PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in “PV-ergic” cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca2+-buffering/sequestration capacity.Parvalbumin (PV) is a cytosolic Ca It is commonly considered as an intracellular Ca2+ buffer or more precisely a Ca2+ signal modulator due to its two high-affinity Ca2+/Mg2+ mixed sites. PV is expressed at high levels in neuron subtypes, e.g. in certain GABAergic interneurons, in fast-twitch muscles, in parathyroid glands and in some epithelial cells in the kidney [2+ buffer modulating the temporal and spatial aspects of Ca2+ transients. PV-deficient (PV-/-) mice develop and breed normally and no obvious alterations in their home cage behavior or physical activity are observed [tibialis anterior (TA) from PV-/- mice was as expected from the absence of a slow-onset Ca2+ buffer, the enhanced resistance to muscle fatigue came as a surprise and was shown to be the result of an upregulation of mitochondria. The relative mitochondrial volume in fast-twitch muscles, e.g. in extensor digitorum longus (EDL) is almost doubled in PV-/- mice [2+ signaling. They are dynamic structures as their relative density and intracellular distribution, morphology and physiology vary in different cells and tissues. The organelle composition is adapted to meet the metabolic and signaling needs of each cell [Parvalbumin (PV) is a cytosolic protein of the family of EF-hand Cae kidney ,2. The pobserved . While t-/- mice . Mitocho-/- mice . Besidesach cell . The invach cell , while each cell . Mechaniach cell . The inv2+, Na+ and Mg2+ [2+ transport are DCT2 and, probably to a lesser extent, CNT. Ca2+ enters the cell at the luminal membrane via the TRPV5 channel and is sequestered by the intracellular “Ca2+ shuttles” calbindin D-28k (CB-D28k) or calbindin D-9k (CB-D9k). At the basolateral side, Ca2+ ions are extruded into the blood via the Na+/Ca2+ exchanger NCX1 and the plasma membrane Ca2+-ATPase PMCA1b [2+ reabsorption. Mg2+ enters the cell through apical TRPM6 channels [2+ shuttle and the extruder protein at the basolateral side has not been disclosed yet. TRPM6, the gatekeeper of transcellular Mg2+ reabsorption, is co-expressed with PV. As PV contains so-called Ca2+/Mg2+ mixed sites, PV is assumed to play a role not only in Ca2+-buffering/shuttling, but also in the regulation of Mg2+ homeostasis. In line, PV expression levels are upregulated in mice subjected to dietary Mg2+ restriction [Besides PV’s expression in excitable cells, it is also expressed in epithelial cells lining certain tubules in distinct regions of the distal nephron of the kidney. In humans and mice, PV is expressed exclusively in the early part of the distal convoluted tubule (early DCT/DCT1). In rats however, PV expression is observed in the thick ascending limb of the loop of Henle, the late DCT, connecting tubules (CNT) and in intercalated cells of the collecting duct ,10. The and Mg2+ . In the e PMCA1b ,13. The channels , while ttriction ,16.2+ levels in the serum and in the urine are normal; PV-/- mice do not waste Ca2+, they even have a positive Ca2+ balance with increased bone mineral density [PV-/- mice exhibit a reduced expression of the thiazide-sensitive sodium-chloride co-transporter (NCC) in the early DCT in the absence of ultrastructural changes. They show increased diuresis and kaliuresis at baseline, with higher aldosterone levels and lower lithium clearance . Of note density . This ph2+ homeostasome in non-excitable, PV-expressing cells. The inverse regulation of PV and mitochondria demonstrated before in muscle cells and Purkinje cells was validated in DCT cells of the kidney. DCT cells without PV manifest an increase in the expression of the mitochondrial marker COX1. The mechanism functions likewise in an in vitro cell culture system, i.e. in Madin-Darby canine kidney (MDCK) cells. Ectopic expression of PV decreased COX1, silencing of PV in the same PV-overexpressing cells via Pvalb shRNA restored the initial situation. The relative mitochondrial volume in PV-positive MDCK cells was strongly reduced and the mitochondrial membrane potential collapsed at lower CCCP concentration compared to PV-negative control MDCK cells. Thus, the previously observed inverse regulation of mitochondria and PV in excitable cells seems to be a general hallmark also prevailing in non-excitable cells of epithelial origin. The PV-induced reduction in mitochondrial volume and changes in the mitochondrial protein composition is proposed to impair the mitochondria’s ability to uphold its membrane potential, when the cytosolic Ca2+ concentration is augmented.Here we used a transgenic mouse line expressing the enhanced green fluorescent protein (EGFP) under the control of the PV promoter crossed with PV-/- mice ,19. Thes1Hmon  expresses EGFP under the control of the PV  promoter thus allowing to identify PV-ergic cells [tm1Swal; [The transgenic mouse line B6,D2F2Tgic cells . The EGFic cells  by crosstm1Swal; ). In thetm1Swal; . Mice we+) tubules mostly consisting of DCTs [For the COPAS sorting, we used the protocol described by Markadieu et al. . Briefly of DCTs  were col™  following the manufacturer’s protocol. Contaminating DNA was removed by incubation with DNase. cDNA was synthesized with ImProm-II™ Reverse Transcriptase . The resulting cDNA was diluted 1:4 prior to use. PCR amplicons from the cDNA (in triplicates) were generated on a Rotor-Gene 6000 thermocycler  using LightCycler® 480 SYBR Green I Mastermix , a one-component hot start reaction mix for PCR containing FastStart Taq DNA Polymerase and DNA double-strand-specific SYBR Green I dye for product detection and characterization. The primer pairs all spanning an exon-exon junction are listed in Total RNA of DCT segments (1600 DCT tubules) was extracted with peqGOLD TriFastFor Western blot analyses, the lysate of 800 DCTs or 20–40 μg protein extract (MDCK cells) were loaded and separated on polyacrylamide gels , then transferred onto nitrocellulose membranes using a semidry transfer protocol. After blocking with 5% solution of non-fat milk in TBS buffer, membranes were incubated with the relevant primary antibodies . Horseradish peroxidase-coupled secondary antibodies  were used at a dilution of 1:10,000. The protein bands were visualized with the Immobilon Western AP Substrate . Imaging and analysis of the blots were performed with the FluorChem E system . Membranes were stained with Ponceau S to check the loading and to enable normalization for densitometry. The integral of all visible protein bands was used in order to prevent a bias from any single reference or housekeeping protein ,23.Kidneys were fixed with 4% PFA and frozen in liquid nitrogen. Cryosections (5 μm) were mounted on microscope glass supports, blocked with 5% nonfat dry milk and then incubated 24 h at 4°C with α-PV25 . For the visualization, we used a fluorescent donkey anti-rabbit secondary antibody . The incubation time was 2 h at room temperature. For the immunostaining of cells, sterile glass coverslips were placed in 6-well culture plates. MDCK cells were then grown directly on the coverslips, fixed with 4% PFA and stained with the antiserum against PV and the fluorescent secondary antibody. Pictures were taken with a Hamamatsu digital camera (C4742-95-12NR) mounted on an upright fluorescence microscope . The data was processed using the Fiji (ImageJ) software.2 in Dulbecco’s modified Eagle’s medium (DMEM high glucose) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were passaged by trypsinization every 2–3 days.Madin-Darby canine kidney cells (MDCK) are derived from adult dog kidney and have properties similar to distal nephron cells . They arPvalb cDNA coding for full length PV. The cDNA fragment was synthesized by PCR using the primers 5’-AGTCGTTTAAACATGTCGATGACAGACTTGCTC-3’ and 5’-AGTCACTAGTTTAGCTTTCGGCCACCAGAGT-3’. The amplicon was digested with PmeI and SpeI and inserted into the PmeI and SpeI unique sites of the pLVTHM vector. Lentiviral vectors were produced by co-transfection of HEK293T cells with three types of plasmids: the transfer vector pLVTHM, the envelope vector pMD2.G-VSVG and the packaging vector pCMVDR8.91 (in a ratio of 10:3:8). Lentiviral vectors in the supernatant of HEK293T cells were harvested 48 and 72 h after transfection. MDCK cells were seeded in 24-well plates. The following day, they were transfected with non-concentrated lentivirus suspension containing 8 μg/ml Polybrene . Clones were selected by serial dilutions, expanded and tested for PV expression by Western blot analysis using the PV25 antibody.pLVTHM-PV lentiviral vector (Addgene plasmid #12247) was used to stably express parvalbumin in MDCK cells. The lentiviral construct and the vector production were described by . BrieflyPvalb  were purchased from Sigma, Buchs, Switzerland. Lentiviral vectors were produced as described above. For the transduction, MDCK cells were seeded into 6-well plates  and grown for 24 h. Lentivirus containing Pvalb shRNA was added at a MOI of 10 and 20. After 3 days of incubation, the medium was removed and replaced with growth medium containing 2 μg/ml puromycin. Cells were kept for 10 days in the selection medium; total proteins were then extracted with RIPA buffer and analyzed by Western blot.PV was downregulated in MDCK cells by short-hairpin RNA (shRNA). Plasmids coding for shRNA against rat ™ X3 2030 multilabel reader .For the detection of mitochondrial membrane potential changes two different fluorescent probes were used: Mitotracker Red (MTR)/Green (MTG)  and JC-1 . Cells were seeded in 96-well plates  and incubated for 24 h at 37°C. The dyes were prepared in culture medium at a final concentration of 50 nM (MTR), 100 nM (MTG) and 5 μM (JC-1). To dissipate the mitochondrial membrane potential, various concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were used. Cells were incubated for 45 min in the dye solution containing increasing amounts of CCCP (37°C), then washed with PBS to remove the free dye. The fluorescence was measured in a Victor6 cells per ml. Cell counts were performed on a BD Accuri C6 flow cytometer . The forward scatter vs. side scatter of untreated cells was used to determine the size gating. Quantitative and statistical analyses were performed with the FlowJo software .To assess the cellular mitochondrial mass, cells were stained with MTG-FM (200 nM), a mitochondrial-specific fluorescent dye showing a distinct fluorescence independent of the mitochondrial membrane potential. Control cells and PV-expressing MDCK cells were trypsinized, normal growth medium was added to stop the reaction. After centrifugation , cells were resuspended in PBS and counted. The cells were stained for 30 min at 37°C. After the incubation, the cells were washed and resuspended in PBS at a concentration of 1x10™ X3 2030 multilabel reader . Bioluminescence signals were normalized to total protein contents  in the samples, the latter reflecting the number of cells in each well. Results are from 4 independent experiments, each one carried out in triplicates. In each experiment the value for control MDCK cells (without PV expression) was defined as 100%.