Urticaria (hives) is a common disorder that often presents with angioedema (swelling that occurs beneath the skin). It is generally classified as acute, chronic or physical. Second-generation, non-sedating H1-receptor antihistamines represent the mainstay of therapy for both acute and chronic urticaria. Angioedema can occur in the absence of urticaria, with angiotensin-converting enzyme (ACE) inhibitor-induced angioedema and idiopathic angioedema being the more common causes. Rarer causes are hereditary angioedema (HAE) or acquired angioedema (AAE). Although the angioedema associated with these disorders is often self-limited, laryngeal involvement can lead to fatal asphyxiation in some cases. The management of HAE and AAE involves both prophylactic strategies to prevent attacks of angioedema  as well as pharmacological interventions for the treatment of acute attacks . In this article, the authors review the causes, diagnosis and management of urticaria (with or without angioedema) as well as the work-up and management of isolated angioedema, which vary considerably from that of angioedema that occurs in the presence of urticaria. Urticaria (hives) is a common disorder, occurring in 15-25% of individuals at some point in life . ApproxiMast cells are the primary effector cells in urticaria and in many cases of angioedema. These cells are widely distributed in the skin, mucosa, and other areas of the body, and have high-affinity immunoglobulin E (IgE) receptors. Mast cell degranulation leads to the rapid release of various inflammatory mediators, such as histamine, leukotrienes and prostaglandins, which, in turn, cause vasodilation and leakage of plasma in and below the skin. There is also a more delayed (4–8 hour) secretion of inflammatory cytokines  that potentially leads to further inflammatory responses and longer-lasting lesions .Urticaria is generally classified as acute, chronic, or physical, depending on the duration of symptoms and the presence or absence of inducing stimuli . As mentioned earlier, angioedema commonly occurs with urticaria, and evaluation and management are similar to those for urticaria. The latter section will review the work-up and management of isolated angioedema, which varies considerably from the diagnosis and treatment of angioedema that occurs in the presence of urticaria.Helicobacter pylori (H. pylori), which has an immunogenic cell envelope, may play an indirect role in the etiology of chronic autoimmune urticaria by reducing immune tolerance and inducing autoantibody formation. However, it is important to note that the limited number of studies conducted in this area have yielded conflicting results [Chronic urticaria is more common in adults, and affects women more frequently than men ,4. In ge results ,8.Patients with chronic idiopathic urticaria do not have evidence of autoimmunity. In this form of urticaria, there appears to be persistent activation of mast cells, but the mechanism of mast cell triggering is unknown. Although rare, chronic urticaria may also be a manifestation of a systemic illness ,3.The most common causes of acute urticaria (with or without angioedema) are medications, foods, viral infections, parasitic infections, insect venom, and contact allergens, particularly latex hypersensitivity. Medications known to commonly cause urticaria ± angioedema include antibiotics , non-steroidal anti-inflammatory drugs (NSAID), acetylsalicylic acid (ASA), opiates and narcotics. The predominant foods that cause urticaria are milk, eggs, peanuts, tree nuts, fish, and shellfish. In approximately 50% of patients with acute urticaria, the cause is unknown (idiopathic urticaria) ,2,9.As mentioned earlier, physical urticaria is triggered by a physical stimulus. The most common physical urticaria is dermatographism , in which lesions are created or “written” on the skin by stroking or scratching the skin. Cholinergic urticaria is also common and results from a rise in basal body temperature that occurs following physical exertion or exposure to heat. Other physical stimuli which can trigger urticaria include exposure to cold (cold-induced urticaria), ultraviolet light (solar urticaria), water (aquagenic urticaria) and exercise. The lesions produced by these physical stimuli are typically localized to the stimulated area and often resolve within 2 hours. However, some patients may experience delayed-pressure urticaria which, as the name implies, comes on slowly after pressure has been applied  and can last several hours or even days. Examples of sites typically affected include the waistline (after the wearing of tight-fitting pants) and the area of the ankles or calves that makes contact with the elastic band of socks .The diagnosis of urticaria, with or without angioedema, is based primarily on a thorough clinical history and physical examination. Based on the history and physical exam, diagnostic tests may also be considered to help confirm a diagnosis of acute, chronic or physical urticaria.The history and physical examination should include detailed information regarding: the frequency, timing, duration and pattern of recurrence of lesions; the shape, size, site and distribution of lesions; potential triggers ; response to previous therapies used; and a personal or family history of atopy ,3.Many conditions can easily be confused with urticaria, particularly urticarial vasculitis and systemic mastocytosis (see Table Skin prick tests (SPTs) and serum-specific IgE tests may help confirm a diagnosis of acute urticaria resulting from allergic or IgE-mediated (type I) reactions to common food allergens, latex hypersensitivity, stinging insect hypersensitivity and certain antibiotics These tests are best performed by allergists with experience in interpreting test results in the appropriate clinical context.H. pylori, antinuclear antibodies (ANA), and erythrocyte sedimentation rate (ESR). SPE can be used to identify increases in IgG and, therefore, may help confirm the diagnosis of chronic autoimmune disease. The presence of thyroid autoantibodies is also suggestive of chronic autoimmune urticaria. An elevated ESR or ANA is often indicative of an underlying systemic condition, such chronic infection or vasculitis [Certain diagnostic tests and assessments can be helpful in the diagnosis and differential diagnosis of chronic urticaria, including: a complete blood count (CBC), serum protein electrophoresis (SPE), the autologous serum skin test (ASST), the basophil activation test, thyroid autoantibodies, sculitis .The ASST is currently one of the most useful tests for confirming a diagnosis of chronic autoimmune urticaria  . It invoChallenge testing, which reproduces exposure to a suspected stimuli in a supervised clinical environment, is often indicated to confirm a diagnosis of physical urticaria. Cold-induced urticaria can usually be confirmed using the ice cube test . Dermatographism can be confirmed by lightly scratching the skin, and aquagenic urticaria can be identified by immersion of a body part into warm water or through the application of warm compresses. Hot bath testing can help identify cholinergic urticaria, and the application of weight/pressure to the patient’s thigh is helpful in the diagnosis of delayed-pressure urticaria .Strategies for the management of acute urticaria include avoidance measures, antihistamines and corticosteroids. For urticaria, antihistamines are the mainstay of therapy. Corticosteroids and various immunomodulatory/ immunosuppressive therapies may also be used for more severe cases, or for those patients who experience a poor response to antihistamines , and avoidance of the offending agent can be an effective management approach. Patients should be provided with clear, written instructions on appropriate avoidance strategies ,3.Second-generation, non-sedating, H1-receptor antihistamines  are the mainstay of therapy for urticaria. These agents have been shown to be significantly more effective than placebo for the treatment of both acute and chronic urticaria . First-gAntihistamine efficacy is often patient specific and, therefore, more than one antihistamine should be tried before assuming therapeutic failure with these agents. Also, antihistamines are most effective if taken daily, rather than on an as-needed basis. If symptoms are controlled with standard antihistamine doses, it is reasonable to continue treatment for several months, occasionally stopping therapy for brief periods to determine whether the urticaria has spontaneously resolved. In patients who do not achieve adequate symptom control at standard doses, it is common practice to increase the antihistamine dose beyond the usual recommended dose. In fact, current European guidelines recommend up to four times the usual recommended dose of antihistamine in patients who symptoms persist with standard therapy . For exaFor some patients with severe urticaria who are inadequately responsive to antihistamines, a brief course of oral corticosteroids  is warranted. However, long-term corticosteroid therapy should be avoided given the well-known side effects associated with prolonged use of corticosteroids and the increased likelihood of developing tolerance to these agents ,3.Various immunosuppressive or immunomodulatory therapies may provide some benefit for patients with severe, chronic urticaria. Double-blind, randomized controlled trials have found cyclosporine (3–5 mg/kg/day) to be effective in patients with chronic urticaria who do not adequately respond to antihistamines ,14. DuriCase reports and other small clinical trials have also found the following treatments to be effective for select patients with severe, refractory, chronic urticaria: sulfasalazine; the antibacterial, dapsone; the anti-IgE monoclonal antibody, omalizumab; and intravenous immunoglobulin G (IVIG) . HoweverLeukotriene receptor antagonists, such as montelukast (Singulair) or zafirlukast (Accolate), have also been shown to be effective in the treatment of poorly-controlled chronic urticaria -17. HoweAngioedema in the absence of urticaria is rare and should alert the physician to alternative diagnoses, such as hereditary or acquired angioedema, idiopathic angioedema, or angioedema associated with angiotensin-converting enzyme (ACE) inhibitors. Hereditary angioedema (HAE) is a rare autosomal dominant genetic disorder resulting from an inherited deficiency or dysfunction of the C1 inhibitor . Two main types of HAE have been defined: type I and type II. Type I HAE is characterized by low C1 inhibitor levels and function (85% of cases) while type II is associated with normal C1 inhibitor levels, but low function (15% of cases). Acquired angioedema (AAE) is another rare C1 inhibitor deficiency syndrome which is most commonly associated with B-cell lymphoproliferative diseases (type 1 AAE). It may also be related to the presence of an autoantibody directed against the C1 inhibitor