Urticaria is a common condition that occurs in both children and adults. Most cases have no specific allergic trigger and the aetiology of urticaria remains idiopathic and occasionally spontaneous in nature. Inappropriate advice such as avoidance of foods  is common place in certain sections of India mostly by nonspecialists that should not be routinely recommended. It is important to look for physical urticarias such as pressure urticaria in chronic cases, which may be present either alone or in combination with other causes. Autoimmune causes for chronic urticaria have been found to play an important role in a significant proportion of patients. Long-acting nonsedating antihistamines at higher than the standard doses is safe and effective. Quality of life is affected adversely in patients with chronic symptomatic urticaria and some may require multidisciplinary management.  Urticaria is a common condition and the chronic form usually has no allergic trigger. Longacting non-sedating antihistamines at higher than the standard doses is safe and effective.Urticaria is characterized by itchy, red, raised , and flared skin reactions that last usually for a few hours . It is classified as chronic urticaria (CU) if it lasts for more than 6 weeks. The chronic spontaneous form of urticaria does not need any stimulus and sometimes it is also referred to as chronic idiopathic urticaria (CIU) –6. It isbefore the drug was started [The wheal has a central swelling surrounded by a reflex erythema that is itchy while the angioedema is associated with pronounced swelling of the lower dermis and subcutaneous tissue with the occasional involvement of mucous membranes  in some patients. Acute urticaria appears more commonly in children and young adults of which common causes are infections, food, drugs , and insect stings. It is important to take a detailed clinical history to identify whether the urticaria is chronic (or acute or chronic), as occasionally a patient may be wrongly labeled as drug allergic when it may be that the urticaria was present  started –3. ThereThis paper aims to discuss existing guidelines of urticaria in the Indian context, with an attempt to demystify some of the myths surrounding this condition based on our collective experience and extensive publications in this field. This paper is, therefore, applicable or relevant to physicians working in India or South East Asia where nonspecialists deal with the majority of cases of urticaria, and higher specialist training in the field of Allergology is yet to begin. This article does not aim to review the urticarias but to discuss the current level of understanding of the patients and treatment options (feasible and otherwise) to the physicians.The EAACI/GA²LEN/EDF/WAO consensus guideline for the diagnosis and management of urticaria was published in 2009. These were based on expert recommendations from the Third International Consensus Meeting on Urticaria (Urticaria 2008), joint initiative of the EAACI Dermatology Section, Global Allergy and Asthma European Network (GA²LEN), European Dermatology Forum (EDF), and World Allergy Organization , 2. SincThe worldwide incidence is 0.1%–3% of the population with women affected twice more likely than men. It is estimated that about 1 in 5 people will have urticaria once in their lifetime and this seems to be the case across all age groups. Up to 1% of the population suffers from chronic urticaria (CU) and all age groups appear to be affected, although the peak incidence is between 20 and 40 years of age. In most cases, the disease lasts between 1 or 5 years, but the duration can be longer for those with severe urticaria, those with concurrent angioedema, those with the physical component, and those with a positive autologous serum skin test.εR1α) which is believed to be the pathophysiological basis of autoimmune urticaria, the role of antithyroid antibodies on persistent cutaneous mast cell and basophil activation remains unproven [Although up to half of the patients with CIU have an IgG autoantibody directed against the alpha subunit of the high-affinity IgE receptor . A small cohort of paediatric patients with CU underwent skin testing to foods that were being avoided based on ELISA allergy results at one centre . None of the 30 children with CU (mean (±SD) age was 10.9 (±4.2) years, 13 males and 17 females) demonstrated skin test reactivity to milk, egg white, egg yolk, prawn, brinjal, and spinach which were the foods on the exclusion list. All patients were able to resume a normal diet on high dose antihistamines that controlled the urticaria. In selected patients with supportive histories, presence of IgE to specific foods or sensitivity (non-IgE mediated reactions) to certain dyes or coloring agents in food  could have a relevance to their chronic urticaria symptoms, but careful elimination and reintroduction are needed to establish the same . Myth 3. Patients with urticaria should undergo testing to exclude specific allergies.Reality. Whilst the concurrent presence of house dust mite  allergy or other aeroallergen sensitivities can be found in some patients [patients , 29–31, Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis), cockroach, pollens, moulds, and animal dander in all patients. Positive control was histamine (10 mg/mL) and positive SPT was defined as >3 mm than the negative control . In view of this perception that allergy testing is mandatory, another observational study at one centre on 43 consecutive patients with chronic urticaria  with skin prick testing to aeroallergens was carried out . Skin prick tests (SPT) were done after a 7-day antihistamine free period to house dust mites . Fisher's exact test was used to see the relationship between CIU, mite reactivity status, and with/without