®Relative ATP levels were determined using the ATP Bioluminescence Assay Kit CLS II (Roche) according to the manufacturer’s instructions. Briefly, 9 volumes of boiling 100 mM Tris/4 mM EDTA (pH 7.75) were added to one volume of the cell suspension. After 2 min of incubation at 100°C, the samples were centrifuged at 1000 x g for 60 s, the supernatant was then transferred to a fresh tube. Samples (50 μl) were pipetted into a 96-well plate and 50 μl luciferase reagent were added; the luminescence was measured in a VictorPvalb promoter [Ucp2, Efhd1, Micu1, Mcu, Mcur1, COX1, Atp5b) were increased. In order to validate the Gene Chip data, total RNA was extracted from a second set of COPAS-isolated DCT fragments of 4 PV+/+ and 4 PV-/- animals and mRNA levels of seven genes coding for mitochondrial proteins were determined by quantitative RT-PCR. Besides prototypical mitochondrial genes including cytochrome c oxidase subunit 1 (COX1), ATP synthase subunit β (Atp5b), uncoupling protein 2 (Ucp2), we focused on genes implicated in mitochondrial Ca2+ handling: mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu) and mitochondrial calcium uniporter regulator 1 (Mcur1). In comparison to PV+/+ mice, mRNA levels in PV-/- DCT were upregulated for all investigated genes (ΔΨm), was increased by 31% ± 7% in the DCT of PV-/- mice . Thus, the changes induced by ectopic PV expression were reverted partially by Pvalb shRNA treatment documenting the bidirectional regulation in an identical setting. The partial reversal of the mitochondrial volume might be the result of the incomplete PV down-regulation by the shRNA approach. ATP synthase subunit β levels were unchanged after PV down-regulation  stably egulated . In ordeelection , while welection . IHC forelection . As the election . Also thgulation , i.e. ne2+ influx-induced membrane depolarization. For this we relied on fluorescent dyes suitable to measure changes in the mitochondrial membrane potential. Since intracellular fluorescence increases as the result of either a rise in ΔΨm or an increase in mitochondrial mass density, a method of controlling for changes in mitochondrial mass is required to monitor changes in ΔΨm [ΔΨm and Mitotracker Green (MTG) that is insensitive to changes in ΔΨm. Normalized ratios for both approaches demonstrated that an increase in the concentration of the uncoupler CCCP in the range of 2.5–10 μM led to a minor decrease in ΔΨm in control cells .The precise mitochondrial protein composition with respect to expression levels of individual protein is assumed to be the result of tissue- or even cell-specific mitochondria function ,28. Bases in ΔΨm . We usedol cells . In PV-eol cells  and JC-1ol cells  fluorescol cells . At the 2+ buffer PV are inversely regulated. Thus, ectopic expression of the slow-onset Ca2+ buffer PV appears to functionally replace mitochondria conceivably based on similar kinetic properties of Ca2+ removal. The decrease in mitochondria volume together with the altered mitochondria protein composition impairs the epithelial cells’ ability to sequester Ca2+ into mitochondria. Putative consequences resulting from PV deregulation are discussed below.Since an inverse regulation of PV and mitochondrial volume was demonstrated in excitable cells ,7,8, we 2+ signals. Among these processes are transcription, cell cycle regulation, differentiation, cell motility/migration, programmed cell death, muscle contraction/relaxation, neurotransmission, secretion, and learning and memory [2+, cells must be equipped with a precisely tunable system regulating the Ca2+ concentration accurately in space and time [2+ signaling toolkit comprises Ca2+ channels, Ca2+ pumps and exchangers, as well as Ca2+ buffers. If a component of the toolkit is defective or missing, compensatory mechanisms are induced, either at the level of the affected cell or within the entire network, where the cell is functionally integrated. In general, the absence of a particular Ca2+-binding protein is not compensated by another EF-hand family member, i.e. one that is normally not expressed in this cell type [2+ binding proteins calretinin and CB-D28k were affected by the lack of PV [2+ buffers in this part of the distal nephron. CB-D28k and CB-D9k, which are present in epithelial cells in close vicinity to the DCT1, exhibited unchanged expression levels and distribution patterns in PV-/- animals. The expression of CB-D28k commences abruptly at the transition early to late DCT and continues at a lower level throughout the connecting tubules. CB-D9k is expressed in late DCT and connecting tubules [Atp5b mRNA coding for the ATP synthase subunit β was increased (Atp5b mRNA [in vitro (this study).Many physiological responses inside a cell and as well communication between cells are regulated by intracellular Cad memory . To corrand time . This Caell type . In the ck of PV . Our exp tubules . As demo tubules  had repo tubules ), an effncreased , no diffncreased . A similPvalb mRNA by shRNA essentially restored the initial situation. Besides energy production, mitochondria are strongly implicated in controlling intracellular Ca2+ signaling and hence cell function by buffering of cytosolic Ca2+ levels [2+ buffer PV by mitochondria might be seen as a homeostatic adaptation based on similar kinetic characteristics. PV is characterized as a slow-onset Ca2+ buffer in essentially all systems investigated. In muscle cells, PV is too slow to affect the rapid rise of the cytoplasmic Ca2+ concentration during the contraction phase, but it helps to increase the initial rate of decay of the transient and thus shortens the relaxation phase [2+ into mitochondria follows changes in the cytoplasm with a slight delay. During a single twitch in TA recorded in vivo, mitochondrial Ca2+ signals reach their maximum with a delay of 10–20 ms compared to the cytoplasmic peak [2+ peak is reached during the early relaxation phase. Thus, like PV, mitochondria promote an increase in the initial rate of cytoplasmic Ca2+ concentration decay. Most probably PV can’t be replaced by cytoplasmic Ca2+ buffers with fast kinetics. The absence of CB-D28k in Purkinje cells strongly affects the shape of dendritic Ca2+ signals [The regulation of COX1 in MDCK cells functions bi-directionally: ectopic PV expression decreased COX1 by 20%, silencing of + levels . The repon phase . Uptake mic peak . The mit signals  as well  signals ; however signals . Mitocho signals .2+ buffers, mitochondria also have the ability to modulate cytosolic Ca2+ signals. This has been demonstrated in many cell types including MDCK cells [2+, store it transiently in the matrix and eventually release it back to the cytosol. These characteristics enable mitochondria to regulate cytoplasmic Ca2+ concentrations [2+ homeostasis under control. Gustavsson et al. [2+ responses in pancreatic β cells. In addition they noticed that the Ca2+ rise was less pronounced in cell areas, where most mitochondria were localized. A buffering effect of mitochondria on cytosolic Ca2+ transients could also be confirmed in cardiomyocytes [2+ signal, but the decay rate tended to be lower. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a regulator of mitochondrial biogenesis and modifies the mitochondrial proteome. PGC-1α overexpression in primary skeletal myotubes and in HeLa cells increased the mitochondrial volume [2+ is distributed in a larger volume, resulting in a reduced increase of the mitochondrial Ca2+ concentration during mitochondrial Ca2+ accumulation. This reduced mitochondrial Ca2+ accumulation might lead to a diminished cellular sensitivity to Ca2+ -mediated apoptosis. Mitochondrial biogenesis is controlled by a Ca2+-regulated signaling pathway. Wu [Pvalb shRNA. The reversal was not complete, likely resulting from the incomplete PV down-regulation after shRNA treatment as shown in Pvalb shRNA treatment , colocalized with PV in the early DCT [SLC12A3 gene coding for NCC [2+ transients via luminal P2Y2 receptors and decreases the expression of NCC at mRNA and protein level. This Ca2+ signal was modulated by PV. NCC down-regulation was abolished in cells overexpressing cytosolic PV, but overexpression of nuclear-targeted or mutated PV unable to bind Ca2+ was not effective [2+ concentrations to stabilize NCC expression within the normal range as observed in cells from wildtype mice.Kidneys of PV -/- mice show a decreased expression of the thiazide-sensitive Naarly DCT . The absarly DCT . Both mo for NCC . Gitelma for NCC ,46. A liffective ,47. The ffective  in addit2+ shuttle facilitating transcellular Ca2+ resorption, is not required for renal Ca2+ uptake. Mice lacking PV even have a positive Ca2+ balance: plasma Ca2+ levels are unchanged, calciuria is slightly reduced and bone mineral density increased. Hypocalciuria might be seen as a consequence of the NCC down-regulation, which leads to renal salt and water loss resulting in contraction of the extracellular volume (ECV). ECV triggers a compensatory increase in proximal Na+ reabsorption. This enhances the electrochemical gradient driving passive paracellular Ca2+ transport in the proximal tubule [2+ absorption and upregulation of the active Ca2+ transport machinery  were observed.PV, a putative intracellular Cal tubule . Additiol tubule , increas2+/Mg2+ mixed sites. It is therefore assumed that PV participates not only in Ca2+-buffering/shuttling, but also in the regulation of Mg2+ homeostasis. TRPM6, colocalized with PV in the early DCT, is considered to be the gatekeeper of Mg2+ reabsorption [2+. This implies that intracellular Mg2+ buffering and Mg2+ extrusion mechanisms strongly impact on the functioning of the channel [2+ , PV is a suitable candidate to act as an intracellular transporter/shuttle [2+ levels in blood/plasma and in urine. A possible explanation for these findings is that the lack of the Mg2+ shuttle PV is compensated by an increased passive paracellular uptake in the proximal tubules and the thick ascending limb of Henle or by intestinal hyperresorption of Mg2+. Alternatively it can’t be entirely excluded that PV is not contributing to the active Mg2+ reabsorption in the DCT. The molecular details of the Mg2+ extrusion across the basolateral membrane, as well as the Mg2+-binding molecules, are not known in detail yet. In a study of de Baaij et al. [2+-sensitive DCT transcriptome was elucidated; 46 genes showing differential expression were identified. Pvalb was among the upregulated genes, which hints at an involvement of PV in renal Mg2+ handling. In addition, yet unknown Mg2+-binding proteins might exist acting (possibly together with PV) as intracellular Mg2+ shuttles.PV contains so-called Casorption . The act channel . Based oj et al. , the Mg22+ uptake evidenced by the increase in proteins implicated in this process. Ectopic expression of PV induced the opposite changes that might be detrimental under some conditions.Our data show that the inverse regulation of PV and mitochondria is not restricted to excitable cells and moreover that it works bi-directionally. Also in kidney DCT1 epithelial cells, PV is replaced by mitochondria as evidenced by the increased mitochondrial volume. Our experiments further show that the mitochondrial membrane protein composition is adjusted to better preserve a high membrane potential and is optimally suited for mitochondrial CaS1 Figtibialis anterior of WT and PV-/- mice. All analyzed genes (mRNA) were increased in mice without PV expression. The increase was statistically significant (Mann-Whitney U test) for Ucp2 (p = 0.042), Micu1 (p = 0.022) and Atp5b (p = 0.026).Total RNA was isolated from (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of  Bacillus anthracis is regarded as one of the most serious of all bioterror threats because of the persistence and ease of dispersion of B. anthracis spores as well as the rapid onset and lethality of disease resulting from spore inhalation .Genes encoding both the native and mutant forms (PA SNKE167-ΔFF-315-E308D) of PA were cloned into expression plasmids and transformed into derivative strains of in MB101 . PurifiewtrPA and mrPA products were formulated  to contain 1.0 mg/mL aluminum, added as Alhydrogel  in Dulbecco’s phosphate buffered saline (DPBS). Dosage forms were prepared as follows: wtrPA formulations with 20 μg/mL, 5 μg/mL, and 1.25 μg/mL rPA protein in Alhydrogel; mrPA formulations with 20 μg/mL,5 μg/mL, and 1.25 μg/mL mrPA protein in Alhydrogel. The dosing solutions were prepared aseptically as 1 mL of total sample in glass vials and stored refrigerated (2–8°C). The positive control article, Anthrax Vaccine Adsorbed , Lot FAV363, was characterized by its manufacturer . Negative control formulations were DPBS containing Alhydrogel. Lots of mrPA and wtrPA vaccines used for this study were characterized for activity by a LBERI Good Laboratory Practice (GLP 21CFR) validated potency assay  utilizing serum from immunized AJ mice ,15.Pasteurella-free , were placed on study. The rabbits weighed between 2.57–3.33 kg  and 2.45–3.29 kg  and were approximately 21–23 weeks old on Day 0 (study start). Rabbits were identified by subcutaneously placed Biometric Data Systems telemetry chips  and randomized into study groups using a validated computerized data acquisition system  based on weight. Body weights of individual animals were ±20% of the group mean for each gender and groups were approximately equal mean body weights at study start prior to vaccination. Rabbits were housed individually in stainless steel/plastic cages in temperature and humidity controlled rooms on a 12h light cycle and fed Harlan Teklad Global Diet 2031C  with free access to water. Prior to pathogen challenge, all test rabbits were conditioned and acclimated to restraint by being placed into rabbit exposure boxes for 10 ± 5, 30 ± 5 and 60 ± 5 minutes on separate days. The last conditioning session occurred within three days of exposure. All animimal procedures were 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 was approved by the Lovelace Respiratory Research Institute Animal Care and Use Committee. Animals were observed at least twice per day (morning and afternoon) prior to B. anthracis spore challenge and three times per day after challenge (see below) for signs of morbidity and mortality. Based on individual animal condition, additional observations for morbity and mortality occurred at the discretion of the Study Director in consultation with technical and veterinary staff. Examinations were oriented toward (1) identifying dead or moribund animals, and (2) documenting the onset of any abnormal clinical signs. The Study Director in consultations with the veterinary and technical staff made decisions regarding the euthanasia of moribund animals. Criteria for moribund status included severe respiratory distress, persistent recumbency and weakness, unresponsiveness to touch or external stimuli, extreme weight loss, or a combination of these observations. When morbidity was observed, all feasible actions were taken to limit pain and suffering by euthanizing animals by intravenous (IV) injection of an overdose of a barbiturate based sedative . Due to the nature of the study investigating the efficacy of a vaccine, no analgesics or anesthetics could be administered.Forty-five male and 45 female New Zealand white (NZW) rabbits, certified 50 of B. anthracis Ames spores (1.1 X 105) -2,5-diphenyltetrazolium bromide  was added to each of the wells at the end of the 4 hour incubation and the plates were then incubated as above for an additional 2 hours. Afterwards, 100 μL of solubilization buffer  was added to each of the wells and the final incubation proceeded as above for 16–20 hours. Plates were read at 570 nm using a Bio-Tek μQuant microplate reader and the data were analyzed using Gen5 Data Analysis Software (Bio-Tek). The ED50, the reciprocal of the dilution that inhibits 50% of the cell death due to the lethal toxin, was obtained by analysis of the 4-parameter curve. The Neutralization Factor (NF50) was determined as the ratio of the test sample ED50 to ED50 of the positive control reference serum.Sera collected pre-vaccination and on Days 14, 28, 42, 65 and 84 were assayed utilizing a Good Laboratory Practice (GLP 21 CFR) validated method for the presence of functional antibodies capable of neutralizing the toxic activity of escribed . Sera coB. anthracis Ames spores used for rabbit inhalation challenge studies were prepared essentially as described -labeled anti-PA antibody). The amount of PA present in the sample was determined by the amount of light that is emitted upon electrochemical stimulation of the STAG initiated at the electrode surface of the kit’s microplate.Sera collected on Days 65, 71 through 74 and at euthanasia was assayed for the presence of anthrax protective antigen in serum using an in-house, GLP validated, qualitative ECL assay . This asB. anthracis colonies was determined. For quantitative assessments, blood and tissue samples were serially diluted in sterile 1% peptone. From each dilution, 100 μL was removed and plated onto sterile 90 mm TSA plates in triplicate and allowed to incubate at 37°C for 16–24 hr. B. anthracis titer (colony forming units [CFU]/mL) was calculated:Blood was collected on Days 65, 71 through 74 and 84 for determination of qualitative or quantitative (Day 84) bacteriology. No baseline serological analyses were reported. All rabbits that were found dead or euthanized post-challenge were necropsied and had select tissues assayed for quantitative bacteriology. For qualitative assessments, collected blood was plated onto a single, sterile 90 mm tryptic soy agar (TSA) plate and incubated at 37°C for 16–24 hr, after which the presence or absence of The total dilution factor included the media plate inoculum volume of 100 μL .Rabbits surviving to the scheduled sacrifice or determined to be moribund were euthanized by intravenous (IV) injection of an overdose of a barbiturate based sedative . Lung, spleen, heart, and tracheal-bronchial