molecule (type II AAE) .Clinically, HAE and AAE are similar, and are characterized by recurrent episodes of angioedema, without urticaria or pruritus, which most often affect the skin or mucosal tissues of the gastrointestinal and upper respiratory tracts. Although generally benign conditions, laryngeal involvement can rapidly lead to fatal asphyxiation if left untreated. Age of onset and the presence of a familial history are distinguishing features of these conditions , appears to play an important role . It is iIsolated angioedema also occurs in approximately 0.1% to 6% of individuals using ACE inhibitors. It tends to occur more commonly in ACE-inhibitor users who are female, smokers or of African-American descent. Like HAE and AAE, ACE inhibitor–induced angioedema is bradykinin-mediated. Most cases of angioedema occur in the first week after starting ACE-inhibitor therapy. However, up to one-third of cases occur months to years after initiating the medication . ACE inhIn the majority of cases of isolated angioedema, the cause is not identifiable (idiopathic angioedema). The exact mechanisms of idiopathic angioedema are unclear; however, based on patient response to medication, some cases are believed to be mediated by IgE-independent mechanisms that lead to mast cell activation ,23. In mThe diagnosis of HAE and AAE is based upon a suggestive clinical history  and the presence of abnormalities in specific complement proteins. Complement studies that should be ordered for patients with suspected HAE and AAE include: C4  level, C1q level, C1 inhibitor antigenic level, and C1 inhibitor functional level . These sIn most patients with AAE, C4, C1q, and C1 inhibitor function levels are low , and C1 inhibitor antigenic levels are low or normal. In type I HAE, C1 inhibitor antigenic and function levels are low ; in type II HAE, C1 inhibitor functional levels are low, but antigenic levels are normal or elevated  that briefly explain the patient's diagnosis, outlines the indicated treatment for acute attacks, and provide contact information for the supervising clinician [The management of HAE and AAE involves both prophylactic strategies to prevent attacks of angioedema as well as pharmacologic interventions for the treatment of acute attacks  and ACE inhibitors. Whenever possible, these triggers should be avoided. Recognition and prompt treatment of oral and dental infections, and screening for and eradication of H. pylori infection may be warranted in some cases [Factors triggering acute attacks of AAE and HAE vary but often include: mild trauma to the face , stress/anxiety, me cases .Attenuated androgens, such as danazol and stanozolol, increase C4 and C1 inhibitor levels and are effective for both the short- and long-term prophylaxis of HAE and AAE. Although generally well-tolerated by most patients, adverse effects with long-term androgen administration may include virilization, abnormalities in serum transaminases, menstrual irregularities, hair growth, decreased libido, weight gain, vasomotor symptoms, lipid abnormalities, and depression. Therefore, the lowest effective dose should be utilized (maximum long-term recommended doses are 200 mg daily for danazol and 2 mg daily for stanozolol), and the patient’s CBC, liver enzymes and lipid profile should be monitored regularly  while on therapy. Contraindications to androgen therapy include: pregnancy, lactation, cancer, hepatitis, and childhood .The antifibrinolytic agent, tranexamic acid, has also been shown to be effective for the prophylactic treatment of HAE and AAE. Evidence suggests that it may be less effective than androgen therapy in patients with HAE, but more effective in AAE . TranexaRegular intravenous injections of plasma-derived C1 inhibitor replacement therapy are also effective for both short- and long-term prophylaxis. These injections can usually be administered at home by the patient or caregiver. The current recommended dose is 20 units/kg; patients with AAE may require higher doses. Since C1 inhibitor replacement therapy is a blood product, annual recipient hemovigilance and vein-to-vein tracking are essential .First-line therapies for the treatment of severe acute attacks of HAE and AAE include: C1 inhibitor replacement therapy, ecallantide and icatibant . C1 inhiEcallantide is an inhibitor of plasma kallikrein . It has recently been approved by the Food and Drug Administration (FDA) in the United States for the treatment of acute angioedema attacks in patients with HAE. The usual recommended dose is 30 mg subcutaneously (adults). Although the side effects of ecallantide are generally mild , this therapy has been associated with rare instances of allergic reactions and anaphylaxis. Therefore, it should only be administered by a clinician in a medical setting equipped to manage anaphylaxis and severe angioedema .Icatibant is a bradykinin receptor blocker that has been approved in the European Union for the treatment of acute attacks of HAE. The usual recommended dose for adults is 30 mg subcutaneously; pediatric experience with this agent is still pending. The most common side effects of icatibant are mild and transient injection site reactions. Other, less common side effects include: nausea, gastrointestinal upset, asthenia, dizziness, and headache .Urticaria is a common disorder that often presents with angioedema. It is generally classified as acute (lesions occurring for < 6 weeks), chronic (lesions occurring for > 6 weeks) and physical . The disorder can usually be diagnosed on the basis of clinical presentation and history, however, diagnostic tests may be helpful for confirming the diagnosis. Second-generation, non-sedating H1-receptor antihistamines represent the mainstay of therapy for both acute and chronic urticaria; first-generation sedating antihistamines may be used as adjunctive therapy in patients with nocturnal symptoms. For severe, refractory chronic urticaria, short courses of oral corticosteroids and certain immunosuppressant and immunomodulatory therapies may be beneficial.Angioedema can occur in the absence of urticaria. The more common causes are ACE inhibitor-induced angioedema and idiopathic angioedema. Rare, but life-threatening causes are HAE or AAE. The work-up and management of HAE and AAE varies considerably from that of angioedema associated with urticaria. Although the angioedema associated with these disorders is often self-limited, laryngeal involvement can lead to fatal asphyxiation. Patients with these disorders demonstrate characteristic abnormalities in certain complement levels and, therefore, diagnostic testing of patients with suspected HAE or AAE should include assessment of C4, C1q and CI inhibitor function and antigenic levels. HAE should be considered in patients with an early age of onset and a family history of the disorder; in patients with AAE, there is no family history and age of onset is usually later. Therapeutic options for the prophylaxis of HAE and AAE include: trigger avoidance, attenuated androgens, tranexamic acid, and plasma-derived C1 inhibitor replacement therapy. First-line therapies for the treatment of acute attacks include: C1 inhibitor replacement therapy, ecallantide and icatibant.• Urticaria is a common disorder characterized by recurrent, pruritic (itchy) lesions with pale centers  that usually subside within 48 hours; it is often associated with angioedema.• Mast cells are the primary effector cells in urticaria.• Urticaria is classified as acute (lesions for < 6 weeks), chronic (lesions > 6 weeks), or physical.• The diagnosis of urticaria, with or without angioedema, is based primarily on a thorough clinical history; however, diagnostic tests may be helpful in some instances.• Second-generation, non-sedating H1-receptor antihistamines are the mainstay of therapy for urticaria. Oral corticosteroids and various immunomodulatory/immunosuppressive therapies may also be used for more severe, chronic cases.• Angioedema can occur in the absence of urticaria, with ACE inhibitor-induced and idiopathic angioedema being the most common causes.• ACE inhibitors should be discontinued in any individual who presents with angioedema as this condition is associated with life-threatening upper airway angioedema.• Idiopathic angioedema responds well to prophylactic antihistamines, however, oral corticosteroids may be required in some cases.• HAE and AAE are rare disorders also characterized by angioedema in the absence of urticaria; they result from a deficiency or dysfunction of the C1 inhibitor , and are associated with life threatening upper airway swelling.• Diagnosis of HAE and AAE should include the assessment of C4, C1q, C1 inhibitor function and antigenic levels.• The management of these disorders involves both prophylactic strategies to prevent attacks of angioedema  as well as pharmacological interventions for the treatment of acute attacks .Dr. Amin Kanani has received consulting fees and honoraria for continuing education from Scherring, GlaxoSmithKline, King Pharma, Merck Frosst, Novartis, CSL Behring and Talecris Biotherapeutics.Dr. Robert Schellenberg is past president of the Canadian Society of Allergy & Clinical Immunology, a member of the Medical Advisory Board of the British Columbia Lung Association, the National Lung Health Steering Committee and the Scientific Advisory Committee for Respiratory and Allergy Therapy of Health Canada. He is also a member of the Data Safety Monitoring Committee for Asthma Treatment. Dr. Schellenberg has received consulting fees and honoraria for continuing education and participation in advisory committees from GlaxoSmithKline, Merck Frosst, Novartis, Talecris, Bayer Biologics, and CSL Behring.Allergy, Asthma & Clinical Immunology. He has received consulting fees and honoraria from Nycomed, CSL Behring and Talecris.Dr. Richard Warrington is the past president of the Canadian Society of Allergy & Clinical Immunology and Editor-in-Chief of