respiratory symptoms .t-test 0.2849). Among mite positive patients with CIU, there was a slight female predominance  that was statistically nonsignificant (P = 0.3586). However, 16 CIU patients with respiratory symptoms had dust mite reactivity as compared to 3 CIU patients with respiratory symptoms but without mite reactivity . The mean (±SD) age was 33.28 (±14.97) years that included 23 males and 20 females. Range of duration of the symptoms of CIU was 6 months to 13 years. SPT demonstrated immediate reactivity to dust mite in 24 patients (55.8%), cockroach 6 (14%), pollens 8 (18.5%), moulds 5 (11.6%), and dander 0 (0%). Five patients were polysensitized . The mean (±SD) age of patients with CIU and mite allergy was 31.1 (±14.7) years compared to 36.1 (±15.2) years in CIU patients without mite allergy  does not mention pregnancy as a contraindication to its use, other than to use it if the benefit outweighs the potential risks. Cetirizine, loratidine, and hydroxyzine have been shown to be safe in pregnancy with no difference in spontaneous or therapeutic abortions, birth weight, mode of delivery, gestational age, and rate of live births, neonatal distress, and major fetal congenital malformations , 35. It is now well recognized that patients with CU have a poor quality of life (QoL)  and psychiatric comorbidity  made this worse even in those without a formal psychiatric diagnosis . In anotStudies on fexofenadine-treated patients (180 mg) have shown significantly greater improvements in mean dermatology life quality index (DLQI) total score than those treated with placebo. These were not only seen in areas such as symptoms and feelings, activities of daily living including less impairment while working, leisure, and personal relationships but also greater improvement in Urticaria Activity Score  when compared with placebo. Indeed it is interesting that while this disease itself causes distress, chronic urticaria is also recognized as a stress-vulnerable disease in which psychological stressors can trigger or increase itching. It is suggested that effective management processes should take into account the psychological factors in some of the patients and the treatment regimen should be tailored to the individual patient's needs and circumstances , 39, 42.Helicobacter pylori induced gastritis and urticaria, and several reports of long-lasting remission of urticaria can be seen in patients after eradication therapy [The consensus guidelines have adopted the management of urticaria into 1) avoidance measures and (2) pharmacotherapy nonspecific and specific. The avoidance approach outlines elimination or treatment of eliciting stimulus or cause  that is not possible in all cases   avoidanc. In line therapy –47. The second approach is lowering or inhibiting mast cell mediator release and the most commonly used drugs (nonspecific approach) that inhibit mast cell release are corticosteroids. Continuous or prolonged use with corticosteroids to treat urticaria is not recommended as the risks and long-term side effects outweigh the benefits. Specific treatment approaches involve the use of nonsedating long-acting antihistamine (anti-H1) drugs such as cetirizine, levocetirizine, loratadine, desloratadine, and fexofenadine that provide both antiallergic and anti-inflammatory effects, such as inhibition of cytokines release from basophiles and mast cells as well as reduction of chemotactic activity of eosinophils. Doxepin, a tricyclic antidepressant, is the only agent that blocks both H1- and H2-receptors, and can be useful in the selected patients who experience significant psychosomatic symptoms of depression and anxiety due to the urticaria. Montelukast is an orally active leukotriene receptor antagonist (LTRA) licensed the maintenance treatment of asthma and to relieve symptoms of seasonal allergies. Montelukast binds to and blocks the action of leukotriene D4 (LTD4) on the cysteinyl-leukotriene receptor CysLT1 in the lungs, with almost no interaction with other antiallergy drugs. This reduces the bronchoconstrictive and inflammatory effects of LTD4 in the airways. Other LTs such as LTC4, LTD4, and LTE4 have important roles in the pathophysiologic mechanisms of allergic inflammation after binding to activating receptors, cystenyl-LT1 (CysLT1) receptor and Cys-LT2 receptor. Hence, LTRAs such as montelukast 10 mg once daily or zafirleukast 20 mg twice daily has been employed either as monotherapy or in combination with H1-receptor and/or H2-receptor antagonists, to treat different forms of CU, including cold urticaria, urticaria related to food additives, chronic autoimmune urticaria, steroid-dependent urticaria, and delayed-pressure urticaria, and CIU and dermographism with varying results –54. Our P = 0.03), and CU index >10 (P = 0.05) predict a favorable response to cyclosporin [Other treatment options that have significant activity on mediator release on basophils include the calcineurin inhibitor cyclosporin A –60, and The most specific and promising therapy for the future appears to be anti-IgE therapy, Omalizumab  –66. A ty1.Chronic urticaria is a relatively common condition in India and most cases have no specific allergic trigger and remain idiopathic.2.Autoimmune causes have been found to be associated with up to 30-40% cases.3.It is important to look for physical urticarias such as pressure urticaria in chronic cases. 4.Avoidance of foods without appropriate testing for food allergy should not be routinely recommended. 5.Long-acting nonsedating antihistamines at even higher than standard doses if necessary are safe and effective. 6.Quality of life is affected adversely in many patients with chronic urticaria.7.Psychological stressors can play an important role in this disease and require special attention.


SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1.An increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2–3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective.We utilized Ca3+-resistant, while the other one is regulated by the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Unlike N- and RCC-ECFCs, the InsP3-dependent SOCE does not drive PMF-ECFC proliferation.Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd We have recently showed that patients with PMF have an increased frequency of circulating CD34+/CD133+/VEGFR2+ cells as compared to the patients with Ph-neg chronic MPNs  and healthy subjects +/VEGFR2+/CD133+ cells are mainly representative of hematopoietic progenitor cells rather than EPCs bona fide EPCs, since they derive from the myeloid lineage, share endothelial and hematopoietic markers, and harbor the JAK2V617F mutation in vivo via the paracrine release of growth factors and cytokines, favouring the recruitment of endothelial cells required for vessel repair and/or endothelial homeostasis. More recently, we have demonstrated that patients with PMF present with an elevated count in the number of circulating endothelial colony forming cells (ECFCs) in vivoJAK2V617F mutation in their hematopoietic cells, do not harbour the mutation JAK2V617F mutated PV patients JAK2V617F positive ECFCs were detectable in patients with Ph-neg chronic MPNs suffering from thrombotic complications + HPCs and CFU-ECs, might be directly involved in the enhanced neovascularisation of both fibrotic BM in vitro tubulogenic activities Besides the increase in circulating CD342+ signalling in ECFC activation 2+ entry (SOCE), the most important Ca2+ entry pathway in mature endothelium 2+-sensitive transcription factor, nuclear factor-κB (NF-κB) 2+ concentration within the lumen of the endoplasmic reticulum (ER), the most abundant intracellular Ca2+ pool 2+-sensitive EF-hand motifs within the luminal NH2-tail: following InsP3-dependent Ca2+ release, Ca2+ dissociates from the canonical EF-hand domain (cEF), thereby stimulating Stim1 to oligomerize and translocate towards ER-plasma membrane junctions, termed puncta. Herein, Stim1 tethers and gates two Ca2+-permeable channels, namely Canonical Transient Receptor Potential Channel 1 (TRPC1) and Orai1, which mediate the pro-angiogenic inflow of Ca2+2+ concentration in non-excitable cells, including human umbilical vein endothelial cells 2+ signalling machinery in ECFCs is extremely plastic and rearranges in response to the environmental conditions. For instance, the amount of Ca2+ stored within the ER is significantly lower in circulating ECFCs harvested from patients suffering from renal cell carcinoma (RCC-ECFCs) relative to their healthy counterparts (N-ECFCs), whereas all the three known InsP3 receptor (InsP3R1–3) subtypes are dramatically down-regulated 2+-ATPase (SERCA) or the physiological production of InsP3, in both N-ECFCs 2+ machinery encountered in ECFCs is provided by umbilical cord derived-cells (UCB-ECFCs): these cells lack InsP3R1 and express TRPC3, a diacylglycerol (DAG)-gated Ca2+-permeable channel which is absent in both N- and RCC-ECFCs 2+ toolkit expressed by human ECFCs is sensitive to both local (e.g. tumor microenvironment) and systemic  influences 3-dependent SOCE controls ECFC proliferation in all the ECFC populations hitherto analyzed 2+ toolkit, it is relevant to assess the involvement of SOCE in cell proliferation in proliferative diseases, as cancer cells may divide even in the absence of Ca2+ entry Recent studies from our group have disclosed the key role served by Ca2+ inflow in ECFCs isolated from peripheral blood of patients affected by PMF (PMF-ECFCs). This was done by exploiting Ca2+ imaging, real-time reverse transcriptase polymerase chain reaction (qRT-PCR), western blot analysis, and functional assays. Our results indicate that PMF-ECFCs undergo a dramatic remodelling of the Ca2+ machinery, which renders them extremely different from any other ECFC subtype so far investigated. The architecture of Ca2+ signalling should, therefore, not be given for granted, but carefully investigated under each pathological condition.The present investigation was undertaken with the aim to analyze the remodelling, if any, in store-dependent Ca2+ levels measured in PMF-ECFCs and ECFCs provided by healthy donors (N-ECFCs) were evaluated upon digital subtraction of the fluorescence background and were not statistically different (p<0.05), the average values of the Fura-2 ratio being 1.463±0.015, n = 353, and 1.470±0.022, n = 233, respectively. Intracellular Ca2+ release and SOCE activation were monitored by first exposing the cells to “Ca2+ add-back” protocol 2+ content in the absence of extracellular Ca2+ (0Ca2+), followed by repletion of the bathing solution with calcium. The height of the transient resulting from Ca2+ mobilization reflects the amount of Ca2+ stored within the intracellular reservoir, whereas the magnitude of the Ca2+ signal induced by Ca2+ restitution depends on the extent of SOCE activation. Both N- and PMF-ECFCs were challenged with agents able to trigger either pharmacological or physiological depletion of their Ca2+ pool. Cyclopiazonic acid (CPA) is a widely employed SERCA inhibitor, which prevents the pump from counterbalancing the passive Ca2+ leak from the stores to the cytosol, thereby leading to a massive drop in the ER Ca2+ content which signals the Stim1-mediated gating of store-operated Ca2+ channels on the plasma membrane. The extent of ER Ca2+ emptying in response to CPA (10 µM) was significantly (p<0.05) higher in PMF-ECFCs as compared to their control counterparts (2Y receptors to trigger InsP3 synthesis and subsequent InsP3-dependent Ca2+ mobilization 2+ release was significantly (p<0.05) lower in PMF-ECFCs relative to N-ECFCs . Overall, these findings suggest that the Ca2+ toolkit is remodelled in PMF-ECFCs as relative to control cells. Moreover, the changes in sub-cellular Ca2+ dynamics are different as compared to ECFCs isolated from patients suffering from solid cancer, such as RCC, which exhibit a coherent decrease in CPA- and ATP-induced Ca2+ release The resting Caterparts . Likewisterparts . The cel N-ECFCs , while S N-ECFCs . The agoM) in these cells as compared to their control counterparts. A more hyperpolarized VM would enhance the electrochemical gradient driving Ca2+ entry into the cytosol, thereby resulting in a larger increase in [Ca2+]iM in ECFCs is mainly set by K+ conductance +) to clamp both types of cells at the same VM of about 0 mV, as indicated by the Nernst equation for K+, EK = (−RT/F)*ln([K+]o/[K+]i). We have previously shown that such a treatment does not affect SOCE in either N- and RCC-ECFCs 2+ entry induced by either CPA (10 µM)   or ATP ( (10 µM)  was not 3 signalling pathway in the physiological activation of SOCE, PMF-ECFCs were first pre-incubated for 10 min in the presence of U73122 (10 µM), a widely employed PLC inhibitor 2+ release nor SOCE were activated by ATP (100 µM) in the presence of U73122 in 98 out of 98 cells. Conversely, both phases of the Ca2+ response to ATP occurred in 76 untreated cells , which argues against the reported inhibition of SERCA in other cell types 3Rs with 2-aminoethoxydiphenyl borate , prevented both ATP-induced Ca2+ release and ATP-induced SOCE , and Gd3+ (10 µM) abrogate SOCE in both N-ECFCs 2+ mobilization in both cases  blocked both CPA- and ATP-evoked Ca2+ inflow without impairing intracellular Ca2+ release  significantly (p<0.05) reduced ATP-induced SOCE . Similar to BTP-2 and La3+, Gd3+ did not influence the extent of ER Ca2+ pool depletion in the presence of either CPA  produced only a modest decrease in the extent of cell proliferation (3+ (10 µM) did not significantly affect PMF-ECFC growth  and donor origin  2+ machinery endowed to ECFCs is not sculpted in the stone and that each ECFC may create its own Ca2+ fingerprint, which could be exploited for both prognostic and therapeutic purposes.Neoplastic transformation is accompanied by a dramatic remodelling in the Ca2+ influx may be conducted by DAG-gated non-selective cation channels, such as TRPC3 and TRPC6 3-dependent depletion of ER Ca2+ reservoir, is up-regulated in RCC-ECFCs as compared to N-ECFCs due to the over-expression of Stim1, Orai1 and TRPC1 3: store-dependent Ca2+ inflow is inhibited by BTP-2, 10 µM La3+ and 10 µM Gd3+ in both N- and RCC-ECFCs 2+ machinery are altered in different types of cancer 2+ entry 2+ inflow Albeit pro-angiogenic Ca2+ add-back” protocol, we found that SOCE is present and up-regulated in PMF-ECFCs in comparison to N-ECFCs. More specifically, SOCE may be triggered by both CPA and ATP, which stimulates purinergic P2Y receptors to activate PLCβ and produce InsP3 to release luminally stored Ca2+. Similar to N-ECFCs 3Rs with 2-APB. U73122 has been found to inhibit SERCA activity and consequently elevate [Ca2+]i in guinea pig colonic myocytes 2+ levels in PMF-ECFCs. Moreover, its inactive structural analogue, U73343, did not impair ATP-elicited SOCE. Therefore, U73122 is unlikely to suppress SOCE by interfering with signalling pathways other than PLCβ/InsP3. 2-APB, in turn, may exert off-target effects by blocking Orai1-mediated Ca2+ entry and stimulating TRPV1–3 channels 2+ response to ATP in the absence of extracellular Ca2+, when only InsP3-dependent Ca2+ mobilization may be affected by the drug. InsP3-elicited store depletion was sufficient to activate SOCE, since Ca2+ entry occurred despite removal of the ligand from the perfusate before Ca2+ re-addition. These initial results, i.e. the higher amplitude of SOCE and its tight dependence on InsP3-induced Ca2+ release, did not differ from those obtained on RCC-ECFCs 2+ peak measured on Ca2+ restoration to PMF-ECFCs exposed to either CPA or ATP was not affected by high-K+ in the extracellular solution. Under these conditions, VM is clamped to 0 mV in all cell types, thereby ruling out the possibility that a negative shift in the resting potential augments the driving force sustaining Ca2+ influx in the latter.By using the “Ca2+ inflow revealed a profound difference in the underlying mechanism(s) as compared to both N- and RCC-ECFCs. First, CPA-induced Ca2+ entry was inhibited by BTP-2 and 10 µM La3+, whereas it was insensitive to 10 µM Gd3+. Conversely, ATP-induced Ca2+ influx was suppressed by BTP-2, 10 µM La3+ and 10 µM Gd3+. These unexpected findings support the hypothesis that, unlike N- and RCC-ECFCs, two distinct signalling pathways are responsible for SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is gated upon the emptying of the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Therefore, the two distinct SOCE mechanisms expressed in PMF-ECFCs may be distinguished based on their differential sensitivity to Gd3+. Second, Stim1, Orai1, and TRPC1 are not the only SOCE-related proteins to undergo a significant up-regulation in PMF-ECFCs. Immunoblot analysis disclosed an increased expression of Orai3 and TRPC4, whose amounts remain unaltered in RCC-ECFCs 3+ modestly inhibit PMF-ECFC proliferation, while Gd3+ does not exert any significant effect on this process. As explained in more detail below, this finding suggests that cell replication evades from the control of the InsP3-dependent SOCE pathway, which is inhibited by BTP-2, La3+ and Gd3+, and switches under the control of the Gd3+-resistant route, which is only sensitive to BTP-2 and La3+.The subsequent pharmacological and molecular characterization of store-dependent Ca2+ upon SERCA inhibition, reside in vicinity of the Gd3+-resistant pathway; on the contrary, InsP3Rs are spatially positioned nearby the Gd3+-inhibitable channel. The structural heterogeneity in the compartmentalization of ER Ca2+ handling/transporting proteins is further supported by the observation that the global Ca2+ content is enhanced in PMF-ECFCs (as indicated by the higher Ca2+ response to CPA in 0Ca2+), while ATP-induced