lymph nodes (when present) were collected for bacteriology and histopathology. Tissue sections were fixed in 10% neutral-buffered formalin (NBF), cut, mounted on slides, stained with hematoxylin and eosin (H&E) and microscopic findings evaluated for incidence and severity by a board certified veterinary pathologist. Findings were given a score from 1 to 4, based upon a subjective assessment of the overall severity in the tissue present on the slide . Where appropriate, an assessment of distribution was also made . A qualitative assessment of bacterial burden was also made where appropriate.Survival, immunogenicity, antibody nuetralizations were the primary outcomes assessed for this study. The number of rabbits dying and mean time to death and/or mean survival time was determined for each group. Survival analysis was tested by log-rank (Mantel-Cox) test analysis [Prism 5.04 ]. In addition, survival at study end for each experimental group was analyzed by Chi-square test. The body weight/change data were statistically analyzed for each time point for differences in treatment group by analysis of variance (ANOVA) and, if appropriate, Dunnett's test. A p value of ≤ 0.05 was considered significant. TNA and ELISA data were log-transformed prior to being statistically analyzed by repeated-measures ANOVA to determine whether any differences exist between the experimental groups. A p < 0.05 was considered to be significant. If significant, a post hoc multiple comparison t-test with Bonferroni’s adjustment was performed. A significant p value was dependent on whether it is smaller than 0.05/ (number of tests). Statistical analyses were conducted in Statview 5.0.1 . Data is presented as a group with a geometric mean of 95% confidence intervals.B. anthracis spore challenge study was designed  as compared to AVA. The mrPA outperformed wtrPA at all comparable doses and the two lower doses of mrPA were equivalent and not statistically different from the highest wtrPA doses or AVA.In addition to the characterization of the appearance of total anti-PA antibodies (described above), anthrax holotoxin neutralizing anti-PA-antibodies were also measured by a toxin neutralization assay Tables  and 2. TIn general, an rPA dose response was observed prior to the Day 28 boost, particularly in the wtrPA dosed rabbits. After the Day 28 boost, the differences between doses of rPA, either wild type or mutant, became less apparent and the levels declined between Day 48 and 65 prior to challenge. Neutralizing antibody levels increased post-aerosol challenge in all vaccine groups as seen in Mortality data and Kaplan-Meier data are shown in Six of ten naïve control rabbits and eight of ten vehicle control rabbits were positive for bacteremia in the blood (data not shown). Of vaccinated rabbits, there were only three rabbits that demonstrated bacteremia at the initial blood (Day 71) draw following challenge, but this resolved in these rabbits by the next blood collection time point (Day 74) and the rabbits survived to Day 84. Of the three wtrPA-vaccinated rabbits that succumbed to disease, none had bacteremia present in any blood sample.4 to 2.21 x 108 CFU/mL. The rabbits who survived to study termination were found to have tissue burden below the detectable limits or, in a few cases, low tissue burden was seen in the lungs. In the three wtrPA vaccinated rabbits that succumbed to disease prior to study termination, high tissue burden was seen in the lung and low levels in the tracheobronchial lymph nodes (TBLN) and spleen. One of the animals that succumbed had high tissue burden in the lung as well as all other tissues collected. The rabbits in the mrPA vaccine groups all survived to study termination and any B. anthracis present in the tissues was at lower levels (less than 30 CFU/100 μL inocula) than wtrPA vaccine group rabbits.Negative control rabbits (unvaccinated and those vaccinated with vehicle) were shown to have high levels of tissue burden for all tissues tested, Most elevated clinical pathology parameters were associated with rabbits that succumbed to challenge in the vehicle control and naïve animal groups . Elevated parameters included blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, triglycerides and gamma glutamyltransferase.Hematology parameters were generally unremarkable prior to challenge . Subsequent to challenge white blood cell (WBC) counts increased in virtually all rabbits. In those rabbits that survived, WBC counts returned to normal by sacrifice.The microscopic findings in the untreated and vehicle control rabbits were characteristic of anthrax and were pooled for baseline data, Microscopic findings in AVA-vaccinated rabbits were consistent with a response to a high dose of inhaled spores in immunized rabbits. Inflammatory cells were scattered throughout the pulmonary interstitium in most rabbits, and there was an increase in the number of alveolar macrophages, many of which contained cytoplasmic particles approximately 1–2 microns in diameter (possible spores). A few rabbits had foci of granulomatous inflammation associated with lymphatic vessels and/or heterophilic  inflammation associated with the conducting airways and lymphatics. All rabbits had an increase in the mucosal-associated lymphoid tissues of the airways and follicles with germinal centers in the tracheobronchial lymph nodes, and 8/10 had an increase in the paracortical zone lymphocytes and/or medullary plasma cells. Spleens and livers were generally unremarkable.B. anthracis spore exposure, the 2.5 μg wtrPA treated rabbit and one of the 0.625 μg wtrPA treated rabbits retained bacteria in the pulmonary lymphatics and alveolar spaces and generalized heterophilic inflammation. The remaining 0.625 μg wtrPA treated rabbit death had findings similar to unvaccinated rabbits.The microscopic findings in rabbits from all 10 μg wtrPA, all 2.5 and 10 μg mrPA, eight of ten 2.5 μg wtrPA, six of ten 0.625 μg wtrPA, and nine of ten 0.625 μg mrPA treated rabbits were generally equivalent to the AVA group and survived to Day 84 necropsy. In addition to an AVA-like inflammatory response, a single 0.625 μg mrPA, one 2.5 μg wtrPA and two 0.625 μg wtrPA treated rabbits additionally retained small inflammatory abscesses in a single lung lobe at Day 84. Of the 2.5 μg wtrPA and two 0.625 μg wtrPA treated rabbits that succumbed to B. anthracis spore-exposed control animals remained PA positive until death. Subjects in vaccinated groups had sporadic, transient PA positive test samples; the 10μg wtrPA (3/10) and 2.5 μg wtrPA (4/10) had the highest number of subjects with a PA positive test sample, but all PA positive test subjects in vaccinated animals returned to a PA negative state by the end of the study.All animals lacked detectable circulating PA in the serum prior to challenge and almost 19/20 control rabbits were PA positive by Study Day 72. Untreated and vehicle-treated B. anthracis protective antigen as a vaccine, the current study was undertaken to compare the efficacy of native, wtrPA and mutant, mrPA vaccines to protect rabbits from an aerosol challenge with B. anthracis spores. Animal numbers were statistically sufficient to answer this question and were consistent with previous literature-based investigations of rPA vaccines [In order to test the feasibility of developing a recombinantly-derived mutant form of vaccines .et al. (2002) have also found an increased yield of this protein compared to the native molecule when prepared from a recombinant B. anthracis host [P. fluorescens and formulated in alum (data not shown). The data that will be derived from this study will reveal any differentiation of the stability of wtrPA vs. mrPA formulated with Alhydrogel.The mutant form of the rPA studied here may present production and functional advantages as a vaccine antigen. The mutant has had two proteolytically sensitive sites removed. These mutations may serve to protect the intact antigen product to proteolytic loss over long periods of storage, making it a better candidate for a stock-piled vaccine. Ramirez cis host , presumacis host . A 24-moImmunogenicity results from the initial preclinical work performed  as well P. fluorescens, and challenged with B. anthracis spores. mrPA generated circulating and neutralizing antibodies that were superior to all comparable doses of wtrPA and superior to the human dose of AVA when administered at the 10 μg/dose level. The mrPA and wtrPA comparative antibody response was reflected in trends observed in survival, general bacteremia and pathology, suggesting that the mrPA conferred superior protection compared to the wtrPA protein. Such mutants that cannot heptamerize have been shown to be more effective antigens. Perhaps this is because they disrupt normal cellular trafficking, making them more susceptible targets for the host’s immune system [B. anthracis lung clearance could be achieved at an associated TNA or anti-PA value.In the current study, ELISA and TNA results from both rPA molecules were consistent with previously reported alum-based rPA anthrax vaccinations and challenge studies . Howevere system . BacteriP. fluorescens are viable vaccine candidates suitable for further development [In summary, these data demonstrate that recombinant mrPA and wtrPA produced in a novel expression system can be formulated with alum to achieve protection that is equivalent to the clinical administration of AVA. Under the conditions of this study, mrPA outperformed the wtrPA formulation and was equivalent to AVA at dose levels as low as 0.625 μg in a two dose regimen. These data combined with ongoing stability studies currently suggest that both the alum-formulated wtrPA and mrPA proteins expressed in elopment .