We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca2+-induced Ca2+-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca2+, whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca2+-wave frequency and decreased the SR Ca2+-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca2+-dependent arrhythmias.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, ‘leaky’ RyR2 channels and Ca Many studies indicate that FKBP12.6 does ‘stabilise’ or reduce sarcoplasmic reticulum (SR) Ca2+-sparks. These results suggested to us that FKBP12 might have a more important cardiac role than previously envisaged, and that a primary target of its action may be RyR2. It is especially interesting therefore, that FKBP12 is thought to be present at much higher concentrations than FKBP12.6 in cardiac cells 2+-induced Ca2+-release (CICR) in isolated cardiac cells. We demonstrate the novel ability of FKBP12 to activate RyR2. Importantly, FKBP12.6 can antagonise activation of RyR2 by FKBP12 and our data suggest that FKBP12.6 may be a partial agonist with negligible efficacy at RyR2. Our results suggest that FKBP12 and FKBP12.6 can regulate the gating of RyR2 by modulating the sensitivity of the channel to cytosolic Ca2+.In contrast to FKBP12.6, there has been less emphasis on the cardiac role of FKBP12. However, the FKBP12 knockout mouse is characterised by severe dilated cardiomyopathy and the RyR2 channels isolated from this model exhibit unusual gating behaviour governed by long-lived sub-conductance state openings 2, and stored at −80°C as previously described cis-chamber corresponded to the cytosolic space and the trans-chamber to the SR lumen. The trans-chamber was held at ground and the cis-chamber held at potentials relative to ground. After fusion, the cis-chamber was perfused with 250 mM HEPES, 80 mM Tris, 10 µM free Ca2+, pH 7.2, unless stated otherwise. The trans-chamber was perfused with 250 mM glutamic acid and 10 mM HEPES, pH to 7.2 with Ca(OH)2 . Experiments were carried out at room temperature (22±2°C). The identity of the channels was confirmed by the single-channel conductance and the application of caffeine at the end of the experiment. The free [Ca2+] and pH of the solutions were determined using a Ca2+ electrode (Orion 93-20) and a Ross-type pH electrode (Orion 81-55) as previously described cis-chamber, the approximate free [Ca2+] was calculated using the program MaxChelator (www.stanford.edu/~cpatton/maxc.html) as 1 nM. The pH of the solutions was maintained constant. Additions of FKBP12 and FKBP12.6 were made to the cis-chamber. Both proteins were stored in a buffer containing 10 mM HEPES/50 mM NaCl, 0.5 mM DTT and volumes added to the cis-chamber were <2% of the total volume. Control buffer solution did not alter Po.Mixed membrane (MM) vesicles were prepared from sheep hearts , as described previously I is the logarithm of the ith time constant and ia is the fraction of the total events represented by that component Channel recordings were displayed on an oscilloscope and recorded on digital audiotape (DAT). Current recordings were filtered at 800 Hz (−3 dB) and digitised at 20 kHz using Pulse . Channel open probability (Po) was determined over ≥3 min of continuous recording, unless otherwise stated, using the method of 50% threshold analysis −1 β-escin for 1 min to permeabilise the surface membrane as previously described 2ATP, 5.4 MgCl2, 25 Hepes, 0.1 K2EGTA, pH 7.0 at 37°C for 5 min. This was to encourage dissociation of endogenous FK-binding proteins from RyR2 as it has been shown that FK-binding proteins dissociate quickly at 37°C 2+] to 234 nM. The waves were recorded at 21°C using a laser-scanning confocal microscope (BioRad MRC 1024) in the linescan mode. Linescan images were analyzed using ImageJ software (wsr@nih.gov). Waves of CICR were thus monitored in each cell before and after the addition of either 3 µM FKBP12 or 200 nM FKBP12.6 to the mock intracellular solution. For experiments to test the ability of FKBP12.6 to antagonize FKBP12, cells were first incubated with 200 nM FKBP12.6 for 5 min at 21°C and subsequently perfused with 3 µM FKBP12 in the continued presence of FKBP12.6. The SR Ca2+-content was examined in the absence and in the presence of FKBP12 after application of caffeine (20 mM) to release Ca2+ stores into the cytosol.Male Wistar rats were killed by cervical dislocation in accordance with the Animal (Scientific Procedures) Act 1986. Rat ventricular myocytes were isolated using a protease and collagenase Langendorff perfusion technique as previously described Escherichia coli BL21(DE3) Codon Plus cells . Protein expression was induced by addition of Isopropyl-β-D-1-thiogalactopyranoside (IPTG 1 mM) to the culture. After 3 hours, cells were pelleted, resuspended in Tris-buffer , and lysed by sonication to obtain soluble protein. The protein extracts were purified by cation exchange and size exclusion chromatography using an HPLC-AKTA machine . The filtered proteins were loaded in a CM Sepharose Fast Flow column  in Tris-buffer, pH 6.8 and pH 6.6 respectively for FKBP12.6 and FKBP12, and eluted by a linear NaCl gradient. The proteins were subsequently purified by gel filtration using a Superdex 75 10/300 GL column  in a buffer containing 10 mM HEPES, 50 mM NaCl, 0.5 mM DTT, pH 8. The purified chromatographic peaks were checked by SDS-polyacrylamide gel electrophoresis. The high-quality samples were mixed and concentrated with Amicon 10 kDa Ultra-4 Centrifugal Filter Units  by centrifugation at 4°C. The final protein concentration of FKBP12 and FKBP12.6 was calculated by a Bradford assay Recombinant DNA techniques were carried out according to standard protocols. The FKBP12 and 12.6 proteins were constructed by PCR amplification using the pCMV6-XL4-true clone  and the PGEX-4T-1 vector  respectively. The two constructs were subcloned in the pET-28a plasmid  and transformed in The protein concentration of the vesicles was calculated by a Bradford assay. Proteins were then size fractionated by SDS-PAGE on either a 4–20% precast gel  or a 15% gel. Following separation, proteins were transferred to a nitrocellulose membrane and non-specific binding sites were blocked for 1 hr at room temperature using 5% dried milk and Tris-buffered saline, 0.1% Tween 20, pH 7.4. Membranes were probed for 2 h at room temperature with a polyclonal primary anti-FKBP antibody ((1∶500) Thermo Fisher Scientific, Leicestershire, UK)). A secondary horseradish peroxidase linked anti-rabbit IgG ((1∶5000) GE Healthcare, Buckinghamshire, UK)) was used in combination with an enhanced chemiluminescent detection system  to visualise the primary antibodies.−1. Peptides were ionized by nano-electrospray ionization at 2.3 kV using a stainless steel emitter with an internal diameter of 30 µm . Tandem mass spectrometry analysis was carried out on a LTQ-Orbitrap Velos mass spectrometer . The Orbitrap was set to analyze the survey scans at 60,000 resolution and the top twenty ions in each duty cycle selected for MS/MS in the LTQ linear ion trap. Data was acquired using the Xcalibar v2.1 software . The raw data files were processed and quantified using Proteome Discoverer software v1.2  with searches performed against the UniProt human database by using the SEQUEST algorithm with the following criteria; peptide tolerance = 10 ppm, trypsin as the enzyme, carboxyamidomethylation of cysteine as a fixed modification and the oxidation of methionine as a variable modification. The reverse database search option was enabled and all data was filtered to satisfy false discovery rate (FDR) of less than 5%.During the procedure for isolation of heavy SR membrane vesicles from sheep cardiac muscle, the MM vesicle fraction, the corresponding supernatant  and the heavy SR vesicles were collected and electrophoresed on a 15% SDS-PAGE. Prior to electrophoresis, the supernatant (S2) was filtered multiple times, each time reducing the pore of the filter from 1.2 µM–0.22 µM and concentrated by centrifugation at 3500× g for 15 h with an Amicon 10 kDa Ultra-15 centrifugal filter unit . Following separation, the proteins were either visualised by staining with Coomassie brilliant blue (CBB) or transferred to a nitrocellulose membrane for Western blot analysis. Where anti-FKBP antibodies indicated the presence of FKBPs on the Western blot, the corresponding band visualised by CBB staining was cut from the gel. The gel slice was subjected to in-gel tryptic digestion using a ProGest automated digestion unit (Digilab UK). The resulting peptides were fractionated using a Dionex Ultimate 3000 nanoHPLC system. In brief, peptides in 1% (v/v) formic acid were injected onto an Acclaim PepMap C18 nano-trap column . After washing with 0.5% (v/v) acetonitrile 0.1% (v/v) formic acid peptides were resolved on a 250 mm×75 µm Acclaim PepMap C18 reverse phase analytical column  over a 120 min organic gradient with a flow rate of 300 nlminwww.math.pitt.edu/~bard/xpp/xpp.html). Steady state Po was computed by taking the trial average of 100 simulations of one channel. For plotting the modelling results we used IGOR Pro (http://www.wavemetrics.com/).Monte Carlo simulations of the two-state RyR ion channel model were performed using XPPAUT . Fluo-5F was obtained from Invitrogen, . Other chemicals were AnalaR or the best equivalent grade from BDH  or Sigma-Aldrich . All solutions were made in deionised water and those for use in bilayer experiments were filtered through a Millipore membrane filter (0.45 µm pore).The endogenous FKBPs associated with RyR2 were dissociated by high salt incubation during the sucrose gradient step of the heavy SR isolation procedure see . For sinp<0.05)). RyR channels are known to exhibit a large variation in their Po levels The FKBP-depleted SR was then incorporated into bilayers and two typical experiments are shown, illustrating that neither a low  nor a hip<0.01). FKBP12 was not able to fully open the channel at any concentration  ntration  indicatintration , to remoThe fact that a wide range of FKBP12 concentrations produce similar increases in Po indicates that when a molecule/s of FKBP12 binds to RyR2, it binds very tightly and dissociates slowly. If this is the case, we would expect Po to remain high after washout of FKBP12 from the cytosolic chamber. Where possible (because bilayers often break with perfusion), we therefore recorded for long periods (9 min) after washing out the FKBP12 in order to investigate if dissociation of FKBP12 from RyR2 could be detected by a sudden drop in Po. Since FKBP12.6 appears to have very little ability to activate RyR2 and since FKBP12 and FKBP12.6 are extremely similar structurally, it is possible that FKBP12.6 could act as an antagonist of FKBP12. Cryo-electron microscopy and 3D reconstruction of RyR1 indicates that FKBP12 and FKBP12.6 bind within the same location on the cytoplasmic face of RyR1 Our washout data  suggests2+ activates RyR2 and the mechanism by which agents such as caffeine that “sensitise” RyR2 to the effects of cytosolic Ca2+ appear to increase Po 2+.To investigate the mechanisms underlying RyR2 activation by FKBP12 we performed lifetime analysis on experiments where only a single channel was gating in the bilayer. . The mean open time was 1.01±0.04 ms in the absence and 1.82±0.24 ms  in the presence of 1 µM FKBP12 and the mean closed time was 27.94±9.98 ms in the absence and 6.93±1.32 ms  in the presence of 1 µM FKBP12. Thus FKBP12 primarily affected the duration of the closed times. This mechanism was confirmed by lifetime analysis and examples of representative open and closed lifetime histograms for control and FKBP12 treated channels are shown in 2+. We found that this was indeed the case. If the cytosolic [Ca2+] was lowered to approximately 1 nM by addition of 10 mM EGTA, then Po was reduced to zero . After addition of 1 µM FKBP12, Po remained at zero (n = 4). Similarly, if cytosolic Ca2+ was reduced