobium japonicum, Aeropyrum pernix, and Sulfolobus solfataricus, respectively)19Pseudomonas putida Kt2440 (PDB code 4H17)  . In thisThe IHL superfamily members are α/β hydrolases, the first several characterized members of which all contained a conserved Cys residue (C111 in MhIHL in this study) which acts as a nucleophile at the active site, and therefore this family was formerly designated as cysteine hydrolases. However, further studies indicated that the cysteine residue is not fully conserved, since several members utilized other nucleophilic residuesIn the present study, we firstly conducted a hydrolysis assay of the racemic γ-lactam by MhIHL in an extended time range. Interestingly, the results showed that MhIHL actually showed both (+)- and (−)-γ-lactamase activity, but with apparently different specificities . To inveKm values of the (+)- and (−)-γ-lactam were 12.9 and 34.2 mM, respectively, indicating that MhIHL exhibited a better affinity towards (+)-γ-lactam than (−)-γ-lactam -γ-lactam (15.77 mM−1 s−1) is around 27 times that of (−)-γ-lactam (0.58 mM−1 s−1), suggesting the better specificity of MhIHL for (+)-γ-lactam. Together, MhIHL exhibited both hydrolytic activities towards (+)- and (−)-γ-lactam and a better specificity for (+)-γ-lactam.In our previous studyγ-lactam . Further121 with one polypeptide chain in the asymmetric unit. The PISA server2, and the decrease in this area upon dimerization is 488.6 Å2 in one protomer.To better understand the different specificities of MhIHL for (+)- and (−)-γ-lactam, we firstly solved the high resolution structure of MhIHL . Like otEscherichia coli ycac gene product. The overall “α-β-α sandwich” fold is highly conserved among MhIHL and other IHL proteins (Oleispira antarctica (PDB code 3LQY), with a root-mean-squared displacement (RMSD) of 1.3 Å between the two proteins. However, they are different in the loop regions between β1 and α1, β2 and β3, and β6 and α4 (Pseudomonas aeruginosa (PDB code 1NF9) represents one of the best characterized IHL family proteins. PhzD, which belongs to the isochorismatase E.C. 3.3.2.1 subgroup, functions in the first step of phenazine synthesis, hydrolyzing 2-amino-2-deoxyisochorismate into trans-2,3-dihydro-3-hydroxyanthranilic acid and pyruvate. The RMSD between the structures of MhIHL and PhzD is 1.44 Å . MhIHL also has this conserved triad (D13-K78-C111), and mutation of either residue to Ala caused sharp decrease or totally abolishment of the activity of this enzyme . SpecifiAcinetobacter baumanii PncAA possible mechanism of MhIHL-catalyzed hydrolysis of (+)-γ-lactam or (−)-γ-lactam, is presented in As both enantiomers of γ-lactam bound to almost the same sites in MhIHL and formed similar interactions with the enzyme, we performed MD simulations to explore the molecular mechanism underlying the different catalytic specificities of the (+)-γ-lactam or (−)-γ-lactam of MhIHL. First, we constructed the structural models of the complex structures of the WT enzyme with (+)- or (−)-γ-lactam separately, based on the complex structures of C111A mutant with (+)- or (−)-γ-lactam using the Protein Local Optimization Program (PLOP). After 50 ns simulation, both complex systems of WT-(+)-γ-lactam and WT-(−)-γ-lactam reached equilibrium, while the average RMSD values of ligands and deviations of proteins were both larger in the WT-(−)-γ-lactam complex than in the WT-(+)-γ-lactam complex . During Specific hydrolysis of (+)-γ-lactam out of racemic γ-lactam to leave optically pure (−)-γ-lactam is of great importance, since (−)-γ-lactam is the key building block of antiviral drugs such as carbovir and abacavir. To date, several (+)-γ-lactamases have been identified, all of which resemble formamidases and acetamidases, but no structure has been described for the complex between (+)-γ-lactam and a (+)-γ-lactamase. The IHLs superfamily is a well-characterized hydrolase family, which is best known in pathogenic organismset al. , out of which the thiol group of the cysteine residue acts as a nucleophile. Second, all structures determined to date from this superfamily exhibit a rare nonproline cis-peptide bond (A106-Q107 in MhIHL) in the active site, which has been proposed to help position the substrate-binding residues appropriatelyet al. .et al. reported that the enzyme SttH from Streptomyces albulus could catalyze the hydrolysis of the amide bond of streptothricins (STs), which are broad-spectrum antibiotics. Streptothricins (STs), produced by Streptomyces strains, consist of a carbamoylated D-gulosamine to which the β-lysine homopolymer (1 to 7 residues) and the amide form of the streptolidine (streptolidine lactam) are attachedInterestingly, Hamano MhIHL exhibited different hydrolysis activities towards (+)- and (−)-γ-lactam, with a better specificity for (+)-γ-lactam. Therefore, in the beginning of the hydrolysis reaction of racemic γ-lactam, (+)-γ-lactam might act both as a substrate of MhIHL, and also an “inhibitor” for (−)-γ-lactam. That explains why it seems that MhIHL first hydrolyzed the (+)-γ-lactam, and then the (−)-γ-lactam . To inveIn all, we have presented three structures of MhIHL and its complex with both (+) and (−)-γ-lactam substrate and conducted biochemical and mutational studies into the catalytic mechanism and enantioselectivity of MhIHL. MhIHL from the isochorismatase-like superfamily, represents a novel class of (+)-γ-lactamases, thus further expanding the (+)-γ-lactamase superfamily. MhIHL displayed different hydrolysis activities towards (+)- and (−)-γ-lactam, and our structural, biochemical, and MD simulation studies demonstrated the underlying mechanisms for the enantioselectivity of MhIHL. Due to the wide applications of (−)-γ-lactam in antiviral drugs, this study provided much needed insight into the catalytic mechanism of a new type of (+)-γ-lactamase and would shed light on the molecular manipulation of (+)-γ-lactamases for higher efficiency and enantioselectivity.M. hydrocarbonoxydans, Paenibacillus dendritiformis C454, Paenibacillus polymyxa SQR-21, Microbacterium testaceum StLB037, and Escherichia coli W3110 were obtained from the China General Microbiological Culture Collection Center, and grown in Luria-Bertani (LB) medium at 37 °C. The gene encoding MhIHL (GenBank accession number AKS37009.1) from M. hydrocarbonoxydans, isochorismatase A from P. dendritiformis C454 (GenBank accession number CP003422.2), isochorismatase B from P. polymyxa SQR-21(GenBank accession number CP006872.1), isochorismatase C from M. testaceum StLB037 (GenBank accession number AP012052.1), and isochorismatase D from E. coli W3110 (GenBank accession number CP013253.1) were amplified by PCR from the corresponding genomic DNA. The amplified genes were purified using a Gel/PCR Extraction kit , digested by NdeI and XhoI , and inserted into pET22b(+) , resulting in plasmids encoding a His6 tag fusion at the C terminal of the recombinant proteins. Mutants were generated by site-directed mutagenesis with a QuikChange Site-Directed Mutagenesis Kit . The expression plasmids for wild-type (WT) and the mutants were introduced into E. coli DH5α and E. coli DMT respectively and verified by DNA sequencing.E. coli Rosetta (DE3) cells harboring recombinant plasmids were grown in LB medium containing ampicillin (50 μg/mL). The cells were induced with isopropyl β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM when the absorbance reached 0.8–1.0 at 600 nm. The incubation was then continued at 30 °C for another 5 h. The cells were harvested by centrifugation at 3,500 × g for 10 min, resuspended in 50 mM Tris-HCl (pH 7.8), and disrupted by sonication. The cell debris was removed by centrifugation at 12,000 × g for 50 min. The cell-free extract obtained was applied onto a nickel nitrilotriacetic acid (Ni-NTA) agarose  column equilibrated with 20 mM imidazole in 50 mM Tris-HCl buffer (pH 8.0). Next, the enzyme was further purified on a Superdex 200 column  with a 50 mM Tris-HCl buffer (pH 7.8) containing 300 mM NaCl and 2 mM β-mercaptoethanol using AKTA FPLC system . The protein concentration was determined spectrophotometrically at 280 nm using the Thermo NanoDrop 2000 and further calibrated according to the theoretical extinction coefficient based on the amino acid sequences. The MhIHL mutants and other isochorismatases were expressed and purified in a manner identical to that used for the WT MhIHL enzyme.Km and Vmax values were calculated by nonlinear fitting. Conversion and enantiomeric excess (ee) were calculated as previously describedThe catalytic activities of MhIHL and mutants were examined by the addition of 0.2 μg pure MhIHL to 1000 μL of the 46 mM substrate solution . The catalytic activities of isochorismatase A, B, C, and D were detected by the addition of 80 μg pure enzyme to 1000 μL of the 46 mM substrate solution . The reactions were incubated at 25 °C for 15 min and then extracted with 1000 μL ethyl acetate. The (+)-γ-lactamase activity was detected as described previouslyAll the crystals were obtained at 20 °C using the sitting drop vapor diffusion method by mixing 1 μL of protein solution (15 mg/mL) with an equal volume of reservoir solution containing 0.2 M sodium malonate (pH 6.0) and 20% (w/v) polyethylene glycol 3,350. Crystals grew to a full size in two or three days. Crystals were cryoprotected in the reservoir solutions supplemented with 25% (w/v) glycerol, flash cooled in the liquid nitrogen, and stored at cryogenic temperatures for data collection. Both co-crystallization and soaking methods were conducted to obtain the enzyme-substrate structure. The final crystals of MhIHL–(−)-γ-lactam complex were obtained by soaking the crystal of the substrate-free C111A mutant enzyme in a cryoprotectant containing 250 mM (−)-γ-lactam for 1.5 h. The final MhIHL–(+)-γ-lactam complex crystals were obtained by soaking the crystal of substrate-free C111A mutant enzyme in cryoprotectant containing 500 mM (+)-γ-lactam for 10 min.Pseudomonas putida Kt2440 (PDB code 4H17) was used as the search model. The final model rebuilding was performed using COOTDiffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF), integrated and scaled using HKL20002 harmonic position restraint applied to all heavy atoms of the protein. The restraints were gradually removed and the production run was performed in MTK-NPT  ensemble for 50 ns (WT enzyme) and 10 ns (C111A enzyme), respectively. The SHAKE algorithmTo further explore the molecular mechanism of enantioselectivity of the enzyme, MD simulations were carried out for the complex structures of the WT/C111A enzyme with (+)-γ-lactam and (−)-γ-lactam respectively. The initial structures of the two WT complexes were modeled based on the C111A mutant-(+)/(−)-γ-lactam crystal structures using Protein Local Optimization Program (PLOP)3536Accession codes: The coordinates and structure factors of the apo-form, (＋)-γ -lactam bound, and (ý)-γ -lactam bound of MhIHL have been deposited in the Protein Data Bank under accession codes 5HA8, 5HWH and 5HWG.How to cite this article: Gao, S. et al. Structural insights into the γ-lactamase activity and substrate enantioselectivity of an isochorismatase-like hydrolase from Microbacterium hydrocarbonoxydans. Sci. Rep.7, 44542; doi: 10.1038/srep44542 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The inhibition of mitochondrial Ca2+ uptake by TSPO is a consequence of the phosphorylation of the voltage-dependent anion channel (VDAC1) by the protein kinase A (PKA), which is recruited to the mitochondria, in complex with the Acyl-CoA binding domain containing 3 (ACBD3). Notably, the neurotransmitter glutamate, which contributes neuronal toxicity in age-dependent conditions, triggers this TSPO-dependent mechanism of cell signaling leading to cellular demise. TSPO is therefore proposed as a novel OMM-based pathway to control intracellular Ca2+ dynamics and redox transients in neuronal cytotoxicity.The 18 kDa translocator protein TSPO localizes on the outer mitochondrial membrane (OMM). Systematically overexpressed at sites of neuroinflammation it is adopted as a biomarker of brain conditions. TSPO inhibits the autophagic removal of mitochondria by limiting PARK2-mediated