However, ET-1 produces a greater response and longer lasting nocifensive behavior than BK in multiple models, indicating a potentially divergent signaling mechanism in primary afferent sensory neurons. Using cultured sensory neurons, we demonstrate significant differences in both Ca2+ influx and Ca2+ release from intracellular stores following ET-1 and BK treatments. As intracellular store depletion may contribute to the regulation of other signaling cascades downstream of GPCRs, we concentrated our investigation on store-operated Ca2+ channels. Using pharmacological approaches, we identified transient receptor potential canonical channel 3 (TRPC3) as a dominant contributor to Ca2+ influx subsequent to ET-1 treatment. On the other hand, BK treatment stimulated Orai1 activation, with only minor input from TRPC3. Taken together, data presented here suggest that ET-1 signaling targets TRPC3, generating a prolonged Ca2+ signal that perpetuates nocifensive responses. In contrast, Orai1 dominates as the downstream target of BK receptor activation and results in transient intracellular Ca2+ increases and abridged nocifensive responses.Endothelin-1 (ET-1) and bradykinin (BK) are endogenous peptides that signal through Gαq/11-protein coupled receptors (GPCRs) to produce nociceptor sensitization and pain. Both peptides activate phospholipase C to stimulate Ca AR), and endothelin-B (ETBR), which are expressed in a tissue-dependent manner  and bradykinin (BK) are two endogenous neuropeptides involved in inflammatory and cancer pain . ET-1 wat manner . The affver ETBR , while Ehan ETBR . Both enhan ETBR , employi1R and B2R, respectively; Gutowski et al., 1991; 2R, which is also the dominant isoform widely expressed in multiple tissue types under normal physiological conditions  and diacylglycerol (DAG) production via activation of phospholipase C  to induce calcium release from intracellular stores. The subsequent decrease of Ca2+ concentration in intracellular stores activates store-operated calcium entry (SOCE) channels, further increasing intracellular calcium accumulation , and U73122 .The following reagents were used: ET-1 , BK , PyR3 , GdClAll procedures utilizing animals were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and were conducted in accordance with policies for the ethical treatment of animals established by the National Institute for Health. Male Sprague Dawley rats, 175 to 200 g in weight , were used for behavior analyses and TG dissection.d-lysine/laminin-coated cover slips  in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 100 ng/ml nerve growth factor. Cultures were maintained at 37 ℃, 5% CO2, and grown for 1 to 2 days for calcium imaging experiments.TG neurons were cultured as previously described . In shorFor single-cell PLC activity experiments, TG neurons were nucleofected with 2 µg of GFP-PLC∂-PH  according to manufacturer recommendations  prior to cover slip placement. Experiments were performed 2 days after procedure.3H]myoinositol for 24 hr before experiments. After labeling cells were washed three times with 500 µl HBSS that contained 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and were preincubated in HBSS containing 20 mM HEPES and 20 mM LiCl for 15 min at 37 ℃ in room air. Cells were incubated with various concentrations of ET-1 and BK  and reactions were terminated by the addition of ice-cold formic acid. Total [3H]IPs were separated by ion exchange chromatography and measured by liquid scintillation spectrometry. Data are expressed as a percentage increase above basal IP accumulation.ET-1- and BK-stimulated inositol monophosphate (IP) accumulation in primary sensory neuronal cultures was measured as previously described . Neurona2+ levels, the dye Fura-2 AM  was incubated with cultured neurons for 30 min at 37 ℃ in the presence of 0.05% Pluronic . Fluorescence was detected with a Nikon Eclipse Ti-U microscope fitted with a 20×/0.8 NA Fluor objective. Fluorescence images from 340 nm to 380 nm excitation wavelengths were collected and analyzed with MetaFluor Software . All Ca2+ measurements were performed at RT. To asses single-cell Ca2+responses after ET-1 or BK stimulation, cells were continuously perfused with standard extracellular solution (SES) that contained (in mM) 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 10 D(+)-glucose. After 60 s baseline was established 100 nM of ET-1 or BK was applied and net changes in Ca2+ were calculated. In recordings where cells underwent double treatment with ET-1 and BK, neurons were washed for 5 min with SES. To assess SOCE stimulated by ET-1 or BK, cultured neurons were perfused with Ca2+ free buffer -glucose, 10 mM HEPES, pH 7.4) and after baseline was established, ET-1 (100 nM) or BK (100 nM) was administered for 30 s. Following 5 min washout, 2 mM Ca2+ was introduced to the extracellular environment. Ratiometric data were converted to [Ca2+]i by using the equation [Ca2+]i = K[(R − Rmin)/(Rmax − R)]. Values of K, Rmin, and Rmax are determined with use of calibration kit . Data collected with calibration kit allow generation of a standard curve, which can then be used to convert fura-2 fluorescence measurements obtained from experimental samples into estimates of free Ca2+ concentration. The plot of the log of [Ca2+]free (x-axis) versus the log of {[(R − Rmin)/(Rmax − R)]} yields a straight line, the x-intercept of which is the log of K. R is the ratio of 510 nm emission intensity with excitation at 340 nm, to 510 nm emission intensity with excitation at 380 nm; Rmin is the ratio at zero free Ca2+; Rmax is the ratio at saturating Ca2+.To measure single-cell intracellular CaAll injections were given intraplantarly in 25 µl volumes via 28-gauge needle inserted through the lateral footpad just under the skin to minimalize tissue damage. Drugs stocks were dissolved in phosphate buffered saline (PBS) or PBS 2% Tween20. After injection (Time 0), animals were freely moving, on a flat surface enclosed by Plexiglas cage. Nocifensive behavior was defined as hindpaw flinching, lifting, and licking. Animals were observed for 40 min and behavior was quantified every 5 min.n referring to the number of analyzed cells per group. Concentration–response data for ET-1 - and BK-mediated IP accumulation were fit to a logistic equation using nonlinear regression analysis to provide estimates of maximal response (Emax) and potency (EC50) using Prism software. The significant difference between groups was assessed by one-way and two-way analysis of variance . Data were given as mean ± standard error of the mean , with thvariance , with Bo2+ accumulation.Inflammatory mediators induce nocifensive behaviors in multiple behavioral models . Here, w2+ responses triggered by ET-1 and BK by real-time Ca2+ imaging, starting with physiological extracellular solution containing 2 mM Ca2+  or BK (100 nM) and changes in intracellular calcium concentration were analyzed. ET-1 triggered significantly higher calcium release from intracellular stores when compared with BK-induced responses  and is broadly used as an index of PLC enzymatic activity  caused a 33% decrease in calcium response , wherearesponse . In cont3+ Orai1 locker ca3+ Orai1 locker cain vitro experiments that identify two ion channels that contribute to ET-1- and BK-mediated responses, nocifensive behavior experiments were repeated with specific ion channel inhibitors, PyR3 was used as a TRPC3-specific inhibitor (3+ was injected to block Orai1 activity (3+) into the hindpaw was followed by 10 nM (25 µl) injection of ET-1 or BK 5 min later. Inhibition of Orai1 with Gd3+ resulted in a significant increase in ET-1-triggered nocifensive behavior through the first 25 min of observation , demonstrating neuronal dependence on SOCE for replenishing Ca2+ stores after a release event  bifurcate to contribute to divergent responses. At the beginning of our investigation, we established that in single-cell measurements, both ET-1 and BK stimulation activate PLC to the same degree (2+ from intracellular stores (In many cell types, BK stimulates transient increases in intracellular Careatment . In conto 60 min . Resultsbehavior . In contbehavior . These o2+ homeostasis, and both are expressed and have been characterized in sensory neurons (2+ signal. Importantly, this divergence in SOCE activation profiles could contribute to the differential nocifensive behaviors caused by ET-1 and BK. To further examine the roles of Orai1 and TRPC3 in ET-1 - and BK-induced behavior, we employed the same pharmacological inhibitors in our nocifensive behavior model.Orai1 and TRPC3 are among many SOCE channels that assist in intracellular Ca neurons . Commerc neurons . In cont neurons . This va3+ (3+ inhibition of Orai1 reduced the time required for ET-1 administration to reach peak nocifensive behavior. These results may simply be due to the quick behavioral response of BK versus the slower response time for ET-1or may involve additional underlying factors.SOCE antagonist results establish parallels between the functional coupling of Orai1 and TRPC3 channels in single-cell responses and nocifensive behavior. Orai1 suppression of neuronal excitability would explain significantly prolonged recovery time after BK injection in rats that were pretreated with the Orai1 blocker, Gd3+ . Further3+  and othe3+ . However3+ prior to ET-1 administration (AR downstream signaling targets Orai1 to a smaller degree than TRPC3, the shift in nocifensive behavior response time shift could be affected. Importantly, TRPC3 inhibition produced greater changes in nocifensive behavior, similar to single-cell studies. Despite the lack of mirrored results between single-cell studies and nociceptive behavior, important findings appear to indicate divergent signaling mechanisms that contribute to our initial behavioral findings.It has been reported that TRPC3 plays vital role in DRG neurons sensitization triggered by exposure to inflammatory mediators . This mastration . Indeed,2+ levels and the perpetuation of nocifensive responses. In contrast, BK triggers short-lived transient increases in intracellular Ca2+ through dominant activation of Orai1, contributing to a rapid, but abbreviated onset of nocifensive behavior. Further studies into this divergent signaling behavior could prove important to alleviating acute hyperalgesia.Taken together , TRPC3 iin vitro and in vivo.Endothelin and bradykinin are inflammatory mediators that bind to receptors on sensory neurons to stimulate various molecular pathways. In this investigation, we identified store-operated ion channels Orai1 and TRPC3 contribute to endothelin/bradykinin effects