to approximately 1 nM following channel activation by FKBP12 in the presence of micromolar cytosolic Ca2+, the channels could be completely shut down. An example of this behaviour can be seen in 2+ to sub-activating (approximately 1 nM) levels.If this is the mechanism by which FKBP12 activates RyR2, then FKBP12 will not activate RyR2 in the absence of activating levels of cytosolic CaFree) and that it can bind FKBP12 or FKBP12.6 forming either the FKBP12 bound state (RyR212) or the FKBP12.6 bound state (RyR212.6). Each binding event leads to a conformational change in RyR2, which in turn modifies channel function in a manner specific to the isoform of FKBP that is bound. Thus, when FKBP12 is bound, the Ca2+-sensitivity of RyR2 is increased (state RyR212*), whereas when FKBP12.6 is bound, any change in Ca2+-sensitivity is much less marked (state RyR212.6*). The model is shown in We constructed a kinetic model to describe the possible channel states and the transitions between states that arise from the binding of FKBP12 or FKBP12.6 to RyR2. We then used this model to simulate our data. We assume the simplest case; that RyR2 can exist in the FKBP-free state  of transition from state C to state O and kC is the rate (probability) of the reverse transition.Equilibrium approximation with respect to FKBP12 and FKBP12.6 binding and the subsequent change in CaO depends on the probability that RyR2 is bound to FKBP12 or to FKBP12.6. Of course, the Po of RyR2 channels also depends on other factors such as the cytosolic and luminal [Ca2+], on the binding of other closely associated proteins and on the redox and phosphorylation states of the channels. We assume, however, that these factors are maintained constant throughout each experiment since Po remains stable , that accounts for this spontaneous background RyR2 activity. Thus, in the presence of FK-binding proteins, RyR2 Po is given by the sum of the probabilities that RyR2 exists in the two possible open states, namely x12*+x12.6*, i.e.ν = x12*+x12.6*. The parameter values of the model are given in In our model, the rate kvice versa. In choosing parameter values for the model, as a starting point we used published estimates of the rate constants of association and dissociation of FKBP12 and FKBP12.6 to RyR2 The model described above allows us to compute the average RyR2 Po for various FK-binding protein concentrations and under different conditions such as the sequential addition of FKPB12 following FKBP12.6 and Our model also allows us to predict how RyR2 gating in the steady-state would be affected if the experimental protocols shown in 2+-release events during diastole and an increased likelihood of developing arrhythmias. There are many mechanisms by which this could occur but one possibility would be that there is simply less FKBP12.6 expressed in the cardiac cells in heart failure. Our model can predict how changes in cellular FKBP12.6 levels could affect RyR2 Po at equilibrium if FKBP12 remained at physiological levels (3 µM). We do not know the physiological level of FKBP12.6 in a cardiac cell except that it is below detection levels and therefore much lower than the micromolar levels of FKBP12. It has been suggested that there is a reduced amount of FKBP12.6 bound to RyR2 channels in heart failure 2+-release under pathological conditions. In heart failure, for example, there is an increased tendency for the generation of spontaneous waves of SR Ca2+-release and the development of delayed after depolarisations (DADs) 2+-release in cardiac cells. FKBP12 would be expected to sensitise RyR2 to a rise in cytosolic Ca2+ thereby potentiating CICR. FKBP12.6 would antagonise this action. We therefore investigated if FKBP12 could augment spontaneous waves of CICR in rat permeabilised cardiac myocytes and whether FKBP12.6 could antagonise this effect. At 5 minutes post-permeabilisation, cells were superfused with Fluo-5F and 234 nM Ca2+. 2+-waves under these conditions. We then perfused with a physiological concentration of FKBP12 (3 µM) (bottom trace) and this caused an increase in wave frequency. 2+-waves but also decreases wave amplitude. Although the unresolved controversy over the single-channel effects of FKBP12.6 2+-content. We examined this by applying 20 mM caffeine to release SR Ca2+ before and after perfusion with 3 µM FKBP12. 2+-transient in control (3.2±0.1 ΔF/Fo) than in the presence of FKBP12  indicating that there is a reduced SR Ca2+-content in the presence of FKBP12.The reduced wave amplitude may result because there is a decrease in the SR Ca2+-release in cardiac cells we perfused cells with FKBP12.6. The levels of FKBP12.6 in cardiac cells are so low that they are undetectable p≤0.001) but after pre-incubation with FKBP12.6, FKBP12 only increases wave frequency by 16.25±0.02% ).Our single-channel experiments suggest that FKBP12.6 behaves as an antagonist of the actions of FKBP12 on RyR2. To investigate if this mechanism is relevant to SR Ca2+, FKBP12 is a partial agonist. We also show that FKBP12.6 has no intrinsic ability to reduce RyR2 Po but perhaps may have extremely low efficacy as an activator of RyR2. It is, however, able to antagonise the effects of FKBP12. These results suggest that FKBP12 and FKBP12.6 may compete for the same binding sites on RyR2, especially since both molecules are structurally so similar. We cannot rule out the possibility that FKBP12.6 acts to reduce FKBP12-induced increases in Po by binding at distinct sites although this seems unlikely since FKBP12.6 does not, alone, reduce the Ca2+-activated openings. There has been rather an over-emphasis of the exclusive nature of FKBP12.6 binding to RyR2 and the possibility that FKBP12 may affect RyR2 function has been somewhat overlooked. Our results should not be too unexpected however, since Jeyakumar et al. (2001) have previously suggested that FKBP12 binds to the cardiac isoform of many species although they also indicate that it may not bind to canine RyR2. Seidler et al. (2007) have also demonstrated binding of GST-FKBP12 to RyR2. Our reductionist approach has revealed new mechanisms underlying the functional interactions of FKBP12 and FKBP12.6 with RyR2. This new information may help to resolve the controversies surrounding the effects of FKBP12.6 on RyR2 channel gating and the many descriptions of the cardiac ‘stabilizing’ properties of FKBP12.6. For example, in models of heart failure, it has been reported that over-expressing FKBP12.6 We demonstrate that FKBP12 can activate single RyR2 channels at very low concentrations. Like many other activators of RyR channels including Caall the endogenous FKBP has been removed before adding back FKBP to observe its effect. In general, depletion of FKBP means simply ‘below the detection level of a Western blot’ rather than complete removal of the protein. This is also true for our cardiac membrane preparations. Although we can ‘deplete’ our cardiac SR of FKBP with a high salt solution we cannot definitively prove that we have removed every molecule. However, since exogenously added FKBP12 always causes a consistent increase in Po, it is clear that, in our experiments, there are vacant binding sites on RyR2 for FKBPs that appear to be functionally important. Many studies have used compounds such as FK506 or rapamycin to lower the levels of FKBPs present in cells or isolated membrane vesicles. There have been reports, however, that FK506 The slow rate of dissociation of FKBP12 from RyR2, evidenced by the washout experiments , means t2+ activates RyR2 is primarily by increasing the frequency of channel opening with only a small lengthening of open lifetime durations 2+. Indeed, the Po of channels that have been activated by FKBP12 can be reduced to zero when cytosolic Ca2+ is reduced to sub-activating levels providing strong evidence for this mechanism of action.We found that the main effect of FKBP12 was to increase the frequency of channel openings. FKBP12 can also increase the duration of channel openings but this effect was less pronounced, probably playing a greater role in the presence of other channel activators. Since the mechanism by which cytosolic Cain situ. In a cardiac cell, we might predict that FKBP12 would increase the frequency of channel openings during diastole; a time when RyR2 Po is normally low. Essentially, FKBP12 would sensitise RyR2 channels to the effects of small rises in cytosolic Ca2+ and the channels would become ‘leaky’ during diastole. This mechanism also provides the best explanation for the FKBP12-induced increased frequency of spontaneous Ca2+-waves that we observe in our permeabilised cell experiments. The increased wave frequency is associated with a lower SR Ca2+-content as shown by 2+-content 2+ following a spontaneous wave of CICR and the SR must refill before another wave is possible 2+-content was sufficiently recovered. If, however, RyR2 was sensitised to the effects of cytosolic Ca2+ , then a wave of CICR would be triggered at a lower SR Ca2+-content, reducing the requirement for the SR to fill. The lower SR Ca2+-content would give rise to smaller waves and since less Ca2+ is released with each wave, the SR would need less time to refill and so the waves would be more frequent. Thus, our results can be explained by an effect on RyR2 gating.This mechanism of action of FKBP12 is important to consider when looking at how FKBP12 would influence the opening of RyR2 channels 2+-release in permeabilised cardiac cells 2+-spark amplitude and width after perfusion with 1 µM 2+-spark amplitude and width after over-expression of FKBP12 2+-spark amplitude and duration are increased in cardiac cells isolated from the FKBP12.6 knockout and it is suggested that the RyR channels are open for longer Two previous studies have investigated how FKBP12 may affect SR Ca2+-release since FKBP12.6 can inhibit the effects of FKBP12. This is confirmed by mathematical modelling of our data (2+ is increased. This would promote a greater tendency for the development of spontaneous waves of CICR and delayed after depolarisations (DADs) and an increased likelihood of fatal arrhythmias. A model of this scheme is shown in 2+-release and could lead to the ‘leaky’ RyR2 channels, reduced SR Ca2+-load and increased likelihood of arrhythmias that occur in heart failure.Our results suggest that the relative levels of FKBP12 and FKBP12.6 in a cardiac cell will be important in the regulation of SR Caour data . For exaFigure S1The effects of high salt incubation on the association of FKBPs with cardiac mixed membrane (MM) vesicles. Western blot analysis following incubation of cardiac mixed membrane vesicles with a high sucrose (0.4 M) solution (left lane) or a high salt (0.4 M) solution (right lane). Vesicles were loaded at 100 µg. Western blots were probed with an anti-FKBP12 antibody (recognising both FKBP12 and FKBP12.6). Size markers are indicated in kDa.(TIFF)Click here for additional data file.