mitochondrial ubiquitination via a peri-organelle accumulation of reactive oxygen species (ROS). Here we describe that TSPO deregulates mitochondrial Ca TSPO interacts with the Acyl-CoA binding domain containing 3 protein (ACBD3)18 that complexes with the cAMP-dependent protein kinase (PKA)19 which, via phosphorylation,20 regulates VDAC activity.The binding of TSPO to VDAC, which is the principal Ca2+ homeostasis and cell signaling by anchoring the ACBD3/PKA complex on the mitochondria and therefore primes VDAC post-translational modifications.We have therefore formulated a working hypothesis whereby the overexpression of TSPO, induced during cellular stress, may be functionally relevant to Ca2+ dynamics, redox transients and cytotoxicity.Here, we have tested this using various approaches to finally propose a model, which sees TSPO as an inducible, OMM-based conduit, to control intracellular Cain situ ubiquitination for autophagic removal.17 The increased accumulation of cytosolic ROS following overexpression of TSPO – previously reported17 – was here confirmed in the neuronal cell line SH-SY5Y overexpressing TSPO , an inhibitor of the Ca2+/Calmodulin-dependent protein kinase (CaMK) II21 which is pivotal for the activation of the NADPH oxidase (NOX)23  and Apocynin (200 nM)24 (The increased ROS levels were reduced by KN93 (10 00 nM)24 .2+-dependent NOX-originated ROS . The NOX family of superoxide (O2•−) and hydrogen peroxide (H2O2) producing proteins are indeed an important source of ROS in signal transduction.25Both inhibitors, consistent with the KN93 data, lowered cytosolic ROS levels in +TSPO conditions thus implying a link with the Ca2+ and Calmodulin-based mechanisms27 as it contains EF-hand Ca2+-binding domains29 and therefore being sensitive to increases in the [Ca2+]i. Interestingly, TSPO has been recently proposed to interplay with NOX proteins.30 To ascertain this and corroborate the pharmacological evidences implying a correlation between TSPO, Ca2+ and ROS producing NOX proteins, we generated a SH-SY5Y cell line stably downregulated for TSPO  – a Ca2+-sensitive photoprotein31 and perfused with ATP (1 mM). The resulting Ca2+ response was calibrated to quantify [Ca2+]m,31 which resulted which resulted in elevated levels of Ca2+ in TSPO knockdown (−TSPO) MEFs  recorded in −TSPO cells (To investigate the role of TSPO in CaPO) MEFs , with a .78±12.2 . Non-sil.78±12.2 . We alsoPO cells .2+ transients, using MEFs transfected with cytoplasmic-targeted aequorin (cytAEQ)33 reporting that the [Ca2+]c was decreased in −TSPO cells (2.43±0.25) compared with Control (4.31±0.14), while significantly higher transients were observed in both +TSPO (6.70±0.10) and +TSPOΔ cells ,34 which inhibits the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), leading to an IP3 independent rise in [Ca2+]c through proton-leakage channels.35 Using a fluorescence-based approach, we found that in −TSPO cells Tg elicited a significantly greater rise in mitochondrial Ca2+ level as determined by Rhod-2 Fmax (1.89±0.11) compared with the Control (0.73±0.12) and +TSPO  compared with Control (0.81±0.04), whereas TSPO knockdown restricted this rise  and mitochondria , TSPO expression level did not alter the level of interaction between mitochondria and ER  exposed to FCCP , while greatly increased when the protein is ablated . Also in this case, NSTSPO was used as control , the mitochondrial network appears larger  is dependent on the mitochondrial Ca2+ uniporter (MCU).42 To understand whether TSPO could be acting at this level, we verified the impact of TSPO expression modulation on the MCU transcription. Quantitative real-time RT-PCR was performed42 on samples extracted from TSPO-silenced MEFs with no differences recorded : 0.21±0.02, +TSPOΔ150–158: 0.23±0.03; The uptake of Caper se, which did not  compared with NSC . In these cells, modulation of TSPO failed to alter the ATP-induced [Ca2+]m signal thus indicating that VDAC1 is essential for its effects on [Ca2+]m . Still, the actual regulatory pattern remained elusive. Though, it was previously demonstrated that VDAC1 can be phosphorylated by the PKA46 leading to reduction in VDAC1 conductance.47 Equally, it was also shown that TSPO forms a macromolecular complex with the ACBD3, PKA and VDAC1 on the OMM.48 To pharmacologically test whether PKA contributes the impairment of the mitochondrial Ca2+ signaling recorded when TSPO is overexpressed, we treated MEFs with a synthetic protein kinase inhibitor peptide (PKI) and re-assessed mitochondrial Ca2+ levels in control and TSPO knockdown cells.49As TSPO interacts with VDAC1, which is the principal Ca49 normalized the differences in mitochondrial Ca2+ transients (The PKA inhibitor (used as 1 mg/ml), which binds the free catalytic subunit of PKA and prevents phosphorylation of the kinase targets,ansients  indicatiIn light of the above results, we examined the mitochondrial recruitment of both the ACBD3 and PKA. In mitochondrial fractions isolated from TSPO-overexpressing cells, we observed an increase of both ACBD3 and PKA, as shown by immunoblotting experiments reported in −/− cells , an increase in ROS production was immediately detected (n=5). Correspondingly, in these cells, glutamate-induced cell death  was far lower (n=3).The aged brain is highly sensitive to glutamate-induced toxicity and we therefore explored the TSPO-coordinated molecular cascade in SH-SY5Y cells challenged with glutamate to provide detected , abolishar lower . In keepThe detrimental effect of glutamate in WT SH-SY5Y was paralleled by TSPO, ACBD3 and PKA accumulation in mitochondrial fractions  suggesti−/−) in which TSPO expression is largely impaired.51 In these cells, glutamate led to negligible TSPO upregulation and abolished mitochondrial accumulation of ACBD3 and TSPO , a highly selective inhibitor of the extracellular signal-regulated kinase (ERK) to prevent stress-induced transcription of TSPO51 ;42 although additional elements can influence the kinetics and efficiency of mitochondrial Ca2+ uptake.55 Among these, the voltage-dependent anion-selective channels, which shuttle Ca2+ and metabolites across the OMM, are of immediate interest given the interaction with TSPO17 and the latter being an indicator of cell and tissue pathology6 recently linked to defective mitochondrial autophagy.17Mitochondrial Ca2+ into the organelle via VDAC1 and this is observed both in human, murine and canine cells, suggesting a conserved mechanism for mitochondrial Ca2+ adaptation. Furthermore, having recorded identical signaling pattern in microglial cells  mastered by the Ca2+ and ROS signaling homeostasis.57Here we demonstrate that mitochondrial TSPO reduces the transfer efficiency of Caal cells , the act2+ uptake capacity and increases the maximum [Ca2+]c following challenge with IP3 generating stimuli 61 thus phosphorylating DRP1 at alternative sites  antagonizing, and not compensating, for PKA-induced inhibitory phosphorylation at ser-656.Interestingly, high PKA activity is reported to promote mitochondrial elongation,tructure . It must2+]c can nonetheless predispose the cells to oxidative stress via activation of NOXs. Experiments performed using the pharmacological NOX enzyme inhibitors DPI, Apocynin, as well as KN93 that instead targets CaMKII , or similar forms of toxicological stress, may prime alternative ways of cell death in which the imbalance of redox homeostasis has a great part. It is indeed intriguing to think that TSPO as a priming element of the oxytosis, of the above-mentioned typologies of cell death, is the prototype.2+]i67 in which mitochondria are implicated as buffering compartments capable of modulating the degree of cellular Ca2+ tonicity.68 Molecules that regulate mitochondrial Ca2+ dynamics may indeed prove fundamental in reducing the severity of cellular insults to prevent or delay onset of neurodegeneration69 as well as regulating other processes, such as Autophagy, which are influenced by the Ca2+ tonicity.70Neuronal demise induced by the overstimulation of glutamate receptors is dependent on a sustained increase in  values using a computer algorithm.73The mtAEQmut transfected cells grown on glass coverslips were incubated with 5 μM rhod-2 AM  and 0.005% pluronic in modified Kreb’s Ringer Buffer  for 35 min at 37 °C.74 Following loading, the cells were washed four times in mKRB before imaging. For cytosolic Ca2+, the cells were loaded with 5 μM Fluo-4 AM  and 0.02% pluronic in mKRB for 30 min at 37 °C. The cells were immediately transferred to a Leica SP5 Confocal microscope for imaging where they were maintained at 37 °C. A field of transfected cells was selected  and using a × 40 objective, a time series was then set up so that a new image was acquired every 1 s to ensure the transient Ca2+ increase is captured. For mitochondrial Ca2+ accumulation, the 543 nm laser was used to excite Rhod-2 (red) and for cytosolic Ca2+, the 488 nm laser was used to excite Fluo-4 (green). After continuous recording for approximately 30 s, ATP was added (100 μM–1 mM). The increase in Ca2+ concentration induces a sharp rise in fluorescence intensity of both Fluo-4 and Rhod-2.74 When the signal returned to near starting values, ionomycin  was added (0.5 μM). The application of ionomycin eliminates any remaining Ca2+ gradient between all membranes, thus saturating available Fluo-4/Rhod-2. Recording was stopped when the intensity of the Ca2+ dye reached a plateau. The data were normalized to the ionomycin response by using the equation (x−min x)/(max x−min x).Alternatively, TSPO knockdown cells on glass coverslips were loaded with 8 17 in this study, the commonly used fluorescent dye, dihydroethidium (DHE), was used. DHE is sensitive to O2− (superoxide) and may be oxidized. Once oxidized, DHE binds to DNA, which results in the amplification of the red signal within the nucleus. The rate at which the signal increases in intensity is dependent on cytosolic levels of O2−, thereby enabling the quantification of ROS levels within the cell. The cells grown on glass coverslips were transfected with mitochondria-targeted GFP (mtGFP) and TSPO expression silenced. A field of healthy transfected cells was selected and 5 μM DHE , diluted in RM, was added. The rate of increase in fluorescence intensity was measured through continuous recording for at least 10 min.To quantify cellular levels of ROS4; 2 mM CaCl2; 10 mM glucose; 10 mM HEPES). The cells were allowed to equilibrate the dye for at least 30 min at room temperature before they were transferred to a Zeiss LSM 510 confocal microscope (× 40 objective) for imaging. Mitochondrial ROIs were demarcated and the corresponding TMRM fluorescence intensities calculated by removing background signal through thresholding as previously explained.74The cells, co-transfected with YFP were loaded with 50 nM tetramethyl rhodamine methyl ester (TMRM)  in HEPES-buffered salt solution  for 30 min. An object-based estimate of the co-localization events between ER-green and mtRFP channel 3D z-stacks was obtained by calculating Manders’ coefficients (M1 and M2).74 These are based on the Pearson’s correlation coefficient but do not consider average intensity values and vary from 0 (non-overlapping) to 1 .The CF35 cells were transfected with mtGFP to visualize mitochondria. To assess mitochondrial morphology, the mitochondrial form factor (FF) and aspect ratio (AR) were calculated as described previously.The 1-, 4-, 12- and 18-month-old C57/BL6 mice were killed by cervical dislocation and brains harvested on ice. The frontal cortex tissue was excised by forceps and lysed in a specific lysis buffer containing 1% Triton X-100 (Applichem Panreac), 320 mM sucrose (Sigma-Aldrich), 50 mM Tris-HCL pH 7.5, 50 mM NaCl (Sigma-Aldrich), supplemented with protease inhibitor cocktail solution (Sigma-Aldrich). Tissue was disrupted by sonication, followed by 30 min incubation on ice to allow protein solubilization. Next, the lysates were cleared by centrifugation for 20 min at 13 000 r.p.m. at 4 °C. The supernatants were then collected and protein concentration measured using Bradford method . The protein samples were stored at −80 °C before western blot analysis.