We describe two canonical Ca2+-binding protein domains (C2 domains and EF-hands) and define criteria that need to be met for a protein to qualify as a Ca2+ sensor mediating STP. With these criteria in mind, we discuss various forms of STP and identify established and putative Ca2+ sensors. We find that despite the multitude of proposed sensors, only three are well established in STP: Munc13, protein kinase C (PKC) and synaptotagmin-7. For putative sensors, we pinpoint open questions and potential pitfalls. Finally, we discuss how the molecular properties and modes of action of Ca2+ sensors can explain their differential involvement in STP and shape net synaptic output.The complex manner in which patterns of presynaptic neural activity are translated into short-term plasticity (STP) suggests the existence of multiple presynaptic calcium (Ca Bursts of action potentials lead to the buildup of residual Ca2+  in the terminal, which outlives neuronal activity, and induce multiple forms of short-term presynaptic plasticity (STP), including facilitation, depression, augmentation and post-tetanic potentiation (PTP) , which acts as the Ca2+ sensor for synchronous vesicle fusion at most synapses , neuronal calcium sensor 1 (NCS-1) and visin-like proteins (VILIPs).The EF-hand is the most common Ca2+-dependent manner  and/or the vesicle fusion machinery, including VGCCs and SM proteins  and pumps, which indirectly affect STP by changing the spatiotemporal distribution of free Ca2+ through binding or extrusion  in order to conclude that a protein is a Ca2+ sensor mediating STP. It could even be argued that a requirement for Ca2+ binding during plasticity must be demonstrated in order to establish a protein as a Ca2+ sensor, but the technology for this type of experiments is currently lacking.With a plethora of C2- and EF-hand-containing proteins in the presynaptic terminal, there are numerous candidate Ca2+]residual  also modulate Ca2+ influx through VGCCs  but also spontaneous events in a Ca2+ signals stretching over an order of magnitude cellular expression and mechanisms of action contribute to the specialization of sensors for different forms of STP. For example, NCS-1 has high affinity for Ca2+ and localizes at the plasma membrane (O’Callaghan et al., 2+ signals. PKCβ, on the other hand, has lower Ca2+ affinity, is cytoplasmic at rest (Newton, 2+increases are likely required for its activation, in agreement with the prolonged stimulation requirement for PTP (Habets and Borst, 2+ affinity can vary as a result of effector binding, phospholipid binding, and post-translational modifications (Xia and Storm, 2+ sensors could help explain why identical activation regimes do not always lead to the same STP across synapses or during development (Rosenmund et al., Different patterns of neuronal activity result in variable Cae Figure . Diverse Newton,  and like2+ sensors interact, and might even compete (Chu et al., 2+-dependent facilitation to explain synaptic output (Tsodyks et al., 2+ sensors, more accurately reflects our understanding of the underlying biology and allows better prediction of synaptic and network behavior (Hennig, Most synapses exhibit multiple forms of STP and the net synaptic output reflects the interaction between these different forms (de Jong and Verhage,  Hennig, .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.


Here, we report that the paralog Orai3 fails to activate NFAT. Orai1 is effective in activating gene expression via Ca2+ nanodomains because it participates in a membrane-delimited signaling complex that forms after store depletion and brings calcineurin, via the scaffolding protein AKAP79, to calmodulin tethered to Orai1. By contrast, Orai3 interacts less well with AKAP79 after store depletion, rendering it ineffective in activating NFAT. A channel chimera of Orai3 with the N terminus of Orai1 was able to couple local Ca2+ entry to NFAT activation, identifying the N-terminal domain of Orai1 as central to Ca2+ nanodomain-transcription coupling. The formation of a store-dependent signaling complex at the plasma membrane provides for selective activation of a fundamental downstream response by Orai1.NFAT-dependent gene expression is essential for the development and function of the nervous, immune, and cardiovascular systems and kidney, bone, and skeletal muscle . Most NF nucleus . In somemembrane . How loc •2+ store depletion leads to the formation of a plasmalemmal signaling complexCa•AKAP79, with bound calcineurin and NFAT1, couples to the N terminus of Orai1 channels•2+ entry though the channels releases activated NFAT, leading to gene expressionCa•These results identify a mechanism for selective activation of a response by Orai1 2+ microdomains near Orai1 channels to activate nuclear gene expression through the transcription factor NFAT. The complex forms after store depletion and brings calcineurin and NFAT, via the scaffolding protein AKAP79, to the Orai1 Ca2+ channels.Kar et al. describe the formation of a plasma membrane signaling complex that enables Ca The molecular basis of this process is becoming clear: store depletion leads to oligomerization of the ER protein STIM1, and the oligomers then migrate to ER-plasma membrane junctions, where they bind to the pore-forming subunit of the CRAC channel Orai1 and trigger the channel to open [Drosophila Orai1 reveals the channel to be a hexamer [Store-operated Cale cells . The bes channel . CRAC ch to open . The cry hexamer .2+ signals in the vicinity of open CRAC channels activate important cytoplasmic signaling molecules, including enzymes [2+-dependent target is closely apposed to the Ca2+ channel, rapidly transducing the local Ca2+ signal into a physiological output. A more complex scenario arises when the activated target is located at a distance well beyond the realm of the CRAC channel Ca2+ microdomain, typically <10–20 nm in spatial extent [fos [2+ nanodomains near CRAC channels are sensed and relayed to cytoplasmic targets such as NFAT.Local Ca enzymes , ion cha enzymes , and ves enzymes . In all l extent . This spl extent  and intrent [fos  and NFAT2+ entry following store depletion with the SERCA pump blocker thapsigargin , the association between the proteins was significantly reduced, although not abolished, in high Ca2+  domain of voltage-gated Cablockers . If calmase pump , which idazolium C, confirigh Ca2+ A and S2Bs buffer C and S2Ds buffer E. Hence,2+ buffer. Pull-down of calmodulin-GFP revealed the presence of Orai1 in lysis buffer containing 4 mM EGTA  provide2+-independent association between Orai1 and calmodulin in low Ca2+, a previous study did not observe such an interaction [2+-calmodulin-dependent fast inactivation of CRAC channels  and with the observation that the mutant Ca2+-insensitive calmodulin protein reduces fast inactivation [Although our studies show Caeraction . We do ntivation   external solution. However, NFAT still did not migrate to the nucleus  destabilize the apocalmodulin region. Alternatively, specific mutations with the calmodulin-binding domain might result in weak association between Orai1 and a bridging protein that brings calmodulin close to the Orai1 pore. In the Y80A Orai1 mutant, calmodulin remains bound in both low and high Ca2+  confer the ability to couple local Caor STIM1 . Stimulaut STIM1 H and 3I.ut STIM1 H and 3I th Orai3 C, but thth Orai3 C. We the2+ channels, where calcineurin can be held close to CaV1.2 [2+ signal evoked by thapsigargin was unaffected by the decreased levels of AKAP79  into close proximity to the intermediary  and executor (NFAT). Calmodulin is tethered to Orai1 or a closely associated protein and is therefore held close to the source of Ca2+. This pool of calmodulin has privileged access to calcineurin because store depletion recruits AKAP79 with attached calcineurin to Orai1. This form of coincidence detection, requiring two conditions to be satisfied , provides a mechanism for ensuring that only local Ca2+ entry via store depletion will activate the pathway. Ca2+ release from stores or local Ca2+ entry through other channels such as Orai3 will not be effective because AKAP79 is not recruited to, and there is less calmodulin associated with, Orai3. This dual requirement for tethered calmodulin and AKAP in mast cells prevents physiological fluctuations in cytosolic Ca2+ from activating NFAT in the absence of store depletion. Large, nonphysiological bulk rises in Ca2+ can activate NFAT, even in the absence of AKAP79 (2+ sufficiently to match the local physiological Ca2+ rise near Orai1. Given that impaired calcineurin-NFAT signaling is linked to various disorders in nonexcitable cells, including autoimmune disease, osteoporosis, Down syndrome, and possibly cancer [A major question in Caf AKAP79 D, by rai