This culminated in the use of the photoprotein aequorin to demonstrate that [Ca2+]i increased after depolarization but before contraction in barnacle muscle. Green fluorescent protein was extracted from the same jellyfish as aequorin, so this work also has important historical links to the use of fluorescent proteins as markers in living cells. The subsequent development of cell-permeant Ca2+ indicators resulted in a dramatic increase in related research, revealing Ca2+ to be a ubiquitous cell signal. High-speed, confocal Ca2+ imaging has now revealed subcellular detail not previously apparent, with the identification of Ca2+ sparks. These act as building blocks for larger transients during excitation–contraction coupling in cardiac muscle, but their function in smooth muscle appears more diverse, with evidence suggesting both ‘excitatory’ and ‘inhibitory’ roles. Sparks can activate Ca2+-sensitive Cl− and K+ currents, which exert positive and negative feedback, respectively, on global Ca2+ signalling, through changes in membrane potential and activation of voltage-operated Ca2+ channels. Calcium imaging has also demonstrated that agonists that appear to evoke relatively tonic increases in average [Ca2+]i at the whole tissue level often stimulate much higher frequency phasic Ca2+ oscillations at the cellular level. These findings may require re-evaluation of some of our models of Ca2+ signalling to account for newly revealed cellular and subcellular detail. Future research in the field is likely to make increasing use of genetically coded Ca2+ indicators expressed in an organelle- or tissue-specific manner.Ever since it was shown that maintenance of muscle contraction required the presence of extracellular Ca The sliding filament hypothesis of striated muscle contraction was established independently (by two different Huxleys) based on structural and functional experiments in the 1950s , while working in the laboratory of Frank Johnson at Princeton. They showed that the amount of light released could be used as a measure of the [Ca2+] in an aequorin-containing solution, opening the way for biological applications of the technique -gated or inositol 1,4,5-trisphosphate receptor (IP3R)-gated release channels. Some of these Ca2+ channels will be mentioned in what follows, but no attempt will be made to provide an overview of this vast field.As is often the case, the availability of new research tools that were relatively easily applied led to a massive explosion in Ca2+ store . Despite2+ imaging itself, it is worth mentioning the close conceptual and historical connections between the development of Ca2+ indicators and other live cell imaging techniques based on fluorescent protein markers. In Shimomura and Johnson's original work on Aequoria, they noticed that the jellyfish produced a second optically active protein, which generated green fluorescence when excited by the light emitted from the aequorin itself  intensity of any emitted fluorescence is recorded. Although a microscope objective is used to collect the light, no fluorescent image is captured. There are several technical reasons for doing this, including increased sensitivity and speed of recording, important with rapidly changing signals. However, the development of confocal microscopy and ever more sensitive CCD-based cameras made true Ca2+ imaging a viable option, in which local changes in [Ca2+]i within individual cells can be visualized. Confocal microscopes combine a scanning laser to excite cell fluorescence with a pinhole to exclude out-of-focus light coming back into the imaging scanhead, allowing clear optical sections to be recorded from much thicker tissues. Initially, this technique was used to improve the resolution of images from fixed specimens stained with fluorescent dyes, and to generate three-dimensional tissue models by imaging different planes in the same sample and combining the data , but at the cost of one spatial dimension. This sacrifice was soon shown to be justified, however, as the resulting linescan images revealed subcellular Ca2+ events previously unsuspected in cardiac muscle cells. These brief, spatially localized increases in [Ca2+]i were dubbed ‘Ca2+ sparks’ and were shown to represent spontaneous release of Ca2+ from the sarcoplasmic reticulum via cardiac ryanodine receptors . Such channels  had already been identified in smooth muscle . The resulting spontaneous transient inward currents (STICs) tend to depolarize the plasma membrane, increasing the steady-state activation of voltage-operated Ca2+ channels and so raising cell [Ca2+]i. Calcium sparks have been shown to generate STICs in a number of different smooth muscle types , a highly potent endothelium-derived vasoconstrictor, regulates Ca2+ signals in retinal arterioles. Recording the average smooth muscle [Ca2+]i using microfluorimetry results in a relatively simple response, with an initial Ca2+ transient lasting approximately 100 s, which then falls back towards the control level but remains elevated above baseline  than the transient seen at the whole vessel level (2+]i recorded using microfluorimetry. Importantly, no individual cell experiences the temporal profile of [Ca2+]i changes recorded in the spatially averaged recording. Any realistic model of cell signalling in response to Et-1 must, therefore, be able to account for the more complex pattern revealed by high-speed imaging. Similar oscillatory responses to constricting stimuli, first reported in vascular tissue in 1994 (2+ oscillations, and that the resulting contractions can be plotted as a function of frequency rather than ‘mean’[Ca2+]i (2+]i in most cells, it is reasonable to argue that Ca2+ imaging has revealed a major ‘digital’, ‘frequency-modulated’ aspect to smooth muscle Ca2+ signalling previously unsuspected from global measurements of average [Ca2+]i (2+ imaging (2+ changes within individual myocytes embedded in their parent tissue using confocal techniques has demonstrated their importance in smooth muscle in a way not previously appreciated from Ca2+ microfluorimetry records. It remains to be seen what functional benefit results from the use of complexly patterned Ca2+ signals rather than simple variations in steady-state [Ca2+]i, although oscillations may allow for intermittent activation of Ca2+-dependent processes with a relatively high Ca2+ threshold while reducing the risk of cell damage (2+ signals may also contribute to signal targeting within the cell just as much as their spatial distribution. This may even play a role in the regulation of protein expression in smooth muscle by Ca2+ signals, a process referred to as ‘excitation–transcription coupling’ (Another aspect of Cabaseline A. This t [Ca2+]i . Calcium’[Ca2+]i a,b;. Alt imaging , but the2+-imaging technology has radically altered the way we have to think about signalling at the cellular level. The complex and highly dynamic responses evoked in smooth muscle by agonists, for example, were not obvious in microfluorimetry recordings of average [Ca2+], and suggest that frequency modulation of phasic Ca2+ waves and oscillations plays an important role in determining the contractile response. Subcellular detail has also been revealed, with brief, spatially localized Ca2+-release events. These Ca2+ sparks can contribute directly to the elevation of cell [Ca2+] and contraction, as they do in cardiac excitation–contraction coupling and in some smooth muscles, or indirectly through activation of Ca2+-sensitive membrane conductances, altering membrane potential and thus regulating Ca2+ influx via changes in activation of voltage-operated channels. As is usual in science, the ability to look at Ca2+ signals in more detail has generated as many new questions as answers, and the challenge of integrating the relevant detail into functional models at the cell and tissue level will be considerable. Future developments will probably include increased use of genetically coded Ca2+ indicators, in which proteins with Ca2+-sensitive fluorescent or luminescent properties are expressed in specific organelles or tissues (The application of Ca tissues . This re tissues .


Ca2+-dependent K+ channels (SK) and the presence of a calcium buffer. Our results show that a variation in the concentration of PV can modulate substantially the intrinsic excitability of the FS interneurons and therefore may be involved in the information processing at the striatal level.Calcium binding proteins, such as parvalbumin (PV), are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly understood. Notably, at the level of the striatum, where PV is only expressed in the fast-spiking (FS) interneurons. FS interneurons form an inhibitory network modulating the output of the striatum by synchronizing medium-sized spiny neurons (MSN). So far the existing conductance-based computational models for FS neurons did not allow the study of the coupling between PV concentration and electrical activity. In the present paper, we propose a new mathematical model for the striatal FS interneurons that includes apamin-sensitive small conductance  Ca2+, as opposed to buffer proteins  which bind Ca2+ as its concentration increases within a cell and are thought to mainly act as passive modulators of the cytosolic calcium level. Nevertheless, it has been suggested that calbindin also acts as a Ca2+ sensor  channels  and Ca2+ with high affinity , the majority of PV’s binding sites (>80%) are occupied by Mg2+. The binding of Ca2+ is determined by the slow Mg2+ off-rate , a = a∞(V). The membrane capacitance was set to 30 pF, the leak conductance leakg to 2.5 nS and the leak reversal potential to −68 mV, to match the known experimental membrane capacitance (25–30 pF), membrane resistance (400 MΩ), and resting membrane potential (−70 mV). The other reversal potentials are NaV = 74 mV, KV = − 90 mV, CaV = 80 mV, and the maximal ionic conductances are Nag = 700 nS, Kv1g = 2 nS, Kv3g = 300 nS, Cag = 30 nS, SKg = 2 nS. The dynamic of the other gating variables (excepted k) are governed by:where x = h, n1, n3. The kinetics of the αx, βx are exactly as published in Mancilla et al.  and off,skk = 0.2 ms−1 . The inward flux is −CaI/2FAd (F is the Faraday constant). The term γ([Ca2+]i − [Ca2+]rest) is the clearance mechanism associated with the Ca2+ fluxes across the plasma membrane or storage organelles . In presence of the slow or fast buffer, the coupling of the calcium dynamic is done via the following equations:where [B]i, [BCa]i are the concentration of free and bound buffer (slow or fast). The total buffer concentration [B]T = [B]i + [BCa]i. The list of model parameters are shown in Table where i increases due to the summation of [Ca2+]i transients. For trains of AP of sufficiently long duration, [Ca2+]i will reach a steady-state plateau level and fluctuate between a lower and upper level. At steady-state, calcium influx and clearance mechanisms compensate . The time course of the lower envelopes of [Ca2+]i transients in the presence of PV is shown in Figure 2+ transients follows a similar time course both for PV and the slow buffer  and 0.3 μM . This value of 0.3 μM is sufficient to activate a significative fraction of the SK channels that have a DK for calcium of 300–700 nM  while it slowly increases from 19 to 35 pA at high PV concentration  both BAPTA and EGTA will interfere with the Ca2+ signaling (Neher, The purpose of our model was to propose a basic mechanism for the regulation of excitability of FS neurons by calcium buffering. Despite the use of a single compartment model with a limited set of conductances and currents, the simplicity of our model and the use of buffers with different kinetics validates our simulations to other neuronal types. Indeed, the proposed mechanism will remain valid providing that the mechanisms of excitability remain the same, as in FS neurons, and that the conductance of the Stocker, . MoreoveStocker, . This spg Neher, .Ca2+ homeostasis, Ca2+-binding proteins appear to play an active role in modulating neuronal intrinsic excitability. Although, information storage is usually believed to be mediated by long-term modifications in the strength of synaptic transmission, activity-dependent changes in the neuronal intrinsic excitability also occur, causing forms of non-synaptic plasticity (Aizenman and Linden, Ca2+ binding proteins. The mathematical model we present here provides a valuable tool for the investigation of the functional role of parvalbumin in the regulation of the activity of the striatal FS interneurons. This study paves the way for further theoretical work to assess the impact of calcium buffering on the activity of the striatal FS interneurons network.In addition to their already documented role in 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.