Recombinant soluble HIV-1 envelope glycoprotein (Env) SOSIP trimers are a design platform for inducing broadly neutralizing antibodies (bNAbs) by vaccination. To date, these and alternative designs of native-like trimers, given singly or in pairs, have not induced bNAbs in test animals such as rabbits or macaques. Here, we have evaluated whether trivalent and tetravalent combinations of SOSIP trimers from clades A, B, and C, delivered simultaneously or sequentially, induce better neutralizing antibody responses in rabbits than when given alone. None of the tested formulations led to the induction of bNAbs. We found that BG505 clade A trimers dominated the autologous NAb responses induced by combinations, which probably relates to the presence of immunodominant glycan holes on the BG505 trimer. Furthermore, autologous NAb responses to all individual trimers were reduced when they were delivered in combinations compared with when delivered alone, suggesting that immunogen interference had occurred. Finally, in a sequential regimen, a heterologous clade C trimer cross-boosted NAb responses that were primed by earlier immunizations with clade A and B trimers. Taken together, these findings should allow us to improve the design of immunization regimens based on native-like HIV-1 Env trimers.IMPORTANCE A successful HIV-1 vaccine most probably requires a trimeric envelope glycoprotein (Env) component, as Env is the only viral protein on the surface of the virus and therefore the only target for neutralizing antibodies. Native-like Env trimers can induce strain-specific neutralizing antibodies but not yet broadly neutralizing antibodies. To try to broaden the antibody response, we immunized rabbits with soluble native-like Env trimers from three different clades using monovalent, multivalent, and sequential regimens. We found that the neutralizing antibody response against each immunogen was reduced when the immunogens were delivered in combination or sequentially compared to the monovalent regimen. In contrast, when the Env trimers from different clades were delivered sequentially, the neutralizing antibody response could be cross-boosted. Although the combination of native-like Env trimers from different clades did not induce broadly neutralizing antibodies, the results provide clues on how to use native-like trimers in vaccination experiments. After more than 30 years of the HIV/AIDS epidemic, an effective vaccine is not yet available. One of the many barriers to overcome is the high diversity of viral strains. The isolation of a considerable number of broadly neutralizing antibodies (bNAbs) from infected individuals, i.e., antibodies that can neutralize multiple highly diverse HIV-1 isolates, has fueled hope that bNAbs can be elicited by appropriately designed vaccine regimens.Soluble native-like envelope glycoprotein (Env) trimers that mimic the native HIV spike and that can induce neutralizing antibodies (NAbs) against relatively neutralization-resistant (tier 2) autologous viruses are a design platform for immunogens intended to induce bNAbs. Since the description of the prototype native-like trimer, BG505 SOSIP.664, many more SOSIP or derivative designs have been described –5. Trime–in silico studies favored the use of antigen combinations to increase breadth  PGT145 affinity-purified SOSIP.v4 or SOSIP.v5 trimers were compared with combinations of trimers given either simultaneously or sequentially . The 40 In total, 25 animals (groups 9 to 13) received three immunizations with a trivalent (groups 9 and 10) or tetravalent groups 11 to 13) trimer combination. In the trivalent groups, three immunizations of BG505, AMC008, and ZM197M SOSIP.v4 trimers were given in either a low dose  or a high dose . The tetravalent groups received three immunizations with BG505, AMC008, B41, and ZM197M trimers, again either at a low dose  or a high dose . In the tetravalent study, the rabbits in group 11 received SOSIP.v4 trimers  than AMC008 and ZM197M , 11 and t1/2) of the titer decays between weeks 6 and 20 were significantly shorter for ZM197M and BG505 (8 and 13 days) than for AMC008 or B41   . In the animals immunized with single trimers, the autologous anti-trimer ELISA titers increased sharply between weeks 4 and 6 and then again between weeks 20 and 22, i.e., after the second and third immunizations, but declined after each of these boosts . The hal 0.0041)  to F. Th 0.0556) .The autologous anti-trimer ELISA titers varied between different immunization regimens. The binding Ab titers induced by BG505 or B41 trimers were similar over time whether they were delivered as individual immunogens or in multivalent combinations, while the responses to AMC008 and ZM197M trimers were slightly weaker for combination regimens than for the monovalent ones, but not to a statistically significant extent . The quaP value of 0.0041 for BG505 and P value of 0.0033 for B41). Thus, when given in combination, these two Env trimers cross-boosted anti-V3 non-NAbs   , but the 0.0079) . OverallP value of 0.0003) and ∼30-fold higher titers when given as part of a combination than those of other trimers in combination pools (P < 0.0001) , the immunogenicity differed among the trimers, consistent with previous reports , 10, 11. 0.0001) , left.P value of 0.0083) than that for the combination groups. One explanation is interference by the other trimers in the mixture, and another is a dose reduction effect in some of the combinations (see below). Similar findings applied to the other trimers .Sera from animals immunized with a single trimer consistently neutralized the corresponding autologous tier 2 virus more potently than sera from the combination groups . Thus, tP value of 0.0045; B41, ∼52-fold difference, P value of <0.0001; ZM197M, ∼3-fold difference, NS). These differences were not seen in the anti-trimer binding antibody analyses described above, implying that the latter assay outcomes were skewed by the presence of substantial amounts of non-NAbs , but greater differences were found for the other trimers     . In cont 0.0360) . Hence,  0.0002) . One posP value of 0.0317)  .P value of 0.0039) and 3.2-fold lower than those in the low-dose trivalent combination groups (NS). For AMC008, the corresponding NAb titer reduction compared with the monovalent regimen was ∼3-fold , but the NAb titer was ∼4-fold higher than that in the low-dose trivalent regimen . For ZM197M, 2 of 5 rabbits in the low-dose trivalent group had measurable autologous NAbs (titers of >40), but 0 of 5 in the sequential group did so (NS), and for the monovalent group the corresponding frequency was 7 out of 10 . In the sequential and combination immunizations, the SF162.LS NAb titers were ∼3-fold greater for the combination groups than those for the pooled monovalent groups (P < 0.0001) and ∼8-fold higher than those for the sequential groups . NAb titers against MW965.26 were moderate for monovalent BG505-, AMC008-, and B41-immunized groups     and then 16 tier 2 Env-pseudotyped viruses (Table S1). The BG505 SOSIP.v4.1 and SOSIP.v5.2 trimers induced a weak NAb response against SF162.LS , consistent with previous reports on these immunogens  3, 11)., 11.3, 1 to 300)  but sign pooled) . The MW9 0.0006) .The monovalent trimer formulations did not induce consistent heterologous tier 2 NAb responses, as described previously , 11, 13.P value of <0.0001 for AMC008 and ZM197M compared to BG505 and B41)  .P < 0.0001)  . The B41Overall, the SOSIP trimers differed in their capacity to induce heterologous NAbs, which may be related to the completeness of their glycan shields. However, simply mixing trimers from different clades is not sufficient to induce neutralization breadth.Sequential and cocktail immunization strategies have previously been explored for HIV-1 Env immunogens using DNA vaccines, recombinant gp120, or uncleaved gp140 immunogens –20. Gene–First, our results suggest that some trimers, particularly BG505, are immunodominant when used in combination with others. This observation is consistent with the strong immunogenicity of the BG505 trimer when used alone , 11, 13.We also found evidence for immune interference when trimers were given in combinations, with the effect being a disproportional reduction in the induction of autologous NAbs by some components. In short, autologous NAb titers were overall lower in combination or sequential immunizations than in monovalent regimens. Models of how GCs respond to immunogen combinations suggest that when individual B cells encounter different antigens, the conflicting selection forces during affinity maturation favor B cell apoptosis, a scenario that might be consistent with our findings . However21–Our third observation is that the B cell responses to trimers in combination are independent of one another, such that multiple autologous NAbs, but not cross-reactive ones, emerge. However, autologous NAbs against all of the immunogen trimers were seen only for the tetravalent combination and not the trivalent one. An explanation may be that the presence of two clade B trimers (B41 and AMC008) in the tetravalent regimen strengthened the NAb response against AMC008. Again, the use of more closely related trimers might be beneficial for driving some degree of neutralization breadth. Trimers derived from evolving Env sequences in the same HIV-1-infected individual might be particularly worth studying in this regard.We also found that the binding titers in the sequential and combination groups at week 22 all reached a similar level, which must have involved cross-boosting. However, since this breadth is not in line with the NAb titers, we can conclude that the cross-boosted responses mostly comprise non-NAbs, which are particularly easy to induce and therefore to cross-boost, as previously reported. Further research should assess whether diminishing the non-NAb responses would increase the breadth of the NAb responses.Finally, in the sequential immunogen group we found that clade C trimers cross-boosted NAb responses active against the clade A and B viruses that had been primed by the earlier clade A and B trimers. For this to happen, some common antigenic determinants relevant to neutralization must be shared on these sequence-divergent trimers; we note that their antigenicities and structures may differ much less than their primary sequences. In principle, such observations could be exploited in the design of future immunization schemes for