We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca2+ waves in cardiomyocytes. Using confocal microscopy, we studied Ca2+ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models , wild-type  mice, and WT rabbits . Consistent with increased RyR2 activity, Ca2+ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca2+ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca2+ waves with significantly higher potency (lower IC50) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca2+ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca2+ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT.Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca Our results also help explain why the contribution of RyR2 block to Ca2+ wave suppression by FLEC is more difficult to identify in myocytes with lower Ca2+ spark frequency and hence RyR2 open probability than Casq2-/- myocytes  was progressively increased to the final Ca2+ of [1 mM]. The rabbit myocytes used in these experiments were harvested from the free wall of left ventricle.Single ventricular myocytes from mice and rabbits were isolated by enzymatic digestion applying a modified collagenase/protease method as previously described. After isolation, cells were washed twice by gravity sedimentation for 20 min in 0.2 mM CaClAn aliquot of about 200 μl of the solution containing the isolated myocytes was placed in a laminin-coated chamber and allowed to settle down for 6 min before starting the permeabilization. In these chambers, fluid was exchanged in bulk for drug application. The detailed procedure for the chemical permeabilization of mouse cardiac myocytes has been previously reported . The samThe permeabilized cells were imaged using an LSM 510 Zeiss inverted microscope and a 40x oil immersion objective lens . Intracellular fluo-4 was excited at 488 nm with a krypton/argon laser. The fluorescent emission was collected through a long-pass filter (>515 nm). All images were acquired digitally in line-scan mode with 0.2 μm and 0.2 ms per pixel resolution. We only collected data from isolated myocytes that showed the classic brick-shape morphology. For each drug concentration, 7 to 10 microscope fields were examined. Wave incidence was calculated as the fraction of cells waving divided by the total number of brick-shaped myocytes per field.http://rsb.info.nih.gov/nih-image). Ca2+ sparks and waves were acquired using the confocal microscope in the line-scan mode. The scan line was placed parallel to the longitudinal (main) axis of the myocyte. LS were obtained only from the population of waving cells. For the purpose of the wave analysis in the present work, only propagated Ca2+ waves were analyzed. Wave amplitude, frequency, and propagation speed were also measured for each drug concentration and concentration-response curves (CRCs) constructed for each wave parameter. The corresponding IC50 was obtained by fitting the CRCs to a Boltzmann function using OriginLab non-linear fitting software. Drug efficacy was defined as the percentage of maximal decrease/suppression in the wave parameter at 100 μM (highest drug concentration tested in each case). All parameter values were normalized to values obtained from cells to vehicle (VEH).In all cases, fluorescence images were analyzed using ImageJ, the public domain NIH Image program , Casq2 null (Casq2-/-), and RyR2-RC4496C+/- mice ] and recorded in line-scan mode using confocal microscopy. Spark-mediated leak was calculated as the product between spark mass and the corresponding frequency. Consistent with an increased RyR2 channel activity, both R4496C+/- and Casq2-/- myocytes exhibited increased spark frequency compared to WT myocytes  exhibit increased intrinsic RyR2 activity. We previously showed that FLEC and RPROP inhibit RyR2 channels in the open state, increase Ca2+ spark frequency but reduce spark mass (“flicker” block), suppress Ca2+ waves and prevents CPVT in mice and humans [2+ waves. To test this hypothesis, we first studied the effects of FLEC and RPROP on Ca2+ wave parameters in permeabilized myocytes isolated from Casq2-/-, RyR2-4496C+/- mutant, and WT littermates  and the maximum percentage of inhibition (efficacy)  and efficacy in Casq2-/- myocytes compared to the other 2 groups studied (2+ wave parameters (2+ waves in Casq2-/- myocytes with higher potency (lower IC50) and efficacy compared to its effect on the other 2 groups studied  . FLEC efCasq2-/- . FLEC ha studied . RPROP arameters . Similar-/- and RyR2-4496C+/- mutant myocytes exhibit higher frequency of sparks and spontaneous Ca2+ waves, we hypothesized that an increased RyR2 opening frequency would make myocytes more sensitive to open-channel blockers like FLEC and RPROP. To directly test this hypothesis, we first studied the effect of caffeine (CAF) 150 μM on the spark frequency of permeabilized WT ventricular myocytes. After 5 min incubation in CAFF 150 μM, CAFF-treated cells exhibited a significantly increased spark frequency compared to the spark frequency observed in vehicle-treated WT cells  on Cas tested . Furtherrameters .2+ sparks in saponin-permeabilized rabbit ventricular myocytes at baseline and examined the effect of RPROP (10 μM) on spark parameters  and TET (non-state dependent RyR2 channel blocker) and IC50 and efficacy calculated for the each wave parameter (2+ waves in rabbit myocytes (50) and efficacy in all other wave parameters analyzed in rabbit myocytes compared to mouse WT cells  determine the potency of open-state RyR2 blockers such as FLEC against Ca2+ waves in cardiomyocytes and could help explain the differences between our results and that of other groups. Consistent with that hypothesis, we found that differences in the rate of Ca2+ sparks, an estimate of RyR2 activity, between WT, CAFF-treated WT, Casq2-/- and RyR2-4496C+/- myocytes directly predicted the Ca2+ wave blocking potency of FLEC and RPROP but not that of the closed channel blocker TET. Also consistent with that hypothesis that RyR2 activity determines FLEC potency, activating Ca2+ sparks with low dose of CAFF rendered WT myocytes more susceptible to Ca2+ wave inhibition by FLEC. Finally, WT rabbit myocytes with intrinsically higher RyR2 Po (higher spark frequency than WT cells) are more sensitive to FLEC and PROP than WT mouse myocytes. Taken together, these results demonstrate that Ca2+ spark rate and hence intrinsic RyR2 channel activity is an important predictor for the potency of RyR2 open channel blockers FLEC and RPROP against Ca2+ waves, which may help explain why the contribution of RyR2 block to Ca2+ wave inhibition by FLEC was not as evident in studies of healthy rat ventricular myocytes [+/- ventricular myocytes [-/- myocytes. Further supporting our hypothesis are reports that flecainide was highly effective against Ca waves in RyR2-R4496C+/- Purkinje cells, which have intrinsically higher spark frequency than RyR2-R4496C+/- ventricular myocytes [The clinical management of CPVT has been improved in recent years with the introduction of FLEC, a drug that effectively suppresses Cain vitro  and previn vitro , 31, 32. in CPVT . The imp in CPVT . On the myocytes . Liu et VT model . BannistVT model . Based omyocytes , 34 or Rmyocytes , which bmyocytes .2+ waves, which was not the case for the state-independent blocker TET: Increasing RyR2 activity increases the potency of FLEC and RPROP action. Interestingly, open channel block and use-dependence  is also a typical feature of FLEC action on cardiac Na+ channels [+ channels, the underlying mechanism of FLEC use-dependence is well-established: FLEC requires Na+ channel opening to gain access to the inactivated state [2+ waves reported here? It seems unlikely that FLEC binds with higher affinity to RyR2 in the open configuration based on our previous studies of single RyR2 channels in artificial bilayers: FLEC block was less potent at pCa4 when RyR2 channels were fully activated compared to pCa7 when RyR2 activity was much lower [2+ spark that triggers a propagated Ca2+ wave. The likelihood of a local Ca2+ release for triggering a Ca2+ wave depends on two main factors: the amount of Ca2+ released  and the Ca2+ sensitivity of a neighboring RyR2 cluster . Increasing RyR2 activity will promote Ca2+ waves, but at the same time reduce SR Ca2+ content and hence Ca2+ spark mass due to the increased SR Ca2+ leak until a new steady state is reached [2+ flux per unit time and hence decreases Ca2+ spark mass. The reduction in Ca2+ spark mass, in turn, reduces the likelihood of activating neighboring RyR2 clusters and therefore propagation of Ca2+-induced Ca2+ release in the form of Ca2+ waves [2+ content. Although more frequent, the resulting Ca2+ waves are smaller and therefore easier to break up by drugs that reduce Ca2+ spark mass. Consistent with that hypothesis is our experimental observation that FLEC and RPROP break up propagated Ca2+ waves into small non-propagated wavelets and macro sparks [2+ leak and reduces SR Ca2+ content, also increased the potency of FLEC and RPROP.In essence, we report that open state blockers FLEC and RPROP exhibit RyR2 “use-dependence” in their action against Cachannels . For Na+ed state , to whiced state . What arch lower . A more  reached . We have reached . The red2+ waves . Hence, o sparks . Further2+ wave block for developing drugs against Ca2+ wave triggered arrhythmias? For one, drugs like FLEC may act preferentially on diseased myocytes with higher RyR2 activity than healthy myocytes. For example, the increased frequency in both Ca2+ sparks and waves observed in myocytes from the two CPVT models studied here are in agreement with previous results reported for these and other CPVT mouse models [2+ leak are also frequently found in myocytes isolated from animal models of heart failure [+ channel blocking properties [2+ waves appears a promising new paradigm for finding better anti-antiarrhythmic medicines [What are the implications of use-dependence of Cae models , 40, 41. failure , 43, andedicines .We would like to emphasize that this study was not designed to address the clinical relevance of RyR2 block for the antiarrhythmic efficacy of flecainide and propafenone in patients. Permeabilized myocytes are a highly reduced system and whether the IC50 concentrations identified in our study are applicable to the clinical care is not known.2+ waves in cardiomyocytes from WT mice, CPVT mouse models and rabbits. The use-dependence of Ca2+ wave inhibition reported here may represent a promising new paradigm for future antiarrhythmic drug development.Our study demonstrates that RyR2 activity determines the potency and efficacy of the open-state blockers FLEC and RPROP for suppressing arrhythmogenic CaS1 ARRIVE Guidelines Checklist(PDF)Click here for additional data file.