intracellular Ca2+ release is significantly reduced .The expression of multiple signalling pathways for SOCE in the same cell type is not unusual, whereas it has reported in LNCaP human prostate cancer epithelial cells 2+ entry routes activated by calcium store depletion is not easy to reconcile with the pattern of expression of Stim1, Orai, and TRPC isoforms in PMF-ECFCs. These cells display higher levels of Stim1, Orai1 and TRPC1 proteins, which are the sole mediators of SOCE in both N- and RCC-ECFCs 3+ and 10 µM Gd3+ block both CPA- and ATP-induced SOCE in these cell types 2+ entry following InsP3-dependent Ca2+ release also in PMF-ECFCs. The molecular makeup of the pathway gating Ca2+ inflow after passive emptying of ER Ca2+ content is far less clear. In addition to Stim1, Orai1, and TRPC1, and unlike RCC-ECFCs 3+-resistant pathway? Orai3-mediated Ca2+ inflow is abrogated by both La3+ and Gd3+ when administrated at 1–10 µM 3+ abrogates TRPC4-mediated SOCE in vascular endothelium 3+-resistant SOCE in PMF-ECFCs. A recent study described a novel mode of La3+-resistant Ca2+ influx which is synergistically activated by B-cell-receptor stimulation and Ca2+ store depletion in DT-40 cells 2+-permeable channels featured by peculiar pharmacological properties 3+-resistant form of SOCE: this is due to the assembly with naïve TRPC channels, including TRPC1 and TRPC4, which occurs under Stim1 control 3+-resistant SOCE in PMF-ECFCs.The pharmacological profile of the two Ca2+ inflow in tumor growth, angiogenesis and metastatization 2+ toolkit in ECFCs under pathological conditions. A global increase in protein synthesis may occur despite the fact that the transcription rate of the underlying genes is identical in both N- and PMF-ECFCs. Changes in the translational machinery drive the overproduction of oncogenic proteins and the underproduction of tumor suppressor genes in several types of cancer 2+ machinery under pathological conditions is further corroborated by the finding that the higher amplitude of SOCE in the human glioblastoma cell line U251 is not mirrored by the up-regulation of Orai1 and Stim1 transcripts Mutations in the genes encoding for Stim1 and Orai1 have long been associated to the development of rare, but lethal, inherited immunodeficiency disorders, such as severe combined immunodeficiency (SCID), where the abrogation of SOCE compromises immune system functions 2+ inflow drives cell cycle progression and DNA synthesis in both non-excitable cells and cancer cells 3+, while it is only slightly affected by BTP-2 and La3+. The pharmacological profile of PMF-ECFC proliferation rules out the involvement of the Gd3+-sensitive InsP3-dependent pathway, while it hints at a modest participation of the additional route activated by passive store depletion. The InsP3-dependent SOCE drives DNA synthesis and cell cycle progression in both N- and RCC-ECFCs 3-dependent pathway to promote cell division in both N- and UCB-ECFCs 3+-resistant pathway suggests that these cells reduce their need for Ca2+ inflow to replicate, as first described in neoplastic cells 2+ influx 2+ inflow accounts for only 20% of cell growth in GBM 2+2+ signals are replaced upstream of the signalling network driving PMF-ECFC proliferation. It, thus, appears that remodelling of the Ca2+ toolkit does not impact on the enhanced neovascularisation of both fibrotic BM + HPCs and CFU-ECs. Consistent with the scarce influence of SOCE on PMF-ECFC proliferation, N- and PMF-ECFCs display no significant difference in both their replication rates and tubulogenic activities. However, the up-regulation of SOCE and the expression of two distinct store-dependent Ca2+ channels in these cells provide the first molecular evidence that peripheral ECFCs isolated from PMF patients differ from their peripheral counterparts. This feature is particularly intriguing when considering that previous work failed to report any difference at molecular level between these two cell types The functional role of SOCE was assessed by evaluating its impact on PMF-ECFC proliferation. SOCE is the ubiquitous mechanism whereby Ca2+ inflow is augmented as compared to N-ECFCs in response to both pharmacological and physiological depletion of the intracellular Ca2+ reservoir. The higher magnitude of SOCE in RCC-ECFCs is associated to the over-expression of its molecular underpinnings, namely Stim1, Orai1, and TRPC1. The scenario becomes far more complex in PMF-ECFCs, where TRPC4, Orai3 and, perhaps, Orai2 proteins are up-regulated as well. A further difference is represented by the pharmacological profile of SOCE. Unlike N- and RCC-ECFCs, where CPA- and InsP3-dependent Ca2+ mobilization activate the same membrane pathway sensitive to BTP-2, La3+ and Gd3+, CPA-induced SOCE is unaffected by Gd3+ in PMF-ECFCs. It is, therefore, conceivable that at least two distinct store-operated channels are present in these cells. Finally, the pharmacological blockade of the InsP3-dependent SOCE does not prevent PMF-ECFC replication. These results start to shed novel light on the mechanisms regulating intracellular Ca2+ homeostasis in ECFCs. It appears that the Ca2+ toolkit is not identical among the different cell populations isolated from distinct blood samples, but is exquisitely sensitive to the extracellular microenvironment. In this view, UCB-ECFCs selectively express TRPC3, which is absent in all the other ECFC types The present investigation demonstrates for the first time that the SOCE machinery is rearranged in endothelial colony forming cells isolated from patients with primary myelofibrosis. Similar to RCC-ECFCs, the amplitude of store-dependent Ca+ CD45− fraction of circulating mononuclear cells, exhibit robust proliferative potential and form capillary-like structures in vitro6 MNCs (range 18–66) was plated on collagen-coated culture dishes (BD Biosciences) in the presence of the endothelial cell growth medium EGM-2 MV Bullet Kit (Lonza) containing endothelial basal medium (EBM-2), 