the elicitation of more broadly active NAbs.When testing cocktail regimens, we found that tetravalent combinations based on SOSIP.v4 or SOSIP.v5 induced comparable autologous NAb responses, suggesting that the SOSIP version and thermostability are not a major determinant for the outcome of our experiments. This is consistent with the observation that SOSIP version and thermostability also were not a determining factor for the induction of autologous NAb responses in immunizations with monovalent vaccines , 11. TheIn conclusion, the induction of bNAbs requires more than the use of randomly chosen clade-divergent trimers delivered sequentially or in trivalent/tetravalent combinations. Nonetheless, we found some clues to guide the design of superior strategies based on the use of more closely related trimers, such as those derived from patient-derived lineages, which could be tested experimentally.Native-like trimers based on BG505 , AMC008 and B41 , and ZM197M (clade C) were used as immunogens 13. Some tn = 5 per group) were immunized at weeks 0, 4, and 20 with 22 μg of trimer (based on peptidic mass) formulated in Iscomatrix adjuvant, as published previously , under permits with approval numbers C0022-15, C0045-15, C0119-15, and C0120-15. The immunization and bleeding schedules are summarized in eviously , 11, 13.Autologous and heterologous NAbs were measured in the TZM-bl cell neutralization assay using sera from week 22, as previously described . Viruses50]) were determined.Titers of trimer binding Abs were assessed using the D7324-capture ELISA described elsewhere , 13. RabAntibodies reactive with V3 peptides were quantified in animals from groups 2, 4, 5, 7, and 9 to 14 as previously described (t1/2) of the binding Ab responses after the second immunization, i.e., between weeks 6 and 20, were calculated with GraphPad Prism using the one-phase unconstrained equation Y = (Y0 − plateau) × exp(−K × X) + plateau. Y0 is the binding titer when time is 0. Plateau is the binding titer when time is infinite. K is the constant rate expressed in reciprocal of days (days−1). The half-life is computed as ln(2)/K and expressed in days.Differences between rabbit groups in anti-trimer and autologous NAb titers, as well as measurements of neutralization breadth, were assessed by two-tailed Mann-Whitney U test. The half-life (


The magnitude of a cellular response is often encoded by frequency modulation of Ca2+ oscillations and correlated with the stimulation intensity. The stimulation intensity highly depends on the sensitivity of a cell to a certain agonist. In some cases, it is essential that neighboring cells produce a similar and synchronized response to an agonist despite their different sensitivity. In order to decipher the presumed function of Ca2+ waves spreading among connecting cells, a mathematical model was developed. This model allows to numerically modifying the connectivity probability between neighboring cells, the permeability of gap junctions and the individual sensitivity of cells to an agonist. Here, we show numerically that strong gap junctional coupling between neighbors ensures an equilibrated response to agonist stimulation via formation of Ca2+ phase waves, i.e. a less sensitive neighbor will produce the same or similar Ca2+ signal as its highly sensitive neighbor. The most sensitive cells within an ensemble are the wave initiator cells. The Ca2+ wave in the cytoplasm is driven by a sensitization wave front in the endoplasmic reticulum. The wave velocity is proportional to the cellular sensitivity and to the strength of the coupling. The waves can form different patterns including circular rings and spirals. The observed pattern depends on the strength of noise, gap junctional permeability and the connectivity probability between neighboring cells. Our simulations reveal that one highly sensitive region gradually takes the lead within the entire noisy system by generating directed circular phase waves originating from this region.Calcium ions (Ca 2+), a universal signaling molecule, is widely recognized to play a fundamental role in the regulation of various biological processes. Agonist–evoked Ca2+ signals often manifest as rhythmic changes in the cytosolic free Ca2+ concentration (ccyt) called Ca2+ oscillations. Stimuli intensity was found to be proportional to the oscillation frequency and the evoked down-steam cellular response. Stochastic receptor expression in individual cells in a cell population inevitably leads to individually different oscillation frequencies and individually different Ca2+-related cellular responses. However, in many organs, the neighboring cells have to overcome their individually different sensitivity and produce a synchronized response. Gap junctions are integral membrane structures that enable the direct cytoplasmic exchange of Ca2+ ions and InsP3 molecules between neighboring cells. By simulations, we were able to demonstrate how the strength of intercellular gap junctional coupling in relation to stimulus intensity can modify the spatiotemporal patterns of Ca2+ signals and harmonize the Ca2+-related cellular responses via synchronization of oscillation frequency. We demonstrate that the most sensitive cells are the wave initiator cells and that a highly sensitive region plays an important role in the determination of the Ca2+ phase wave direction. This sensitive region will then also progressively determine the global behavior of the entire system.The calcium ion (Ca Cells possess sophisticated machinery to precisely regulate the free Ca2+ concentrations in the cytoplasm (ccyt), the endoplasmic reticulum (cER) and the mitochondria (cmito). Maintaining the low concentrations of Ca2+ in the cytoplasm against a 10,000-fold higher extracellular Ca2+ concentration, i.e. the strong trans-membrane electrochemical gradient of Ca2+ ions needed for proper cell signaling Xu(t) and Xv(t) are perfectly synchronized, that is, all their peaks occur simultaneously. When the phases vary over time, so do their differences. Therefore, in order to quantify synchronization, one has to consider the distribution over time of the phase differences. The more the distribution is concentrated around 0, the more synchronized Xu(t) and Xv(t) are. A uniform distribution corresponds to the null case of no synchronization.Synchronization means here that two randomly selected neighboring cells tend to adjust the times at which they produce Canization ,31. Firsribed in . Denote Su, v is estimated by Smax,u,v = log). The number of class intervals is nb and pk denotes the relative frequency that Δϕu,v lies in the kth interval. The index Qu,v lies between 0 (no phase-synchronization) and 1 (perfect synchronization). Finally, we define a synchronization measure of the network by taking the mean of Qu,v over all neighboring cells u ~ vε| is the number of edges, i.e. pairs of neighbors in the network. When msync = 1, the synchronization between all cells is perfect and in particular no wave nor any pattern would appear. For medium ranges of msync, synchronization between neighboring cell is efficient enough to enable waves to emerge. When msync is close to zero, all cells tend to behave independently from each other. Our synchronization measure msync indicates the degree of phase synchrony focusing on time periods at which peaks are produced within the whole network. Amplitudes of the peaks are not taken into account for the parameter msync.To quantify the concentration of the phase differences distribution Tass et al.  and CazeTwo graph topologies were used: homogeneous, fully dense graph and heterogeneous sparse graph topologies .V0 = {vij, 1 ≤ i ≤ n, 1 ≤ j ≤ m} and let ε0 denote the set of links. Let V0. These nodes are connected in I) The homogeneous, fully dense graph topology consists of a grid graph to which diagonals were added G0, . In thisp(v) is built with a colored noise in space, in order to create spatial correlations among the network. Let F be a Gaussian random field on the graph with standard deviation set to one and with a correlation function of the Gaussian form, that isp(v) are as followsp ∈  in a way that in regions with nodes having low values of F, the probability to remove links is high, creating sparse areas, whereas regions with large values of F tend to be more strongly linked. Choosing a small quantile q of F, it is possible to create holes in the network by excluding nodes v from V0 whenever F(v) < q. The resulting graph is denoted by II) Heterogeneous sparse graph , .Xv(t), Yv(t)) the free Ca2+ concentrations in the cytosol (ccyt) and in the ER (cER) of the cell v at time t. The corresponding dynamical system on the graph v ∈ V with initial conditions Xv(0) = ccyt,ini = 110nM and Yv(0) = cER,ini = 260μM according to  is not precisely bell-shaped  also has an effect on the loading of the ER. Increased [InsP3] reduces the amount of Ca2+ ions stored in the ER. We simulated this effect by changing the parameter Ri2. The functions M, Ri,max and Ri,2 have similar forms, given byThe shape of pendence . In our l-shaped . For sim2+ oscillations: (i) it increases the levels of InsP3 by G-protein-regulated phospholipase C and (ii) it increases the Ca2+ flux characterized by JINF, mainly due to the opening of plasma membrane Ca2+ channels. We assume that both processes are positively related to the stimulus intensity, but two cells with the same [InsP3] can have different JINF values. Hence, we used two input parameters:t1 is the time point of the onset of increase in intracellular InsP3 levels and of the increase of the Ca2+ flux characterized by JINF. KIP3 and KJINF are positive constants. The functions InsP3 and JINF can be inhomogeneous in space, hence depending on the cell v. Initially, they are set to small values, which are constant in time and identical for all cells. At time t1(v), the processes start and both functions rapidly increase to approach limiting values iIP3,max(v) and iJINF,max(v), respectively. This leads to different frequencies of single cell Ca2+ oscillations. In accordance with experiments  is the minimum of (εv) and ψ[1] the maximum of (ψv). For a given node v ∈ V, if εv = εi][ and φv = φi][. The resulting noises (ψv) and (φv) are white, but (φv) is negatively correlated with (εv) and (ψv) is positively correlated with (εv). Finally, sett1(v) and iJINF,max(v) for all v ∈ V. With this construction, large values of iIP3,max correspond to small values of t1. Indeed, the