However, whether functional gaps are also involved in the regulation of normal Ca2+ signaling, in particular the basal [Ca2+]i activities, is unclear.It has been found that gap junction-associated intracellular Ca2+ signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca2+ transients and local Ca2+ sparks in monolayer NRVMs and Ca2+ sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP3 butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca2+ signal and LY uptake by gap uncouplers, whereas blockade of IP3 receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca2+ signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibody against Cx43 demonstrated apparent increases in membrane labeling of Cx43 and non-junctional Cx43 in overexpressed cells, suggesting functional hemichannels exist and also contribute to the Ca2+ signaling regulation in cardiomyocytes.Global and local Ca2+ signaling in normal ventricular myocytes, in which IP3/IP3 receptor coupling is involved. This finding may provide a novel regulatory pathway for mediation of spontaneous global and local Ca2+ activities in cardiomyocytes.These data demonstrate that Cx43-associated gap coupling plays a role in the regulation of resting Ca Hemichannels are precursors of gap junctions, assembled by six connexin subunits that span the lipid bilayer. Like conventional ion channels, hemichannels do not remain continuously open, instead, they flip between open and closed states regulated by multiple stimuli. For instances, reduction in extracellular Ca+2+ and Na+It has been demonstrated that functional connexin hemichannels also exist in isolated ventricular myocytes 2+ ([Ca2+]i) transient represents the global intracellular Ca2+ signaling, while Ca2+ sparks are the building blocks of intracellular Ca2+ activity that derive from local, rapid and transient Ca2+ release from a cluster of ryanodine receptor (RyR) activation in the sarcoplasmic reticulum 2+ activities that potentially generate lethal arrhythmias and hyperconstriction in ventricles 2+ signaling in diseased heart. Yet whether gap junction and hemichannels are also involved in the modulation of Ca2+ signaling, particularly, in the basal Ca2+ activities in normal heart, is presently unknown.Intracellular Ca2+]i activities and compared them with those found in monolayer myocytes that already form typical gap junctions. We found that both confluent and single myocytes exhibited downregulated Ca2+ signaling in response to gap uncouplers and interference of connexin43 (Cx43) expression the predominant connexin in the ventricles, while overexpression of Cx43 displayed enhanced Ca2+ activities in both densities of the cells. Therefore, this study demonstrates that Cx43-associated coupling plays a fundamental role in the mediation of local and global Ca2+ signaling in ventricular myocytes.In this study, we used single cardiac myocytes to determine the effects of hemichannel on the i activities as gap uncouplers in neonatal and adult myocytes; and (v) like in monolayer cells, the changes in Ca2+ signals and LY uptake were well correlated with the level of cell surface Cx43 labeling and non-junctional Cx43 in single NRVMs.The present study demonstrated that functional Cx43-relevant gap junction/hemichannels play an important role in the regulation of spontaneous Ca2+ signaling accordingly. In fact, a direct impact of Ca2+ spreading among adjacent cells has already been demonstrated in Cx43-expression manipulated cells in other studies. For instances, it has been shown that Cx43-expression in other cell types is correlated with both intercellular dye transfer 2+ waves through gaps 2+ transients that further hampers the synchronous beating 2+ signals among connected cells. The present study further depicts a role of Cx43-associated gap coupling in the regulation of intrinsic Ca2+ signaling inside the ventricular myocytes. Such effect of Cx43-coupling should be unrelated with the instant electrical exchange between myocytes, because similar observations found in monolayer myocytes were also spotted in single unconnected cells, suggesting a potential role of Cx43-relevant hemichannels in the regulation of spontaneous Ca2+ signaling in normal cardiac myocytes.In agreement with the chemical inhibitors of gap junction, specific overexpression or knockdown of Cx43 in NRVMs by adenovirus infection, the predominant connexin in ventricular myocytes, could upregulate or downregulate the gap permeability as well as the spontaneous Ca3 has been found to be responsible for the hemichannel-mediated intercellular Ca2+ wave initiation and spreading in non-muscle cells 2+ activities of adjacent unconnected cells by ATP release to the buffer and entrance into these unconnected cells via hemichannels 2+ signaling in this study, for suramin, an antagonist of purinergic 2z/2x receptor, did not affect the spontaneous Ca2+ transients in the wild type NRVMs, nor did on the potentiated spontaneous Ca2+ oscillations in the wt-Cx43 NRVMs (data not shown). In contrast, inhibition of IP3R with XeC or 2-APB inhibited the global spontaneous Ca2+ signals as well as the gap exchange in NRVMs, whereas blockade of L-type Ca2+ channel or RyR, two important effectors of Ca2+ signaling, did not show such parallel effects on Ca2+ activity and intercellular communication. Importantly, both membrane-permeable IP3 and PE, to produce endogenous IP3, could completely recover Ca2+ oscillations inhibited by heptanol or Gap 27. Moreover, knockdown of the endogenous IP3R mimicked the 2-APB effects on NRVMs and, interesting, affected the phosphorylation level of Cx43 (unpublished data in a separate manuscript). Therefore, these data indicate that IP3/IP3R-associated pathway contributes significantly to Cx43-regulated Ca2+ signaling, a proposal is also supported by other studies that demonstrate a direct IP3 release mediated by Cx43 Interestingly, a release of ATP or IP2+ signaling is severely compromised, e.g. lower amplitude and longer duration of Ca2+ transient compared with those in normal myocardium 2+ signaling in cardiomyocytes. Therefore, the present study demonstrates that in addition to contributing for Ca2+ spreading between adjacent cells in normal heart and abnormal Ca2+ signal formation in diseased heart, Cx43-associated coupling may also play a role in the regulation of basal intracellular Ca2+ activities in normal ventricular myocytes.In pathological situations, gap junction remodeling characterized by Cx43 lateralization and down-regulation are typical features in failing myocardium


Treatment of chronic urticaria consists of antihistamines as the first-line treatment. For more severe symptoms, combinations can be necessary as well as dose augmentations. The recent guidelines suggest the possibility of using omalizumab in resistant cases. We treated 2 patients with cold-urticaria (CO), 1 with cholinergic urticaria(CH) and 11 with chronic spontaneous urticaria (CSU) with omalizumab, who had not benefited from the recommended first-line, second-line and third-line treatments.Patients were required to document their CU symptoms once daily with urticaria activity scores for 7 days (7UAS). Briefly, the symptoms were monitored in terms of numbers of wheals , and the intensity of their pruritus , for a total of 42 points. To evaluate the efficacy of the omalizumab treatment, 7UAS obtained at baseline was compared to that at the third and sixth month of the therapy. Omalizumab was given at 150 μg/month irrespective of IgE levels and increased to 300 mg if needed (no response). The concomitant medication was slowly reduced according to clinical response.The 7UAS improved significantly in all severe urticaria patients with omalizumab as early as one month (not shown) after initiation of therapy and was sustained for the 6 month observation Figure . 7UAS inOur results show that omalizumab improves significantly recommended treatment-resistant urticaria patients (13/14) in terms of clinical symptomatology (7UAS) and drug reduction in a real life setting. None of the patients reported any adverse effect.


CIITA, a master regulator of MHC class II gene expression and truncated in B-cell lymphoma and Hodgkin disease, is found to fuse with DEXI in 48% of normal karyotype AML cases. The fusion transcripts formed between adjacent genes highlight the possibility that certain such fusions could be involved in oncological process in AML, and provide a new source to identify genetic markers for normal karyotype AML.Genetic aberrations contribute to acute myeloid leukemia (AML). However, half of AML cases do not contain the well-known aberrations detectable mostly by cytogenetic analysis, and these cases are classified as normal karyotype AML. Different outcomes of normal karyotype AML suggest that this subgroup of AML could be genetically heterogeneous. But lack of genetic markers makes it difficult to further study this subgroup of AML. Using paired-end RNAseq method, we performed a transcriptome analysis in 45 AML cases including 29 normal karyotype AML, 8 abnormal karyotype AML and 8 AML without karyotype informaiton. Our study identified 134 fusion transcripts, all of which were formed between the partner genes adjacent in the same chromosome and distributed at different frequencies in the AML cases. Seven fusions are exclusively present in normal karyotype AML, and the rest fusions are shared between the normal karyotype AML and abnormal karyotype AML.  