5% foetal bovine serum, recombinant human (rh) EGF, rhVEGF, rhFGF-B, rhIGF-1, ascorbic acid and heparin, and maintained at 37°C in 5% CO2 and humidified atmosphere. Non-adherent cells were discarded after 2 days and thereafter, medium was changed three times a week. The outgrowth of ECs from adherent MNCs was characterized by the formation of a cluster of cobblestone-shaped cells. That ECFC-derived colonies belonged to endothelial lineage was confirmed as described in in vitro Matrigel assay.Blood samples (40 ml) were obtained from seven healthy human volunteers and seven patients affected by primary myelofibrosis at time of diagnosis  with the purpose to avoid  any potential bias due to cell differentiation. However, in order to make sure that the phenotype of the cells did not change throughout the experiments, in preliminary experiments we tested the immunophenotype of ECFCs at different passages and we found no differences 2, 1 MgCl2, 10 Glucose, 10 Hepes. In Ca2+-free solution (0Ca2+), Ca2+ was substituted with 2 mM NaCl, and 0.5 mM EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The high-K+ extracellular solution was prepared by replacing 100 mM NaCl with an equimolar amount of KCl. The solution was then titrated to pH 7.4 with KOH. Media with reduced osmolality (290 mOsm) was prepared by reducing extracellular NaCl to 126 mM. Control experiments were performed by using isotonic medium prepared by substituting 48 mM NaCl with 48 mM sucrose. Increased osmolarity (430 mOsm) was achieved by adding 92 mM sucrose to PSS. The osmolality of PSS as measured with an osmometer  was 338 mmol/kg.Physiological salt solution (PSS) had the following composition (in mM): 150 NaCl, 6 KCl, 1.5 CaCl2+]i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed “ratio”). An increase in [Ca2+]i causes an increase in the ratio ECFCs were loaded with 4 µM fura-2 acetoxymethyl ester  in PSS for 1 hour at room temperature. After washing in PSS, the coverslip was fixed to the bottom of a Petri dish and the cells observed by an upright epifluorescence Axiolab microscope , usually equipped with a Zeiss ×40 Achroplan objective . ECFCs were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. A first neutral density filter  reduced the overall intensity of the excitation light and a second neutral density filter  was coupled to the 380 nm filter to approach the intensity of the 340 nm light. A round diaphragm was used to increase the contrast. The excitation filters were mounted on a filter wheel . Custom software, working in the LINUX environment, was used to drive the camera  and the filter wheel, and to measure and plot on-line the fluorescence from 10 up to100 rectangular “regions of interest” (ROI). Each ROI was identified by a number. Since cell borders were not clearly identifiable, a ROI may not include the whole cell or may include part of an adjacent cell. Adjacent ROIs never superimposed. phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2) was purchased from Calbiochem . All other chemicals were obtained from Sigma Chemical Co. .Table S1Phenotypic characterization of passaged endothelial colony forming cells. Endothelial colony forming cells (ECFCs) were phenotipically characterized at the beginning and during the study. In keeping with previously published data (DOC)Click here for additional data file.


TRPC3 was recently demonstrated as a player in pathogenesis of cardiac hypertrophy, while the potential pro-arrhythmogenic role of TRPC3 is incompletely understood. Using a TRPC3 transgenic overexpression mouse model, we examined the involvement of TRPC3 in cardiac actions of angiotensin II (AngII).2+ transients. Furthermore, L-type Ca2+ channel current, action potentials and non-selective ion currents were analyzed electrophysiologically.AngII effects on cardiac functions were characterized in Langendorff perfused hearts. Single ventricular myocytes were isolated and field-stimulated to measure effects on sarcomere shortening and Ca2+ transient was significantly higher  in myocytes from TRPC3(+/−) mice ([Ca2+] F/F0 0.354 ± 0.024) as compared to WT ([Ca2+] F/F0 0.262 ± 0.021). Also, the time constant (τ) of Ca2+ decline was different between WT  and TRPC3(+/−) . Sarcomere shortening showed no significant difference between the two groups  whereas the SR-loading estimated from rapid application of caffeine (20 mM) revealed an increased SR loading of up to 40% in TRPC3(+/−) myocytes as compared to WT . The time constant of Ca2+ decline during caffeine challenge was also significantly changed (p < 0.05) in TRPC3(+/−) myocytes  as compared to WT cells . Importantly, AngII (100 nM) induced a rise in diastolic Ca2+ levels, which was accompanied by irregular contractions in TRPC3(+/−) but not in WT myocytes. The rise in the diastolic Ca2+ levels was significantly suppressed by Pyr3 , SEA 0400  and KN-93 . Electrophysiological characterization of L-type voltage-gated Ca2+ currents and action potentials revealed that baseline electrophysiological parameters were not affected by TRPC3 overexpression, while AngII induced a transient prolongation of action potential duration only in TRPC3(+/−) myocytes. This TRPC3-dependent response was associated with a higher incidence of delayed afterdepolarizations.AngII (100 nM) reduced left ventricular pressure (LVP) within 2 min to 64%, +dP/dt to 50% and −dP/dt to 55% of control in TRPC3(+/−) hearts, while even producing a positive inotropic effect in wild-type (WT) hearts. Simultaneously, ECG recordings demonstrated AngII-induced episodes of acute arrhythmogenicity in all TRPC3(+/−) hearts (n = 6), whereas rhythm of WT hearts (n = 6) remained unaffected. The AngII-induced impairment of cardiac functions in TRPC3(+/−) hearts was partially reversed by Pyr3 (30 µM). The amplitude of CaOur results demonstrate that AngII modulation of cardiac functions is strictly dependent on TRPC3 expression and suggest a key role of TRPC channels in AngII-mediated arrhythmogenicity.