largest iIP3,max(v) has the smallest value t1(v), the second largest iIP3,max(v) has second the smallest t1(v), etc.This model aims at considering the natural stochastic properties observed in living systems. Instead of fixing particular values for GR, we define v ∈ V and where j ∈ {0,1}. Define a small zone of size v, j = 0. The other parameters are defined analogously, with the correlation assumptions fulfilled as in the random model GR.Similarly as in the model All analyses were performed in MATLAB version 2013b  and the code for the scripts is available in the Supplementary Material .G0) oscillates in response to a given input encapsulated into the functions JINF and InsP3 for well-chosen parameters . Unlesssaturate . The modGD) with gap junctional coupling d > 0 and one particular region at the center of the homogenous, fully dense graph iJINF,max,1 >iJINF,max,0, iIP3,max,1 > iIP3,max,0 and t1,1 < t1,0). In this setting, a Ca2+ wave emerges from the center of the graph, indicating that the most sensitive cells are the wave initiators  outside of the range that would allow them to oscillate independently  leads to independent Ca2+ oscillations in each cell  Ca2+ coupling is active, but there is no InsP3 coupling (3 coupling is active but there is no Ca2+ coupling (t1(v), InsP3 coupling is very efficiently involved in the synchronization process, but afterwards acts poorly as synchronizing agent  Incoupling  and (iiicoupling . There a2+ wave propagation i.e. the heterogeneous sparse graph was used d), the density of the connection p(v) influences the wave propagation. Remember, the parameter p(v) indicates, whether two neighboring cells are connected or not, while the parameter d represents the strength of the coupling if such a connection exists. Hence it is not surprising that increasing the link removal probability p(v) has a similar effect on the wave propagation as decreasing the strength of the gap junctional coupling d. Highly connected cells (regions) enable waves to spread easily, while poorly linked regions will act as a barrier deviating the wave front to another direction. In the extreme case, holes were added to the network  play key roles in spiral generation. Independently of its strength, noise acts as a catalyst for producing spirals. For a low level of noise, moderate gap junctional coupling enables the appearance of transitions between circles and spirals. In highly disordered systems, the level of gap junctional coupling needed to smooth the spirals and obtain stable circles increases. Indeed, a high gap junctional coupling allows waves to spread rapidly among the cells and thus enables a fast synchronization of their behavior . As suchGR is used with a relatively high sensitivity. Due to the noise, the system is inhomogeneous enough to produce spirals, as locally explained in GR,C, by adding a small central zone with higher sensitivity regulates the behavior of the system and concentric circles develop  was found to substitute for the plasmalemmal Ca2+ exchange function, thus rendering the oscillations independent of extracellular Ca2+ [2+ transport into mitochondria was also found to influence the Ca2+oscillation frequencies [3R dynamics. The model proposed in our previous works [2+ waves, the key limitations are the size of the system , restritem  To generate a long distance Ca2+ wave, Ca2+ ions do not need to travel for long distances; it is enough to diffuse to the next InsP3R or RyR. More precisely, since these receptors are organized in clusters, Ca2+ ions only have to diffuse from one cluster to the neighboring cluster. Since the ER consists of a network in the cytoplasm filling almost the entire cell, the cluster-to-cluster distances between adjacent cells should be of similar magnitude than that of intracellular cluster-to-cluster distances, i.e. based on the images presented in Chalmers et al. [2+ buffer proteins not only take up Ca2+ ions, but to the same extent, also release Ca2+ ions. Depending on their Ca2+-binding parameters (fast or slow kinetics) they can promote or inhibit intracellular Ca2+ wave formation [2+ buffer, calretinin, on wave propagation. Our model predicts that calretinin promotes intercellular synchronization. (iv) As presented in the introduction, there are two types of Ca2+ waves: Ca2+ trigger waves and Ca2+ phase waves. If we complete our model with the gap junctional transport of InsP3, this results in Ca2+ trigger waves, since only few cells get activated before activation of the large majority of the other cells. In this case the gap junctional transport of InsP3 is the main agent harmonizing the initial Ca2+ signal, in agreement with previous experimental results [2+ oscillatory machinery and the individual stimulation of each cell, as in our model, allows the generation of phase waves in which the emergence of the waveform is due to Ca2+ release from adjacent oscillating cells slightly differing in their oscillation phase.It has been known for a long time that gap junctions are permeable both to InsPa2+ ions . Howeverof InsP3 . Thus In2+ waves . Althoug2+ waves  measureds et al. , approxiormation . Most pr results . However2+ ions and InsP3 are likely the synchronizing agents, possibly in a synergistic way. InsP3 is more involved in the formation of the initial Ca2+ trigger waves, while Ca2+ ions serving as a coupling agent are more implicated in the later ones indicating that Ca2+ phase waves are dominant at the later stages of a model experiment  required to generate the full range of oscillatory patterns and spreading Ca2+ waves [2+ signals are mostly the consequence of different strengths of coupling via gap junctions. Non-synchronous oscillations in individual cells is favored when gap junctional coupling is weak as was observed in cultured primary mesothelial cells [2+ bursts  [2+ oscillations, while high Cx26-expressing GT1-1 cells showed spontaneous intercellular Ca2+ waves, completely in line with our model. The occurrence of Ca2+ waves strongly depends on cell-cell contact probability and moreover the strength of gap junctional coupling. Our model also predicts that in a noisy system, in which each cell has an individual sensitivity to stimulation, the most sensitive cells act as the wave initiator cells.Stimulation of primary mesothelial cells induces Ca2+ waves . Our modal cells . Moderat+ bursts . Neverth+ bursts –65. This+ bursts . Strong 2+ waves ,68 are a6 (Cx26) ,70. Low 2+ signaling are cell-type dependent: Ca2+ signals elicit contraction in muscle cells [2+ signals in immune cells participate in the regulation of cell differentiation, gene transcription and effector functions [2+ signaling, frequently in the form of Ca2+ oscillations, activates specific enzymes and transcription factors in a cell, which are often involved in cell proliferation [2+ signals are usually not restricted to individual cancer cells, but are propagated to neighboring cells in the form of intercellular Ca2+ waves, usually Ca2+ trigger waves are observed [2+ signaling is the major way by which cells encode and transmit information, it is likely that during the passageway of a Ca2+ wave, several Ca2+-dependent targets in affected cells would be activated and/or deactivated. Our model predicts that Ca2+ waves play an important role in the harmonization of evoked responses i.e. Ca2+ wave initiator cells are capable of activating neighboring cells, even when those cells would not oscillate by themselves; however they start to oscillate and hence support waves when coupled to other cells. Thus, a highly sensitive cell may trigger cell division even if the neighboring cells are not sensitive enough to mitogenic stimuli. The exploration of the details on Ca2+ wave-dependent harmonization of Ca2+-related cellular responses remains an interesting topic to be investigated experimentally in the future.Physiological cellular responses to evoked Cale cells , neurotrle cells  and e.g.le cells . Ca2+ siunctions . They arunctions . Intraceferation . Ca2+ siobserved . Assumin2+ spirals are observed in “overstimulated” conditions. Frog oocyte overexpressing muscarinic acetylcholine receptor produce spiral waves in ccyt upon stimulation [Our model is capable of producing both circular waves and spirals within a specific range of parameters. Systems producing spirals, such as the famous Fitzhugh-Nagumo equations have been mathematically analyzed ,78–80 anmulation .2+ waves are required, i.e. Ca2+ phase waves propagating along a certain direction. For instance, rhythmic Ca2+ phase waves are required for the contraction of the gastric pylorus [2+ phase waves, but in this case the gap junctional transmission of action potentials is thought to be the relevant synchronizing process [2+ and InsP3 also plays role in the synchronization. This might explain the phenomena of delayed after-depolarization [3R [3 [During normal physiological function of many organs, directed rhythmic Ca pylorus , uterus  pylorus ,68, inte pylorus  or urina pylorus . Heart c process . Neverthrization  or arrhytion [3R . In our n [3R [3 . This con [3R [3 ,88.From a general viewpoint, noise can arise in any other excitable oscillating network, in which the individual units are connected by different synchronizing agents. Examples for coupled excitable units exist in the field of Biology (synchronization of cellular clocks in the suprachiasmatic nucleus ), PhysicS1 Movie(AVI)Click here for additional data file.S2 Movie(AVI)Click here for additional data file.S3 Movie(AVI)Click here for additional data file.S4 Movie(AVI)Click here for additional data file.S5 Movie(AVI)Click here for additional data file.S6 Movie(AVI)Click here for additional data file.S7 Movie(AVI)Click here for additional data file.S8 Movie(AVI)Click here for additional data file.S9 Movie(AVI)Click here for additional data file.S10 Movie(AVI)Click here for additional data file.S11 Movie(AVI)Click here for additional data file.S12 Movie(AVI)Click here for additional data file.S13 Movie(AVI)Click here for additional data file.S14 Movie(AVI)Click here for additional data file.S15 Movie(AVI)Click here for additional data file.S16 Movie(AVI)Click here for additional data file.S17 Movie(AVI)Click here for additional data file.S18 Movie(AVI)Click here for additional data file.S19 Movie(AVI)Click here for additional data file.S1 Code MATLAB Code(ZIP)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S3 Text(PDF)Click here for additional data file.