Genetic aberrations including translocation, amplification, inversion, insertion, and deletion, are well-known to contribute to leukemia Acute myeloid leukemia (AML) is a major type of leukemia, with estimated 13,780 new cases and 10,200 death in the United States in 2012 . A total of 1,374,219,760 paired-end reads , or on average 30,538,217 reads  per sample, were collected in the study .RUNX1-RUNX1T1 fusion in two cases , MLL-MLLT1 fusion in one case  and MLL-MLLT3 fusion in one case . The detection of these three known fusions shows the sensitivity of the paired-end RNAseq and the mapping programs used for the analysis. Under the threshold that a fusion must be detected by at least two pairs of fusion sequences and in at least two AML cases, a total of 134 fusions were identified in the 45 AML samples used in the study (GGCT-BC041636 (3 cases), R3HDM2-INHBC (3 cases), LY6G5C-ABHD16A (2 cases), CRNKL1-NAA20 (2 cases), and HSPA14-SUV39H2 (2 cases)] were detected only in normal karyotype AML. Twenty-six fusions were only detected in the abnormal karyotype AML and/or the AML without karyotype information  were confirmed as true fusion transcripts in the 45 leukemia samples, of which 30 fusions were in the 29 normal karyotype AML cases . Of the partners . Six fusCIITA-DEXI fusions were expressed in 7 out of 8 cell lines  shows that all the detected novel fusions are formed between the genes adjacent each other in the same chromosome. Complicated orientations of upstream and downstream partner genes are present, including 3′-5′ tail to head, 5′-3′ head to tail, 3′-3′ tail to tail, and 5′-5′ head to head . Considell lines . Testing samples .IL17RB-ACTR8 fusion is 4,044 bp in the reference genome sequences whereas the size of the amplified genomic fragment in AML is about 4 kb, suggesting that this fusion was generated by post-transcriptional process rather than genomic structural change; in the case of CLR-CLEC2D fusion, the size in the reference genome sequences is 31 kb but the size of the amplified genomic fragment has only 8 kb. This fusion transcript is likely caused by genomic DNA deletion between the two partner genes. The results indicate that both genomic structural change and post-transcriptional processing can contribute to the fomation of the detected fusion transcripts.Both genomic structural changes between the partner genes and post-transcriptional process can cause the formation of fusion transcripts. We used long-range PCR to amplify the mapped genomic region between the partner genes to know if there is a genomic structural change as reflected by the size difference of the amplified region comparing to the reference genome sequences. Of the 11 successfully amplified genomic DNA fragments, 6 have the sizes similar to and 5 have the sizes shorter than their corresponding regions in the reference genome sequences . For exaADSL-SGSM3, ARHGAP27-LOC201175, C11orf79-C11orf66, CS-CNPY2, ETFB-CLDND2, FLOT2-DHRS13, GALT-IL11RA, GPN3-ARPC3, MGC72080-ASNS, RAB24-MXD3, RRM2-C2orf48, SCO2-TYMP, STX16-NPEPL1, TNFAIP8L2-SCNM1, VAMP8-VAMP5, and VPS72-TMOD4, XPA-NCBP1, HARS2-ZMAT2, HSP90B1-DKFZp547P055, IL17RB-ACTR8, ANKRD23-ANKRD39) are identified to share with the fusions identified in prostate cancer but no fusions are shared with breast cancer, colorectal cancer, ovarian cancer, and lung cancer. Further classification of the remaining 113 fusions shows that 7 fusions  are present only in normal karyotype AML, 18 are present only in abnormal karyotype AML or AML without karyotype information, and 88 fusioins are shared between normal, abnormal karyotype, and no karyotype AML groups  bound at MHCII promoters and other transcriptional factors . The involved gene CTCF, INHBC and SUV39H2 are particularly interesting. CTCF encodes a zinc-finger transcriptional regulator protein with 11 zinc fingers. It regulates transcription both positively and negatively. Mutations in CTCF have been observed in breast, prostate, and Wilms' cancers INHBC encodes the beta C chain of inhibin, a member of the TGF-beta superfamily involving hormone secretion FAM65A-CTCF and R3HDM2-INHBC both removed the first exon and translational starting site of CTCF and INHBC, likely caused loss of the function of CTCF and INHBC; SUV39H2 encodes a histone-lysine N-methyltransferase HSPA14-SUV39H2 fusion removed the last 3 exons of HSPA14 and connected the remaining HSPA14 part to the first exon of SUV39H2.There are seven fusions detected only in the normal karyotype AML  are shared between AML and prostate cancer. This raises an interesting question whether certain fusions could be common genetic aberrations contributing to different types of cancer. The HARS2-ZMAT2 fusion was formed in-frame between the 3′ UTR of HARS2 and the 3rd exon of ZMAT2 in 3′-5′ tail to head, sense orientation. HARS2 (histidyl-tRNA synthetase 2) is a nuclear-encoded mitochondrial protein involving in the synthesis of histidyl-transfer RNA HARS2 cause Perrault syndrome with ovarian dysgenesis and hearing loss ZMAT2 is a matrin-type 2 zinc finger protein with unknown function. The HSP90B1-DKFZp547P055 fusion was formed between the 17th of 18 exons of HSP90B1 before its stop codon and the 2nd exon of a functionally unknown gene DKFZp547P055, fused in a 3′-3′ tail to tail orientation between the two genes. HSP90B1 is a member of HSP90 proteins located in the endoplasmic reticulum. It is involved in folding Toll-like receptors involving in regulating innate and adaptive immunity, maintaining hematopoietic stem cell development, and potentially to be used as a vaccine for cancer treatment HSP90B1-DKFZp547P055 fusion disrupted the normal structure of HSP90B1, and possibly led to loss of function of HSP90B1. IL17RB-ACTR8 fusion is formed in a 3′-3′ tail to tail opposite orientation before the stop codon of IL17RB and the 3′ UTR of ACTR8. IL17RB is involved in the activation of the NF-kappaB pathway IL17RB, suggesting that the fusion could be translated into the two original proteins. Prostate cells are derived from endoderm and hematopoietic cells are derived from mesoderm. Therefore, the shared fusions between prostate cancer and AML are unlikely attributed to the same developmental origin. It will be interesting to further study the biological basis of the shared fusion transcripts between the two very different cancer types. MHCII is generally expressed in T cells, B cells and dendritic cells involving antigen presentation CIITACIITA in normal karyotype AML could contribute to its escape of the host's immune-surveillance.Five fusions . The fusions identified from prostate cancer, breast cancer, ovarian cancer, lung cancer and colorectal cancer were obtained from the references Total RNA was extracted from each AML sample using TRIZOL reagent (Invitrogen). RNAseq library was prepared using PolyA+ mRNA isolated from each RNA sample following Illumina RNASeq protocol. Paired-end sequencing at 2×50 was performed by using Illumina GAIIx sequencer. Raw sequence data were processed by the standard GAIIx quality control program. FusionSeq 6 cells) using the RNeasy Protect Cell Mini Kit (QIAGEN).Eight cell lines of AML origin were used for validation study. Cell lines of KG-1, THP-1, MV4-11, HL-60, Kasumi-1 and Kasumi-6 were obtained from the American Type Culture Collection; Molm-13 and Molm-14 were provided by John Kersey (University of Minnesota). All cell lines were cultured in RPMI-1640, 10% fetal calf serum, 2 mM L-glutamine, 50 units/ml penicillin and 50 µg/ml streptomycin (Invitrogen). Total RNA was isolated from each cell line (5×10http://frodo.wi.mit.edu/). Strand-specific reverse transcription was performed with either antisense primer or sense primer (2 pmole), total RNA (100 ng), M-MLV Reverse Transcriptase  in a 20 µl volume at 65°C for 5 minutes, 37°C for 50 minutes, and 70°C for 15 minutes. Five µl of the resulting cDNA were used for PCR amplification in 50 µl containing sense primer (10 pmole), antisense primer (10 pmole), Taq polymerase , MgCl2 (1.5 mM) in an ABI 9700 thermal cycler under cycling conditions of 95°C 7 minutes, 38 cycles of 95°C 30 seconds, 56°C 30 seconds, and 72°C 30 seconds, and final extension at 72°C 7 minutes. PCR products were loaded on 2% agarose gels. The bands with expected size were excised from gel, purified with MinElute Gel Extraction Kit (QIAGEN), and sequenced by BigDye reagents (Life Technologies). Sequences were mapped to hg18 by Genetyx program .Strand-specific RT-PCR and Sanger sequencing were used to validate the fusion transcripts identified by sequence mapping analysis. For each fusion selected for the test, at least one AML case was used for the validation. For primer design, sequences longer than the paired-end sequences covering the fusion were generated by extending the fusion–mapped location upstream and downstream within the exon region. Sense and antisense primers were designed using Primer3 program (The same sense/antisense primer pairs used for RT-PCR were used to amplify the genomic regions by using a LongRange PCR kit (QIAGEN) with cycling conditions of 93°C 3 minutes, 35 cycles of 93°C 15 seconds, 55°C 30 seconds, and 68°C 4 minutes, and final extension at 68°C 7 minutes. The amplified products were checked on 0.8% agarose gels and the sizes were estimated by comparing with 1 kb DNA ladder (BioLabs).String 9.0 program was used to identify the interaction proteins for CIITA Figure S1RNAseq mapped fusion.(PDF)Click here for additional data file.Figure S2Genomic patterns of fusion partner genes.(PDF)Click here for additional data file.Table S1Full fusion transcript list.(XLS)Click here for additional data file.Table S2Validated fusion transcript list.(XLS)Click here for additional data file.