HIV-1, via the transcription factor Tat, for instance, potentiates Ca2+ influx through dihydropyridine-sensitive voltage-gated L-type Ca2+ -alpha cytokine, an important compound for HIV-1 replication and pathogennesis  and NMDAr channel antagonists . In contrast, Tat, similar to verapamil, inhibits cytotoxic release of serine esterases by blocking the phenylalkylamine-binding site of voltage-gated Ca2+ channels  protein HBx activates caspase-dependent cleavage of host Ca2+-modulating factors exist for bacteria, fungi, and protozoa as well  mycolactone and lipoarabinomannan (Rojas et al., Repurposed medications which target pathogen capacities to alter host Ca


Francisella tularensis. Because of concerns that tularemia could be used as a bioterrorism agent, vaccines and therapeutics are urgently needed. Animal models of pneumonic tularemia with a pathophysiology similar to the human disease are needed to evaluate the efficacy of these potential medical countermeasures.Pneumonic tularemia is caused by inhalation of the gram negative bacterium, Francisella tularensis strain SCHU S4 developed a rapidly progressive fatal pneumonic disease. Clinical signs became evident on the third day after exposure with development of a fever (>40.5°C) and a sharp decline in both food and water intake. Blood samples collected on day 4 found lymphopenia and a decrease in platelet counts coupled with elevations in erythrocyte sedimentation rate, alanine aminotransferase, cholesterol, granulocytes and monocytes. Radiographs demonstrated the development of pneumonia and abnormalities of intestinal gas consistent with ileus. On average, rabbits were moribund 5.1 days after exposure; no rabbits survived exposure at any dose . Gross evaluation of tissues taken at necropsy showed evidence of pathology in the lungs, spleen, liver, kidney and intestines. Bacterial counts confirmed bacterial dissemination from the lungs to the liver and spleen.Rabbits exposed to aerosols containing F. tularensis.The pathophysiology of pneumonic tularemia in rabbits resembles what has been reported for humans. Rabbits therefore are a relevant model of the human disease caused by type A strains of  Francisella tularensis, a gram negative coccobacillus that can cause disease in a number of animals including amoeba, is the causative agent of tularemia F. tularensis are divided into four subspecies; the most virulent are subspecies tularensis , followed by holarctica  mediasiatica and novicida are not thought to cause human disease. In humans, F. tularensis can cause a spectrum of disease that is largely dictated by the route of infection; the most virulent form is pneumonic tularemia, which can cause severe morbidity/mortality at even very low doses as low as 10 CFU F. tularensis was among the most common laboratory-acquired infections F. tularensis as a biological weapon F. tularensis is considered a Category A select agent by the U.S. Department of Health & Human Services holarctica subtype which is named the live vaccine strain (LVS); LVS provided partial protection of humans and nonhuman primates against aerosol challenge with a type A strain of F. tularensisholarctica subtype. Outbreaks of pneumonic tularemia involving a type A strain are fairly rare. Human clinical trials to determine efficacy sufficient to support licensure of a vaccine or therapeutic will therefore be ethically and logistically impossible. Licensure of vaccines or therapeutics for protection against F. tularensis will only be possible through the use of the FDA's Animal Rule. The Animal Rule requires the use of at least one well-characterized animal model that is relevant to the human disease and in which the mechanism of protection is understood and shown to be similar in humans F. tularensis, including strains that are attenuated in humans and nonhuman primates such as F. novicida and the live vaccine strain (LVS) F. tularensis strains at robust challenge doses. These findings suggest that different mechanisms may be involved both in the pathogenesis of F. tularensis infection in mice and the protection elicited by vaccines compared to what occurs in humans. Nonhuman primates (NHP) are a relevant model of the human disease and are protected against respiratory challenge with virulent strains of F. tularensis after vaccination with LVS. However, they are expensive and difficult to work with in numbers sufficient to generate statistical significance. Particularly for tularemia vaccines, animal models are needed that are more relevant than the mouse to screen candidate vaccines prior to studies with NHP.Although cases of tularemia are reported every year, the number of cases is typically quite small and most outbreaks involve the more ubiquitous but less virulent F. tularensis was first isolated from a rabbit. An outbreak of pneumonic tularemia in Martha's Vineyards was termed ‘lawnmower tularemia’ after it was revealed that the outbreak resulted from mowing over rabbit dens and possibly infected carcasses F. tularensis infection or the relevance to the human disease. A British study in the 1970's established that the pathology of experimental pneumonic tularemia in rabbits was similar to that reported in humans and nonhuman primates F. tularensisTularemia is also known as rabbit fever and F. tularensis strain SCHU S4 at doses ranging from 190 cfu up to 5.4×104 cfu .To develop a rabbit model of pneumonic tularemia, 20 young female NZW rabbits were exposed to aerosols of ×104 cfu . RabbitsA febrile response (designated as body temperature >40°C) was first seen in rabbits 2–3 days after exposure . On averp = 0.05) but did not rise to the level of significance (p = 0.06) between the low and high dose groups.Similarly, weight loss was first detected in rabbits regardless of dose group on day 3 . RabbitsSix rabbits used in these studies underwent more extensive monitoring to determine other parameters associated with the outcome of pneumonic tularemia. As shown in F. tularensis; all of the rabbits were bled on day 4 and then again immediately prior to being euthanized on day 5 (n = 5) or day 6 (n = 1). Bacteremia was assessed by plating on CHA plates. Surprisingly, only one of the