Although either iron chelators or antioxidants provide cardioprotection, a comparison of the efficacy of deferoxamine (DFO), deferiprone (DFP), deferasirox (DFX), N-acetyl cysteine (NAC) or a combination of DFP plus NAC on cardiac [Ca2+]i homeostasis in chronic iron overload has never been investigated. Male Wistar rats were fed with either a normal diet or a high iron (HFe) diet for 4 months. At 2 months, HFe rats were divided into 6 groups and treated with either a vehicle, DFO (25 mg/kg/day), DFP (75 mg/kg/day), DFX (20 mg/kg/day), NAC (100 mg/kg/day), or combined DFP plus NAC. At 4 months, the number of cardiac T-type calcium channels was increased, whereas cardiac sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) was decreased, leading to cardiac iron overload and impaired cardiac [Ca2+]i homeostasis. All pharmacological interventions restored SERCA levels. Although DFO, DFP, DFX or NAC alone shared similar efficacy in improving cardiac [Ca2+]i homeostasis, only DFP + NAC restored cardiac [Ca2+]i homeostasis, leading to restoring left ventricular function in the HFe-fed rats. Thus, the combined DFP + NAC was more effective than any monotherapy in restoring cardiac [Ca2+]i homeostasis, leading to restored myocardial contractility in iron-overloaded rats.Intracellular calcium [Ca Divalent metal transporter 1 (DMT1) has an important role in the uptake of iron into the heart26171731332+]i homeostasis as well as calcium cycling and iron regulatory proteins in iron-overloaded rats has never been investigated. In addition, growing evidence indicates that N-acetyl cysteine (NAC) has an iron chelating property402+]i homeostasis as well as the impact of this treatment on calcium cycling and iron regulatory proteins in iron-overloaded rats have never been investigated. The aim of this study was to investigate the hypothesis that DFO, DFP, DFX or NAC alone can improve cardiac [Ca2+]i homeostasis and left ventricular (LV) function in iron-overloaded rats, as well as whether the combined DFP plus NAC treatment can synergistically provide beneficial effects for these conditions.At the present time, three frequently used iron chelators; subcutaneous deferoxamine (DFO), oral deferiprone (DFP) and oral deferasirox (DFX), are used to treat patients with iron overload cardiomyopathy3536Plasma NTBI level was significantly increased in the HFeV rats when compared with the NDV rats, indicating that an iron overload condition occurred in the HFe-fed rats . On the Chronic iron overload led to cardiac dysfunction in iron-overloaded rats. The percentage of LV ejection fraction (%LVEF) decreased markedly in the HFeV rats at 3 months , and 4 m2+]i transients occurred in iron-overloaded rats when compared with the NDV rats  DFO, DFP, DFX or NAC monotherapy could lead to the restoration of the level of SERCA protein, improvement in cardiac [Ca2+]i transient amplitude, rising rate as well as restoring [Ca2+]i transient decay rate and diastolic Ca2+ level leading to improved LV function in iron-overloaded rats; (3) only a combination of DFP plus NAC resulted in a restoration of all parameters of Ca2+ homeostasis leading to restored normal LV function in iron-overloaded rats; (4) chronic iron overload and all pharmacological interventions had no effect on the levels of DMT1, ZIP14, hepcidin, ferroportin, LTCC and NCX in iron-overloaded rats. The summary of major findings of this study is shown in The main conclusions which could be drawn from this study were as following: (1) chronic iron overload led to increased levels of TTCC, decreased level of SERCA, and impaired cardiac [Ca2+]i dysregulation in rats. It is also significant that our results show that combined DFP plus NAC exerted synergistically beneficial effects on cardiac [Ca2+]i transients, and the combination therapy was more effective than monotherapy in iron-overloaded rats. Ca2+ function as secondary messengers and play an important role in many signaling pathways in all cell types43442+]i regulate electrical signals which play a role in cardiac rhythm and excitation–contraction coupling leading to cardiac contraction and relaxation2+ triggers Ca2+ release from the sarcoplasmic reticulum through ryanodine receptor2 (RyR2), which is called Ca2+ -induced Ca2+ release leading to directly activated cardiomyocyte contraction432+ is removed from the cytosol by four types of Ca2+ transporters: (1) Ca2+ is taken up into the sarcoplasmic reticulum by SERCA; (2) Ca2+ is removed from cytosol by NCX, and (3) sarcolemmal Ca2+-ATPase; (4) Ca2+ is taken up into mitochondria by mitochondrial Ca2+ uniporters1516182+), Ca2+ current could decrease, indicating that Fe2+ might compete with Ca2+ influx into the heart through LTCC which may contribute to systolic dysfunction in cases of iron overload cardiomyopathy472+ current was slower when Fe2+ concentration was increased in isolated ventricular cardiomyocytes of rats, which might possibly contribute to impaired diastolic function472+ entry into the cardiomyocytes through TTCC in iron overload conditions may be similar to LTCC. As expected, a previous study found that CaV3.1 TTCC was a significant portal for Fe2+ entry into cells leading to an iron overload condition2+ may compete with a Ca2+ influx into the myocardium through Ca2+ channels by both LTCC and TTCC2+ entry into the heart of thalassemic mice under iron overload conditions171879107This is the first study to demonstrate the cardioprotective effects of DFO, DFP, DFX, NAC or combined DFP plus NAC treatment on iron overload-induced cardiac i transients and cardiac contractility in the present study. Impaired cardiac SERCA function could prolong [Ca2+]i transient decay rates, leading to reduced Ca2+ reuptake into the sarcoplasmic reticulum and resulting in increased diastolic Ca2+ level in the present study. In addition, a previous study found that Fe2+ could directly inhibit the binding of [3H] ryanodine to its high affinity sites on RyR2 which led to reduced sensitivity of cardiac ryanodine receptors towards activation by Ca2+ 2+ competed with Ca2+ at the activating sites on RyR2 leading to decreased Ca2+-induced Ca2+ release in rat hearts which might contribute to impaired cardiac [Ca2+]i homeostasis, and cardiac dysfunction under conditions of iron overload2+ release by the sarcoplasmic reticulum leading to decreased [Ca2+]i transient amplitude and rising rate, and caused impaired cardiac contractility in the present study. Finally, %LVEF was significantly decreased in the HFe-fed rats leading to LV dysfunction in our study. In addition, our previous study and others demonstrated that an increase in cardiac iron store led to increased cardiac oxidative stress which caused increased mitochondrial dysfunction, resulting in cardiac dysfunction and iron overload cardiomyopathy72+ into the sarcoplasmic reticulum through a concentration gradient which leads to intracellular Ca2+ homeostasis2+ homeostasis as shown in our present study. Therefore, the restoration of Ca2+ homeostasis in iron-overloaded heart could be an indicator of functional restoration, which might have resulted from improved ATP production and respiration, due to both iron detoxification and an improved reductive capacity by either iron chelators or antioxidant(s). Nevertheless, the effects of iron chelators and/or antioxidant(s) on cardiac mitochondrial ATP production and respiration in iron-overloaded rats should be further investigated.Iron-catalyzed Haber-Weiss and Fenton’s reactions led to increased ROS production in iron overloaded rat’s plasma and heart tissueHowever, in our present study the levels of DMT1 and ZIP14 did not alter in the HFe-fed rats when compared with the NDV rats. The results support the findings in previous studies which demonstrated that DMT1 and ZIP14 were not the common pathways for iron entry into the heart under iron overload conditions18582+]i homeostasis leading to restored normal cardiac contractility and LV function by increased %LVEF to a normal condition in iron-overloaded rats. Although previous studies reported that neither DFO nor DFX have shown to be efficient in improving %LVEF in thalassemia major or TDT patients602+]i transients could be clarified by the following reasons. First, DFP has the lowest molecule weight of iron chelators when compared with DFO and DFX, and has a high lipophilicity which means it can rapidly penetrate into cells2+]i homeostasis. Second, NAC is an antioxidant that can decrease ROS levels, increase SERCA2a activity as well as enhance cytosolic Ca2+ removal2+]i dysregulation in this study. Moreover, NAC has been considered and used as a promoter of GSH synthesis and thereby cell reductive power2+]i homeostasis in the present study. The effects of all pharmacological interventions on cardiac [Ca2+]i homeostasis and LV function in iron-overloaded rats are summarized in 2+]i homeostasis leading to improved LV function. Although these monotherapy treatments showed similar efficacy to combined DFP plus NAC in restoring [Ca2+]i transient decay rate and diastolic Ca2+ level, only combined DFP plus NAC could restore all parameters of [Ca2+]i transients, including [Ca2+]i transient amplitude, rising rate, decay rate as well as diastolic Ca2+ levels. These factors led to a restored normal LV function in rats with iron-overload. For this reason, combined DFP plus NAC therapy may provide these similar cardioprotective results in patients with iron overload and so may be used as a future therapeutic strategy for treatment of iron overload cardiomyopathy particularly in hemochromatosis and TDT patients.Interestingly, our recent studies found that the combination of an oral iron chelator DFP and an antioxidant NAC could improve and restore normal brain and heart function effectively and provided more robust results than either DFO, DFP, DFX or NAC alone in the iron-overloaded rat model40ad libitum and were acclimatized for 7 days prior to the experimental protocols.The Institutional Animal Care and Use Committee (IACUC) at the Faculty of Medicine, Chiang Mai University authorized the animal study protocols (Permit number: 21/2557), in compliance with NIH guidelines, and in accordance with the ARRIVE guidelines for reporting experiments involving animals10H10Fe; Sigma-Aldrich, Co., St. Louis, USA) (w/w) was prepared as previously outlined®, Novartis Pharma Stein AG, Stein, Switzerland), (HFeDFO) 25 mg/kg/ day via subcutaneous injection, (3) deferiprone , (HFeDFP) 75 mg/kg/day, (4) deferasirox , (HFeDFX) 20 mg/kg/day, (5) N-acetyl cysteine , (HFeNAC) 100 mg/kg/day, or (6) combined DFP 75 mg/kg/day plus NAC 100 mg/kg/day (HFeDFP + NAC) through gavage feeding for 2 months, and all iron loading groups were regularly fed with the high iron diet. Since DFO is a parenteral iron chelator, it may cause suffering in patients with cardiac iron overload who are continuously treated with this drug. Previous studies have shown that DFP was significantly more potent than DFO in diminishing cardiac iron overload in TDT patients35352+]i homeostasis. Dosage of all iron chelators are the lowest therapeutic range that can be used to treat patients with iron overload712+]i transients.A standard laboratory rat diet containing 0.2% of ferrocene (CCardiac function was determined using echocardiography (GE Vivid I)422+ transient level in rat ventricular myocytes was measured using a fluorimetric ratio technique712+ indicator Fura-2 was loaded by incubating the cardiomyocytes at room temperature for 30 minutes with 25 μM of Fura-2-AM . Ultraviolet light wavelengths of 340 and 387 nm were used for the excitation of the Fura-2 from a xenon arc lamp controlled by a microfluorometry system  and the excitation light beam was directed into an inverted microscope . The ratio of emitted fluorescence signals from the Fura-2/AM loaded cardiomyocytes at 510 nm was recorded. The Ca2+ transient parameters including the [Ca2+]i transient amplitude, rising rate, and decay rate, as well as the diastolic Ca2+ level were recorded during electrical pacing . The ratio of the emission wavelengths is directly related to the amount of cardiac [Ca2+]i71The Ca3VO4, 5 mM NaF, and 1% proteinase inhibitor) at 4 °C, and subsequently centrifuged at 13,500 rpm for 10 minutes. The total protein of 40 mg was mixed with the loading buffer (1 mg/mL), boiled for 10 minutes and loaded onto 10% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels. Proteins were transferred to a nitrocellulose blotting membrane . Immunoblots were blocked with either 5% of bovine serum albumin or 5% of skimmed milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 (TBST) for 1 hour at room temperature. Then, immunoblots were exposed to the primary antibodies including the iron transporter protein DMT1  and Zip14 , iron regulatory protein ferroportin  and hepcidin , Ca2+ cycling protein LTCC , TTCC , SERCA  and NCX , and β-actin  for a loading control at 4 °C overnight. After that, the membrane was washed five times with TBST and then incubated with the horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. After washing the membrane again five times with TBST, the blots were visualized with an enhanced chemiluminescence (ECL) reagent and exposed by using ChemiDocTM Touch Imaging System . The images of the western blots were analysed using the ImageJ (NIHimage) analysis software17The heart tissues were extracted in a homogenizer in a lysis buffer . The data were processed using SPSS  release 22.0 for Windows. Differences between groups were tested for via One-way ANOVA analyses. Significant difference between groups was assumed if How to cite this article: Wongjaikam, S. et al. Restoring the impaired cardiac calcium homeostasis and cardiac function in iron overload rats by the combined deferiprone and N-acetyl cysteine. Sci. Rep.7, 44460; doi: 10.1038/srep44460 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