Several genes are implicated in controlling mitochondrial Ca2+ uptake , however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type , differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.Uptake of Ca Fmax was obtained when cells were treated with the cell-permeable Ca2+ ionophore ionomycin which both inserts into the plasma membrane and passes into the cell, inserting into mitochondrial membranes, leading to saturation of the indicator when in regular medium (2 mM Ca2+). Fmin was obtained under the same conditions except in zero Ca2+ medium. The linear relationship between [Ca2+] and (F-Fmin)/(Fmax-F) has been previously confirmed by calibrating the indicator as expressed in neurons, exposing them to ionomycin in the presence of sequentially different solutions of precise [Ca2+], obtained by mixing K2EGTA and CaEGTA solutions  at different ratios [This was performed essentially as described . Neuronst ratios .Paired t-tests were used to compare non-independent data pairs, while student t-tests were utilized when the two groups were not related. Error bars represent standard error of the mean.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.


Troponin I (TnI) and Myosin Binding Protein C (MyBP-C) are the prime targets in the myofilaments. TnI phosphorylation lowers myofibrillar Ca2+-sensitivity and increases the speed of Ca2+-dissociation and relaxation (lusitropic effect). Recent studies have shown that this relationship between Ca2+-sensitivity and TnI phosphorylation may be unstable. In familial cardiomyopathies, both dilated and hypertrophic (DCM and HCM), a mutation in one of the proteins of the thin filament often results in the loss of the relationship (uncoupling) and blunting of the lusitropic response. For familial dilated cardiomyopathy in thin filament proteins it has been proposed that this uncoupling is causative of the phenotype. Uncoupling has also been found in human heart tissue from patients with hypertrophic obstructive cardiomyopathy as a secondary effect. Recently, it has been found that Ca2+-sensitizing drugs can promote uncoupling, whilst one Ca2+-desensitizing drug Epigallocatechin 3-Gallate (EGCG) can reverse uncoupling. We will discuss recent findings about the role of uncoupling in the development of cardiomyopathies and the molecular mechanism of the process.Contraction in the mammalian heart is controlled by the intracellular Ca Contraction is initiated by Ca2+ release from the sarcoplasmic reticulum via the Ryanodine receptor and is terminated by Ca2+-uptake by the ATP-powered sarcoplasmic Ca2+ pump (SERCA). Ca2+ binds to troponin C (TnC), the Ca2+ receptor of the contractile apparatus to switch on contractile interactions between actin and the myosin motor protein in the thick filaments   and troponin I (TnI) within the cardiac myofibril. TnI is the inhibitory component of the trimeric troponin molecule that makes up the CaEngland, , that TnEngland,  and that2+-sensitivity by TnI phosphorylation is quite a labile system and that mutations associated with cardiomyopathies in particular, can lead to disruption of the system. This was first noted with mutations in TnI that caused hypertrophic cardiomyopathy (HCM)  it was found that in all of these DCM-causing mutations the myofilament Ca2+-sensitivity is independent of the level of TnI phosphorylation. Therefore, by analogy with the S22/23A transgenic mice, it was proposed that this uncoupling was necessary and sufficient to cause the DCM phenotype  phosphorylation levels in situ is very important. Quantitative methods such as Top-down mass spectrometry and phosphate affinity SDS-PAGE has clearly established that serines 22 and 23 are the main amino acids phosphorylated in native heart tissue in rats, mice or humans  phosphorylation and Ca2+-dependent specific chelators of phosphoproteins, when added to standard SDS-PAGE, phosphoproteins are retarded in proportion to the number of phosphates per mole of protein  generally have a low level of phosphorylation (0.1–0.4 mols Pi/mol TnI)  or increased by PKA treatment  with isolated human cardiac Tn in reconstituted thin filaments. By manipulating the level of Tn phosphorylation and then measuring the level using phosphate affinity SDS-PAGE (Phos-Tags)  phosphorylation, that phosphorylation of troponin (Tn) modulates Cas Figure   was not affected by PKA phosphorylation of cTnI  tropomyosin  and troponin T (TnT) (4 mutations recorded to date) in addition to 5 mutations in cTnC and 5 mutations in TnI. The currently known mutations causing uncoupling are summarized in Table These seminal studies on uncoupling investigated mutations in the regions of TnI and TnC that could be directly involved in the phosphorylation-dependent interaction that modulates Ca2+-sensitivity. It was found that when TnI was fully phosphorylated, the mutations had different effects on Ca2+-sensitivity of thin filaments compared to non-failing; some increased Ca2+-sensitivity , some decreased it  whereas for α-actin E361G and α-Tropomyosin E54K there was no change in Ca2+-sensitivity. This confirmed that the simple hypothesis that Ca2+-sensitivity is always reduced by DCM-causing mutations, that we and others had proposed, is no longer tenable . The HCM phenotype has been generally thought to be due to mutations increasing myofilament CaMarston, . In addiMarston, , cTnT K2Marston, , α-TropoMarston, , cardiacMarston,  and cTnIMarston,   the hypertrophied interventricular septum causes LVOTO (left ventricular outflow tract obstruction) and pressure overload. Several abnormalities in the contractile proteins in septal tissue from HOCM patients have been observed including; low phosphorylation levels of TnI and MyBP-C . Of particular interest are the Ca2+-sensitizing drugs EMD57033 and Bepridil -Epigallocatechin 3-Gallate], is a Cag Figure . Most stg Figure .2+-sensitivity of isolated cardiac myofibrils. It also blunted the PKA-dependent calcium desensitization induced by phosphorylation at Ser 22/23 and uncoupled the effects of phosphorylation from β-adrenergic stimulation  increased Ca2+ regulation is due to the interaction of a 30 amino acid N-terminal extension of TnI, containing the PKA-specific phosphorylation sites at Ser 22 and 23, with cTnC   than surrounding residues. When the Ser 22 and 23 were phosphorylated in silico, the two serines become more mobile relative to arginines 20 and 21 suggesting a weakening of their interaction with TnC. In addition, Ca2+ becomes more exposed to solvent and the interaction of the “switch peptide” with TnC is altered. Thus, coupling can be accounted for by the formation of a weak ionic complex between TnC and TnI Ser 22 and 23 that is destabilized by phosphorylation.The molecular dynamics simulations indicate a possible structure of TnI N-terminus interacting with TnC   can be found that increase Ca2+-sensitivity but do not uncouple.Whilst it is recognized that the uncoupling phenomenon provides a satisfactory molecular mechanism for thin-filament based mutations that cause DCM, the role of uncoupling in HCM is not as clear. Since CaMarston, , it is lUncoupling inevitably leads to blunting of the response to β-adrenergic agonists but the lack of response to dobutamine is of course not only due to uncoupling. Heart failure is associated also with desensitization of β-receptors, such that the activation of PKA is attenuated, or the activity of phosphatase increased whilst coupling is intact (Houser and Margulies, Patient studies from the 80 and 90 s using echocardiography showed that IDCM and HCM patients could be classified into dobutamine responders and non-responders and that the non-responders have a poor prognosis whilst the responders can respond to treatment (Borow et al., 2+-sensitivity coupling mechanism. Our recent finding that EGCG is capable of recoupling in vitro, although it has different effects in vivo (Feng et al., This dichotomy would suggest that different treatments would be optimum for the two cases. Drugs are available that impact on β-receptors but so far no drugs act positively on the TnI-phosphorylation-CaThe 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.