We investigated the role of NHE in inducing changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and alterations in ventricular function during ischemia-reperfusion in type 2 diabetic mouse hearts.A higher increase in intracellular Nadb/db (12-15 weeks old) and age-matched control db/+ mice were subjected to Langendorff perfusion and loaded with 4μM of the Ca2+ indicator fura-2. The hearts were exposed to no-flow ischemia for 15 minutes and then reperfused. [Ca2+]i was measured by monitoring fura-2 fluorescence at 500 nm (excitation wavelengths of 340 and 380 nm), while left ventricular (LV) pressure was simultaneously measured.Hearts from male type 2 diabetic db/db hearts exhibited a lower recovery of LV developed pressure than db/+ hearts during reperfusion following ischemia. Diastolic [Ca2+]i was increased to a greater level in diabetic hearts than in the control hearts during ischemia and reperfusion. Such an increase in cytoplasmic Ca2+ overload during ischemia-reperfusion in diabetic hearts was markedly reduced in the presence of the NHE inhibitor cariporide. This was accompanied by a significantly improved recovery of ventricular function on reperfusion, as shown by a lower increase in diastolic pressure and increased recovery of developed pressure.2+ overload during ischemia-reperfusion and severely impairing post-ischemic cardiac function in hearts from type 2 diabetic db/db mice.NHE plays a key role in enhancing cytoplasmic Ca We previously reported that a highly increased intracellular Na+ concentration ([Na+]i), mainly via NHE, caused enhanced sensitivity to ischemia in type 2 diabetic db/db mouse hearts i and Ca2+ transient amplitude during ischemia and reperfusion in isolated type 2 diabetic db/db mouse hearts loaded with fura-2, a fluorescent dye [2+]i and cardiac function was investigated, as well as the importance and role of NHE in [Ca2+]i changes.The purpose of the present study was to determine changes in diastolic i, and was independent of the fura-2 concentration. Background autofluorescence was subtracted to assess diastolic [Ca2+]i. We evaluated changes in diastolic [Ca2+]i by using the following equation to equalize the differences in fura-2 loading conditions in each heart.Intracellular Ca, Japan) ,9. Follopre is the pre-ischemic diastolic ratio and Apre is the amplitude of pre-ischemic ratio. Each ratio was calculated from the value of fluorescence intensity at 340 nm (autofluorescence subtracted) divided by fluorescence intensity at 380 nm (autofluorescence subtracted). Each ratio was expressed in arbitrary units. Calibration of fura-2 was not performed. Throughout the experiment, we simultaneously monitored fura-2 fluorescence and hemodynamic parameters.Where, R is the diastolic ratio, RBased on results from pilot experiments, the experimental protocol consisted of 10 minutes of control perfusion, followed by 15 minutes of no-flow ischemia and 15 minutes of reperfusion. In each group, diabetic and non-diabetic hearts either received or did not receive the NHE inhibitor cariporide  ,13,17,18Blood samples (fed dietary status) were taken from the body cavity after excision of the heart. Plasma glucose was measured using a glucose oxidase method.p < 0.05.Results are presented as the means ± SEM. The data were analyzed using either Student's t test for unpaired data or ANOVA, followed by the appropriate post hoc test, using Scheffe's test to locate differences between groups. Significance was set at db/db mice were significantly higher than those in non-diabetic db/+ littermates  compared with that in control db/+ hearts . Cariporide improved the recovery of LVDP in both groups of hearts  was smaller compared with the loaded hearts. However, the increase in autofluorescence at 340 nm was larger than that at 380 nm after induction of ischemia, though the value for each remained constant during ischemia. The average values (arbitrary units) from 4 hearts in each groups were as follow: (i) 0.12 and 0.20, at 340 and 380 nm, respectively, during control perfusion and reperfusion, (ii) 0.16 and 0.21, at 340 and 380 nm, respectively, during ischemia. Therefore, the change in diastolic level of fluorescence ratio might have been overestimated during the early phase of ischemia when autofluorescence was not subtracted. It has been previously found that autofluorescence at both 340 and 380 nm increased in parallel during ischemia, with the result that the ratio remained constant i in hearts from diabetic db/db mice have been reported by several investigators. Belke et al. noted increased Ca2+ leakage from the sarcoplasmic reticulum (SR) [2+]i cycling due to reductions in expressions of both sarcolemmal Ca2+ channels and SR Ca2+ release channels were demonstrated by Pereira et al. [2+ transient were significantly prolonged in db/db hearts -dependent manner in normal cardiomyocytes [2+ handling in diabetic db/db mouse hearts. Basal alterations in [Ca2+]i might precipitate cytoplasmic Ca2+ overload during ischemia-reperfusion, as we observed in the present db/db hearts. On the other hand, cytoplasmic Ca2+ overload in these hearts was largely prevented by the presence of the NHE inhibitor cariporide  -6, we folum (SR) , while ia et al. . In the myocytes . Howevermyocytes  generateerfusion . In thiserfusion  and ranoerfusion  have als2+]i and Ca2+ signaling in cardiomyocytes [+]i increase during ischemia and severely impaired post-ischemic cardiac function in hearts from diabetic db/db mice. In that study, NHE inhibitor cariporide reduced ischemia-induced [Na+]i increase and improved post-ischemic cardiac function in db/db hearts i i, due to increased Ca2+ leakage i ,32 and ady [2+]i -35. In v leakage . Future 2+ ([Ca2+]m) overload by one of the NHE blockers during ischemia-reperfusion have been reported [2+]m in the present study. In addition, though these effects of cariporide might be different between diabetic and non-diabetic hearts, [Ca2+]m cannot be selectively measured with the methods employed in this study. Finally, NHE inhibitors have recently been shown to affect mitochondria by blunting MPTP formation and ROS release i: Cytoplasmic Ca2+ concentration; [Na+]i: Intracellular Na+ concentration; LVDP: Left ventricular developed pressure; LVEDP: Left ventricular end diastolic pressureDM: Diabetes mellitus; NHE: NaThe authors declare that they have no competing interests.RA conceived of the study, carried out the experiments, and drafted the manuscript. SS and TN participated in the fluorescence measurement study. IT, DF, and MY conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final version of the manuscript.


Valvulogenesis and septation in the developing heart depend on the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). These cushions are invaded by a subpopulation of endocardial cells that undergo an epithelial-mesenchymal transition in response to paracrine and autocrine transforming growth factor β (TGFβ) signals. We previously demonstrated that the RNA binding protein muscleblind-like 1 (MBNL1) is expressed specifically in the cushion endocardium, and knockdown of MBNL1 in stage 14 embryonic chicken AVC explants enhances TGFβ-dependent endocardial cell invasion.In this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGFβ3 after it is no longer required to induce basal levels of invasion. TGFβ3, but not TGFβ2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGFβ3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGFβ3 production in the endocardium. More TGFβ3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGFβ3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGFβ3 secretion, and early exposure to excess TGFβ3 induces precocious invasion. MBNL1 expression precedes TGFβ3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGFβ3 signaling. The stimulatory effects of MBNL1 knockdown on invasion are lost in stage 16 AVC explants. Knockdown of MBNL1 in OFT explants similarly enhances cell invasion, but not activation. TGFβ is necessary and sufficient to mediate this effect.Taken together, these data support a model in which MBNL1 negatively regulates cell invasion in the endocardial cushions by restricting the magnitude and timing of endocardial-derived TGFβ3 production. Epithelial-mesenchymal transition (EMT) is a critical process in the formation of the valves and septa of the heart. Prior to the onset of EMT, endocardial cushions form in the atrioventricular canal (AVC) and outflow tract (OFT) regions when localized deposition of extracellular matrix separates the endocardial lining from the myocardial wall of the heart tube. In response to inductive signals from the adjacent myocardium, a subset of endocardial cells within the AVC and OFT cushions undergoes EMT and invades the cushion matrix -4. FusioEndocardial cell EMT can be separated into two distinct events: activation, in which cell polarity and cell-cell contacts are lost, and invasion, in which the transformed mesenchymal cells migrate into the matrix. In the chick, both steps are induced by members of the transforming growth factor β (TGFβ) family. TGFβ2 is required for activation, while TGFβ3 is required for invasion ,7. TGFβ2Muscleblind-like 1 (MBNL1) is a known regulator of pre-mRNA alternative splicing in the developing heart, driving fetal-to-adult transitions in alternative splicing ,12. MBNLIn this study, we investigate the relationship between MBNL1 and TGFβ3 in the AVC and OFT cushions. We provide evidence that knockdown of MBNL1 leads to precocious and elevated endocardial-derived TGFβ3 levels in AVC explants, and that early exposure to excess TGFβ3 leads to precocious and elevated levels of mesenchymal cell invasion. Likewise, TGFβ is necessary and sufficient to stimulate excess invasion in response to MBNL1 depletion in OFT explants. Taken together, these data support a model in which MBNL1 acts as a negative regulator of endocardial cushion EMT by restricting the timing and amount of autocrine TGFβ3 production in the AVC and OFT endocardium.TGFβ3 is required for AVC mesenchyme invasion ,7,9,10. One possible explanation for the continued dependence on TGFβ3 for the enhancement of invasion in MBNL1 siRNA-treated explants is that loss of MBNL1 leads to an increase in TGFβ3. To determine whether TGFβ3 levels are affected by MBNL1 knockdown, secreted TGFβ protein levels were compared in conditioned media collected from mock- and MBNL1 siRNA-treated stage 14 AVC explants by ELISA Figure A. MediumWithin the AVC, TGFβ3 is expressed in the myocardium before, during, and after EMT takes place, but is not expressed in active form in the endocardium until the onset of invasion ,8,9. MBNTo further confirm that TGFβ3 levels are elevated in endocardial cells following MBNL1 knockdown, immunofluorescence was performed on mock- and siRNA-transfected stage 14 AVC explants using an anti-TGFβ3 antibody. In control explants, TGFβ3-positive cells were detected within the endocardial monolayer Figure C. This iTGFβ3 transcripts, RNA was collected from the non-myocardial cells in mock and MBNL1 siRNA-treated stage 14 AVC explants and TGFβ3 levels were measured by real time RT-PCR  and fibrillin 2 (FBN2), two EMT markers known to be induced by TGFβ3  at 65°C for 2 hrs after which they were incubated with RNA probe in hybridization buffer diluted 3:1000 at 65°C overnight. Sense and antisense MBNL1 probes were previously described [2) at room temperature for 10 min. Slides were stained with BM Purple (Roche) at 4°C in the dark until desired strength of staining was reached. Slides were then placed in Stop Solution  at 4°C in the dark for 15 minutes before being dehydrated in a 25, 50, 75, and 100% ethanol series and xylene and finally mounted with Permount (Fisher Scientific). Sections were imaged on a Leica DM2500 microscope using QCapture Pro 6.0 software. Brightness, contrast, and color balance were adjusted using Adobe Photoshop software to match the appearance of the sections when viewed through the microscope by eye.Embryos were fixed in 4% paraformaldehyde in PBS at 4°C overnight and dehydrated the next day by sequentially washing with 25, 50, 75, and 100% methanol in PBSw (PBS with 0.1% Tween-20). To embed, embryos were incubated in the following sequence of solutions: isopropanol at room temperature for 15 min, fresh isopropanol at 68°C for 15 min, isopropanol/paraplast (1:1) at 68°C for 30 min, liquid paraplast at 68°C twice for one hour each and once for 30 min, and solidified at room temperature. Ten micron thick sagittal sections mounted on charged slides were de-waxed and rehydrated in xylene and a 25, 50, 75, and 100% ethanol series then rinsed in 2X SSPE (300 mM NaCl, 20 mM NaHescribed . The nexData are reported as the mean plus the standard error of the mean unless otherwise noted. Real-time RT-PCR data are reported as the mean flanked by the upper and lower 95% confidence intervals. Comparisons between means were performed via one-tailed t-tests assuming unequal variances. Comparisons between proportions of explants with significant invasion (Figure MBNL1: Muscleblind-like 1; TGFβ: Transforming growth factor β; AVC: Atrioventricular canal; OFT: Outflow tract; EMT: Epithelial-mesenchymal transition; H&H: Hamburger and Hamilton.The authors declare that they have no competing interests.in situ hybridization experiments and helped with some of the explant experiments. NAV performed some of the explant experiments. ANL performed the ELISA experiments, helped with explant experiments, participated in the design and coordination of the entire study, and prepared the draft manuscript. All authors helped analyze experimental results and edit the manuscript. All authors read and approved the final manuscript.KEL carried out the majority of explant experiments. YB-H performed immunofluorescence and real-time RT-PCR experiments, and helped with statistical analyses. SJS performed