While, KChIP2 enhances the expression and stability of the Kv4 family of potassium channels, leading to a more pronounced transient outward K+ current, Ito,f, the guinea pig myocardium is unique in that Kv4 expression is absent, while KChIP2 expression is preserved, suggesting alternative consequences to KChIP2 loss. Therefore, KChIP2 was acutely silenced in isolated guinea pig myocytes, which led to significant reductions in the Ca2+ transient amplitude and prolongation of the transient duration. This change was reinforced by a decline in sarcomeric shortening. Notably, these results were unexpected when considering previous observations showing enhanced ICa,L and prolonged action potential duration following KChIP2 loss, suggesting a disruption of fundamental Ca2+ handling proteins. Evaluation of SERCA2a, phospholamban, RyR, and sodium calcium exchanger identified no change in protein expression. However, assessment of Ca2+ spark activity showed reduced spark frequency and prolonged Ca2+ decay following KChIP2 loss, suggesting an altered state of RyR activity. These changes were associated with a delocalization of the ryanodine receptor activator, presenilin, away from sarcomeric banding to more diffuse distribution, suggesting that RyR open probability are a target of KChIP2 loss mediated by a dissociation of presenilin. Typically, prolonged action potential duration and enhanced Ca2+ entry would augment cardiac contractility, but here we see KChIP2 fundamentally disrupts Ca2+ release events and compromises myocyte contraction. This novel role targeting presenilin localization and RyR activity reveals a significance for KChIP2 loss that reflects adverse remodeling observed in cardiac disease settings.Pathologic electrical remodeling and attenuated cardiac contractility are featured characteristics of heart failure. Coinciding with these remodeling events is a loss of the K The development of heart failure, whether from atrial fibrillation, hypertrophy, or myocardial infarction, culminates in compromised contractility and the insufficient ability to pump blood for the demands of the body. While current therapeutics have done much to stem and treat the progression of heart failure, we are still unable to stop the progression, let alone reliably reverse adverse remodeling. Given the ever growing rise in number of heart failure patients, the need for greater understanding of the molecular targets for disease onset and progression are required.Ito,f, critical for early phase 1 repolarization during the cardiac action potential i produced by stimulation. Ca2+ transient parameters were defined referring to the methods described previously [2+ was defined as cytosolic Ca2+ level just prior to the onset of the Ca2+ transient or just prior to the action potential upstroke in the cases where there was no obvious Ca2+ transient. Amplitude of intracellular Ca2+ transient was calculated from the difference between peak and diastolic Ca2+. Time to peak was measured from the onset to the peak of Ca2+ transient. The duration of intracellular Ca2+ transient was measured as the onset of the Ca2+ transient to the point of time when the transient decayed by 50%. SR content was compared between groups of myocytes after 10 mM Caffeine pulse induced SR Ca release using high speed solution exchange system. Sarcomere shortening was assessed using a video-based sarcomere length detection system (IonOptix Corporation) [Myocytes were bathed in a chamber with Tyrode’s solution composed of (mmol/L) NaCl 137, KCl 5.4, CaCl2 2, MgSO4 1, Glucose 10, HEPES 10, pH to 7.35 with NaOH. Intracellular Ca2+ transient and sarcomere shortenings were initiated by field stimulation at 2Hz at 35eviously . Cells woration) . Data acFreshly isolated guinea pig ventricular myocytes were cultured for 24 h at 37°C in M199 medium under control GFP or with KChIP2 antisense virus. Upon collection, cardiomyocytes were washed in ice-cold PBS and resuspended in a RIPA lysis buffer , 50 mM Tris, pH 8.0, plus Roche Inhibitor tablet) and then sonicated on ice to disrupt cell membranes. 30–40 μg of whole cell extract was loaded into SDS-PAGE gels, transferred to nitrocellulose membrane, and western blotting performed using antibodies against RyR , Serca2a , Phospholamban , NCX , GAPDH , beta-actin , pan-cadherin , and presenilin 1 . Protein concentrations were determined by the BCA method (Pierce).2+ transients were recorded by confocal line scanning (Leica DMi8). Myocytes were preloaded with 10 μM Rhod-3 dye (Thermofisher) in a tyrode solution consisting of (mmol/L) NaCl 137, KCl 5.4, CaCl2 2, MgSO4 1, Glucose 10, HEPES 10, pH to 7.35 with NaOH and 0.1% Pluronic® F-127 (Thermofisher). Mycoytes were paced at 1 Hz for 10 beats and then the pacing was halted to record Ca2+ spark activity at rest. Cells were scanned longitudinally along the long axis of the myocyte. Line scan images were processed and analyzed with ImageJ software utilizing the SparkMaster plugin [Line scan Car plugin .Isolated guinea pig myocytes were plated on laminin coated coverslips for 2 hrs, after which the media was changed with fresh M-199 and treated with Ad.GFP or Ad.KChIP2 KD for 24 hrs. Cells were fixed in 4% formaldehyde diluted in PBS for 15 minutes. Cells were permeabilized for 10 min in PBS + 0.03% Triton X-100 and blocked for 2 hrs in a solution of PBS, 5% normal goat serum, and 1% BSA. Cells were incubated overnight with primary antibody  in PBS with 2% normal goat serum and 1% BSA. Cells were rinsed 3x in PBS then incubated with secondary antibody (Alexa-568 1:1000 against Presenilin 1 and Alexa-647 1:500 against RyR) in PBS with 2% normal goat serum and 1% BSA for 2 hrs at room temperature. Labeled cardiomyocytes were scanned with a Leica DMi8 confocal microscope. Fluorescence intensity profiles were generated by ImageJ using the plot profile function for the declared regions of interest. A single region of interest was evaluated per myocyte. The peaks of all resulting plot profiles within each treatment group were aligned to create a representative average trace.The experimental data were expressed as mean ± SEM. A Student’s t-test or a paired Student’s t-test in the case of immunoblot analysis was performed. The paired test was applied given that each set of Ad.GFP or Ad.KChIP2 KD myocytes were derived from the same cell isolation. All tests were two-sided and a significance level of p < 0.05 was defined as statistically significant .This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for tissue isolation from adult guinea pig (Protocol Number: 2012–0175) samples was approved by the Committee on the Ethics of Animal Experiments of Case Western Reserve University. 14 male Hartley guinea pigs 5–6 weeks old from Charles River were used to conduct our studies.2+ transients and contractility. In myocytes treated with KChIP2 KD we observed a significant reduction in Ca2+ transient amplitude by 18.8% (Ca2+ transient amplitude: control 1.11 ± 0.06 vs KChIP2 KD 0.90 ± 0.07 AU)  or an antisense sequence for KChIP2 (KChIP2 KD) to acutely suppress expression as previously confirmed . Followi0.07 AU)  at 0.5 Hduration  but no cduration . Remarka2+ release and contraction are weakened. Notably, SR Ca2+ content was assessed by caffeine stimulation showing no change between treatment groups , SR Ca2+ ATPase (SERCA2a), phospholamban, and sodium calcium exchanger (NCX) expression, however, were all unchanged in response to KChIP2 KD  [N-methyl-D-aspartate (NMDA) receptor [2+ current. Moreover, these gain of function mutations to presenilin 1 are shown to enhance the amplitude of Ca2+ release through RyR, and have also been associated with the development of dilated cardiomyopathy and heart failure [2+ release, as we see in our investigation.Precedence for the ability of KChIP to modulate Casenilins , 14. Thesenilins . Notablyrations, –28 whichr (IP3R) , the N-mreceptor , and mosreceptor –17. As a failure . Togethein vitro through yeast-2-hyrbid assays [Notably, KChIP2 shares a high degree of sequence homology with KChIP3/calsenilin, further supporting that its ability to modulate presenilins is a conserved function. Indeed, KChIP3 is capable of modulating Kv4 channel expression and kinetics in the same manner as KChIP2 , 32, andd assays  and natid assays , we can d assays , or if t2+ release as well as slowed channel inactivation [2+ release, reflected in our time-to-peak measurements Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


PASMC from patients with idiopathic PAH (IPAH) have increased resting [Ca2+]cyt and enhanced Ca2+ influx. Enhanced Ca2+ entry into PASMC due to upregulation of membrane receptors and/or Ca2+ channels may contribute to PASMC contraction and proliferation and to pulmonary vasoconstriction and pulmonary vascular remodeling. We have shown that the extracellular Ca2+-sensing receptor (CaSR), which is a member of G protein-coupled receptor (GPCR) subfamily C, is upregulated, and the extracellular Ca2+-induced increase in [Ca2+]cyt is enhanced in PASMC from patients with IPAH in comparison to PASMC from normal subjects. Pharmacologically blockade of CaSR significantly attenuate the development and progression of experimental pulmonary hypertension in animals. Additionally, we have demonstrated that dihydropyridine Ca2+ channel blockers , which are used to treat PAH patients but are only effective in 15–20% of patients, activate CaSR resulting in an increase in [Ca2+]cyt in IPAH-PASMC, but not normal PASMC. Our data indicate that CaSR functionally couples with transient receptor potential canonical (TRPC) channels to mediate extracellular Ca2+-induced Ca2+ influx and increase in [Ca2+]cyt in IPAH-PASMC. Upregulated CaSR is necessary for the enhanced extracellular Ca2+-induced increase in [Ca2+]cyt and the augmented proliferation of PASMC in patients with IPAH. This review will highlight the pathogenic role of CaSR in the development and progression of PAH.Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary vascular resistance (PVR) leading to right heart failure and premature death. The increased PVR results in part from pulmonary vascular remodeling and sustained pulmonary vasoconstriction. Excessive pulmonary vascular remodeling stems from increased pulmonary arterial smooth muscle cell (PASMC) proliferation and decreased PASMC apoptosis. A rise in cytosolic free Ca Under normal physiological conditions, the pulmonary circulation is maintained in a high flow, low pressure, and low resistance state. During conditions of increased blood flow or cardiac output , vasodilation and recruitment (opening of closed blood vessels) of pulmonary arteries are two important mechanisms for reducing pulmonary vascular resistance (PVR). However, under pathological conditions, increased PVR is a major cause for the development of pulmonary hypertension. Elevated PVR over time leads to right ventricular hypertrophy and right heart failure.Pulmonary hypertension is defined by a mean pulmonary arterial pressure (PAP) ≥25 mmHg at rest, as measured by right heart catheterization. Pulmonary arterial hypertension (PAH) describes a subpopulation of patients with pulmonary hypertension, and is hemodynamically defined by a resting mean PAP ≥25 mmHg, pulmonary arterial wedge pressure ≤ 15 mmHg, and PVR >3 Wood units   is upregulated in PASMC and lung tissue from patients with IPAH , and pulmonary arterial endothelial cells is essential for maintaining normal structural and functional integrity of the pulmonary vasculature. If the balance is tipped in favor of cell proliferation, thickening of the wall, luminal narrowing, and eventual obliteration can occur. These structural changes that lead to hypertrophy and/or luminal occlusion are referred to as pulmonary vascular remodeling  and a member of family C of the GPCR acids. It has a large extracellular domain that is approximately 600 amino acids long. This large extracellular domain is composed of a bi-lobed Venus-flytrap-like domain which is connected to the seven-transmembrane-domain by a cysteine rich region. The intracellular domain of CaSR contains the COOH domain which is 216 amino acids long  resulting in conversion of phosphatidylinositol 4,5 bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) , which then activates the mitogen-activated protein kinase (MAPK) signaling cascade ultimately resulting in phosphorylation and activation of ERK1/2  leading to decreased conversion of ATP to cAMP and decreased protein kinase A activity  activity. RhoGEF then activates RhoA by catalyzing the exchange of GDP for GTP  on the sarcoplasmic reticulum (SR) releasing Ca2+ from the SR to the cytosol. In PASMC, depletion of Ca2+ from the SR induces store-operated Ca2+ entry (SOCE) through store-operated Ca2+ channels (SOC). DAG activates receptor-operated Ca2+ channels (ROC) in the plasma membrane resulting in receptor-operated Ca2+ entry (ROCE). A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation. Several studies have demonstrated that resting [Ca2+]cyt, SOCE, and ROCE are all increased in PASMC isolated from IPAH patients  compared to vehicle injected control rats. Additionally, resting [Ca2+]cyt. The increased [Ca2+]cyt results from Ca2+ release from intracellular stores and Ca2+ influx through ROC and SOC. Transient receptor potential conical (TRPC) channels have been shown to function as both ROC and SOC in PASMC   to chronic hypoxia results in attenuated RVSP, Fulton's Index, and pulmonary arterial wall thickness compared to chronically hypoxic wildtype mice  have been successfully administered as a long term therapy to a number of PAH patients who respond positively to acute vasodilator challenge , we have identified several miRNAs that may potentially target CASR. Further efforts should focus on in vitro and in vivo approaches to examine the potential involvement of upregulated/downregulated miRNAs in the upregulation of CASR shown in PASMC from IPAH patients and animals with experimental PH.While it has been demonstrated that both CaSR and TRPC6 are upregulated in PASMC from IPAH patients and animals with experimental pulmonary hypertension and play a critical role in the pathogenesis of PAH, it remains unknown how this is achieved. The human CaSR gene, CASR, can be transcriptionally regulated by transcription factors, post-transcriptionally regulated by microRNAs and epigenetically regulated by DNA methylation .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Molecular mechanisms underlying this survival defect are not yet clear. We have shown that lyp/lypGimap5 T lymphocytes display reduced calcium influx following T cell antigen receptor (TCR) stimulation that was associated with impaired buffering of calcium by mitochondria. Here, we investigated the subcellular localization of GIMAP5 and its influence on Ca2+ response in HEK293T cells and T lymphocytes. The more abundantly expressed GIMAP5v2 localizes to the lysosome and certain endosomal vesicles. lyp/lypGimap5 T lymphocytes showed increased accumulation of calcium in the lysosomes as evidenced by Gly-Phe β-naphthylamide (GPN) triggered Ca2+ release. As a corollary, GPN-induced Ca2+ flux was decreased in HEK293T cells expressing GIMAP5v2. Strikingly, TCR stimulation of rat, mouse, and human T lymphocytes increased lysosomal calcium content. Overall, our findings show that lysosomes modulate cellular Ca2+ response during T cell activation and that GIMAP5 regulates the lysosomal Ca2+ compartment in T lymphocytes.T lymphocytes from  IP3 binds to its receptor IP3R on endoplasmic reticulum (ER) and triggers Ca2+ release from the ER store, resulting in a conformational change in the ER-localized STIM1 protein (2+ release-activated Ca2+ (CRAC) channel on the plasma membrane, inducing capacitative Ca2+ entry from the extracellular milieu  activates the Ca2+ uniporter on the mitochondrial membrane that induces a slow, membrane potential-driven uptake of Ca2+, which is released later via the Na+/Ca2+ exchanger (2+ uptake by mitochondria ([Ca2+]m) is necessary to prevent feedback inhibition of the SOC channel activity by the raising cytosolic Ca2+ concentration  stimulation modulates up to 75% of genes implicated in survival and proliferation through activation of calcineurin and NFAT, leading to IL-2 gene expression . TCR engsphate IP and diacr milieu . CRAC chr milieu . TCR stiferation . Followixchanger . This Cantration , 12.lyp mutation causes a 5- to 10-fold reduction in CD4+ T lymphocyte numbers and absence of CD8+ T lymphocytes in secondary lymphoid organs  gene, resulting in a hypothetical protein lacking 223 amino acids at the C-terminus that is not expressed  domain, but possess a coiled-coil domain and a transmembrane (TM) domain -induced release of Ca2+ from the ER was comparable in cells expressing GIMAP5v1, GIMAP5v2, or the ΔTM constructs phenylhydrazone (FCCP), which uncouples the mitochondrial membrane potential. As shown in Figure 2+ released from the mitochondria in lyp/lypGimap5 T lymphocytes was slightly lower than that observed in T lymphocytes from control rats. These observations suggest that the mitochondrial Ca2+ content may be reduced in T lymphocytes due to the lyp mutation. It is also possible that this reduction in the mitochondrial Ca2+ content is a refection of the modest loss in mitochondrial membrane potential ex vivo  within the lysosomes by cathepsin C results in osmotic lysis of the acidic compartment  GIMAP5 prevents lysosomal Ca2+ accumulation in the absence of TCR activation.Given that GIMAP5 appears to regulate lysosomal Cachondria . Mitocho surface , 48. To 2+ homeostasis by modulating lysosomal Ca2+ stores. Lysosomes store significant amounts of Ca2+ and contribute to vesicular trafficking, autophagy, endocytosis, and exocytosis in most of the cell types, including immune cells  and lysosomal Ca2+ are important as Ca2+ influx following GPN and TG addition was higher in HEK293T cells expressing the GIMAP5ΔTM as well as in cells expressing GIMAP5v1 phenylhydrazone (FCCP) were from Sigma-Aldrich . Gly-Phe-β-naphthylamide (GPN) was obtained from Santa Cruz Biotechnology . Nocodazole was obtained from Calbiochem. R73 (anti-rat TCR), H57 (anti-mouse TCR), and IP36 (anti-human TCR) monoclonal antibodies were obtained from Affymatrix-eBiosciences . Dynalbeads®-M450 tocylactivated beads, Lysotracker Red, and Fura-2 were from Molecular Probes . Anti-LAMP2 (H4B4) mAb was obtained from Developmental Studies Hybridoma Bank at the University of Iowa . Secondary antibodies for immunoblotting were purchased from Jackson ImmunoResearch Laboratories. Secondary antibodies for fluorescence microscopy were purchased from Thermo Fisher Scientific .Tissue culture media, fetal calf serum, CaClPlasmids pmEGFP_α_tubulin_IRES_puro2b (Addgene plasmid # 21042) and pmCherry_α_tubulin_IRES_puro2 (Addgene plasmid # 21043) were kind gifts of Dr. Daniel Gerlich . EGFP-IC+ T lymphocytes. HEK293T or HeLa cells were transfected using polyethylenimine Max  and the stable transfectants were selected using puromycin (2 µg/ml). Protein expression was confirmed by immunofluorescence and by immunoblotting with anti-Flag antibody. The cell lines were cultured in DMEM containing 5% FCS, 1% pen/strep at 37°C in 5% CO2 containing atmosphere.Full-length or transmembrane-deleted GIMAP5v2 constructs tagged with an N-terminal Flag-tag, or EGFP tag were subcloned from pCDNA3.1  into pIR+ T lymphocytes were isolated from human peripheral blood and rat and mouse lymph nodes by negative magnetic selection as described previously . All reactions were run in duplicates along with no template controls for each primer sets. Changes in mRNA expression were calculated using the difference in CT values when compared to housekeeping gene (Gapdh) and expressed relative to controls.Total RNA was extracted using Trizol reagent  following the manufacturer’s instructions. cDNA was synthesized from 1 µg of RNA using QuantiTect Reverse Transcription Kit . Quantitative RT-PCR amplification reactions were carried out in iCycler iQ™  PCR detection systems using iQ™ SYBRStable HEK293T cell lines expressing the GIMAP5 constructs were grown on 15 mm coverslips and were transiently cotransfected with EGFP-α-tubulin, EYFP-KFC5 (Kinesin), or EGFP-IC2-FL (Dynein). Forty-eight hours post-transfection, cells were washed with PBS, fixed with 2% PFA for 13 min, washed, permeabilized with 0.2% saponin for 12 min, washed, and blocked for 20 min with 2% milk in PBS. The slides were incubated with the appropriate primary Ab overnight at 4°C. After washing, the slides were stained with flurochrome-conjugated secondary Abs along with DAPI that stains the nucleus. After the final wash, the slides were mounted with VectaShield (Vector Labs) prior to confocal microscopy. Images were acquired using an Olympus 1 × 81 FV1000 microscope with a 60× oil-immersion objective, and image analysis and Pearson coefficient quantification were done using Fluoview Olympus software 3.1.d-lysine for 2 h at 37°C.For live cell microscopy, HEK293T were grown in coverslips for 48 h before experiments. For purified CD4 T lymphocytes, cells were incubated in RPMI 5% at 37°C for 1 h in previously coated 25 mm coverslips with 100 µg/ml of poly-l-lysine coated coverslips were loaded with 1 µM Fura 2-AM for 30 min at 37°C. Coverslips were washed and deesterified for 20 min at room temperature in Ca2+ loading buffer . Coverslips were then placed in a chamber in an Olympus IX71 microscope  equipped with a Lambda-DG-4 illuminator . Cytosolic Ca2+ [Ca2+]c measurement was done over an average of 50–70 cells per coverslip per experiment. [Ca2+]c was measured at room temperature using two different excitation wavelengths of 340 and 387 nm, and fluorescence emission was monitored at 510 nm through a 415–570 nm dichroic mirror using an Evolve™ EMCCD camera . Image digitalization was done using MetaFluor software . In experiments where there was no Ca2+ in the extracellular medium, 0.5 mM EGTA was add to the loading buffer instead of 2 mM CaCl2 to chelate any extracellular Ca2+. Free [Ca2+]i was calculated using the Grynkiewicz method (HEK293T cells grown on 25 mm coverslips for 24–72 h and purified primary T lymphocytes plated on poly-z method .Proteins were extracted from cell lysates by homogenizing in RIPA buffer containing protease inhibitors and carried out as described in Ref. .p < 0.05) between two groups was determined by unpaired t test.Statistical analyses were performed using GraphPad Prism 6.0 . Data are presented as mean ± SEM. Statistical significance (considered to be relevant when DS and SR planned the experiments, interpreted the data, and wrote the manuscript. FG performed the experiments involving human and murine lymphocytes. SI and CL contributed to the analysis of the data and manuscript revision. GB helped with Fura-2 measurements.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.