six rabbits was positive and that was the rabbit that survived to day 6; it was only positive on day 6 and the levels were very low, 6.1×102 cfu/ml of blood (data not shown). No difference was seen by incubating the blood in 0.1% sodium deoxycholate prior to diluting and plating on CHA. White blood cell counts dropped on day 4 relative to the pre-exposure baseline and then more dramatically on day 5 postexposure (p = 0.003)  . Of the p = 0.002). The ESR average increased still further, peaking on day 5 at 60 mm. Similarly, serum cholesterol and alanine aminotransferase (ALT) levels were elevated on day 4 and even more noticeably on day 5 . Other liver parameters  evaluated did not show remarkable or consistent differences between baseline and post-exposure blood samples in all animals (data not shown).Erythrocyte sedimentation rate (ESR) was performed on blood samples as a non-specific measure of inflammation and C-reactive protein levels . Pre-expon day 5 ; these dThe progression of pneumonic tularemia in the rabbits was also assessed by x-rays and gross pathological evaluation of euthanized rabbits. Radiographs of the abdominal regions show considerable changes 4 days after infection, including abnormal gas distribution with distension of the stomach as well as of the small and large intestines  suggesti7 cfu/g for euthanized rabbits as compared to an average of 6.0×104 cfu/g for rabbits that were found dead. In spleens from three of the animals found dead no counts were obtained. In the liver the average was 2.5×105 cfu/g in euthanized rabbits compared to 2.1×103 in rabbits found dead. In the lungs, in contrast, there was considerably more overlap between concentrations from rabbits that were euthanized or found dead; the average cfu/g from the lungs were 2.5×105 for euthanized rabbits and 6.0×104 for rabbits found dead. However, because of the variability in the concentration of F. tularensis, none of the differences were statistically significant between rabbits that were euthanized or found dead for any of the organs assayed.Samples of liver, spleen and lung taken at necropsy were homogenized and dilutions plated to determine the extent of bacterial dissemination to these tissues; the results are shown in The data presented here detail the disease course and outcome of pneumonic tularemia following infection with type A strain SCHU S4 in New Zealand White rabbits, a potential model of the human disease. The value of the current experimental model is predicated on its ability to reproduce the key patho-physiological events of the human disease. Thus, our findings here for the rabbit model match well with what has been reported in human cases of pneumonic tularemia, including fever, weight loss, elevations in ALT and ESR, leukopenia, and radiographs detailing the development of bibasilar pneumonia F. tularensis was first isolated from a rabbit and tularemia is also known as ‘rabbit fever’, there have been few studies detailing experimental infection with F. tularensis in rabbits, particularly for infection via the respiratory tract. Baskerville & Hambleton examined the pathogenesis of pneumonic tularemia in New Zealand White rabbits after exposure to aerosolized F. tularensis strain SCHU S4 4–4×108 cfu; however, they did not report how the ‘calculated inhaled retention dose’ was calculated. Our doses are calculated as presented doses, which are represented as the total amount of bacteria inhaled 10 cfu higher than the challenge doses we delivered in these studies. Importantly, our study demonstrates that rabbits are susceptible to very low doses of tularemia without appreciable increases in time to death. Over the course of the study Baskerville & Hambleton noted pathological changes first in the lung, progressing into the cervical and bronchial lymph nodes, the liver, and the spleen. Similar to the results reported here, they noted elevations in body temperature on day 2, fever >40.2°C by day 3, and a reduction in food intake. A subsequent publication reported leukopenia and an increase in neutral fats 2–3 days after infection F. tularensis in rabbits constitutes a relevant model of the human disease. Further work is needed, including detailed pathogenesis and immunological studies, to elucidate the pathophysiology of tularemia in rabbits to aid in determining whether rabbits are a suitable model to meet the requirements of the Animal Rule.Although F. tularensis by the intracutaneous or aerosol routes induced at least partial protection in humans against aerosol challenge with the SCHU S4 strain of F. tularensis; humans were protected against aerosol challenge with 200–2,000 cfu but not 10-fold higher doses 5 cfu ) but no rabbits survived challenge An additional requirement of the FDA's Animal Rule for selection of animal models to use in pivotal efficacy studies is that the mechanism of amelioration/protection must be the same in the animal model as in the human. While ethical and logistical reasons impede the ability to conduct an efficacy study of medical countermeasures against pneumonic tularemia in humans, there exists data from studies conducted in the 1950's and 1960's that can be used to guide the selection of an animal model. In particular, vaccination with the live vaccine strain (LVS) of F. tularensis that are attenuated in NHP and humans and vaccination with LVS poorly protects against respiratory challenge with F. tularensisFrancisella infection in mice relative to humans or other species. Nonhuman primates have generally been considered good models of human tularemia; however, the FDA's Animal Rule has commonly been interpreted to mean that testing in more than one animal model is necessary to satisfy the FDA's requirements for pivotal efficacy studies to support vaccine licensure. There have also been recent reports detailing tularemia studies in rats The animal models most commonly used to study pneumonic tularemia are mice and nonhuman primates (NHP). Many aspects of the human disease are recapitulated in these models. There are concerns about the use of the mouse model as mice are susceptible to strains of F. tularensis, rabbits are resistant to LVS F. tularensis will be necessary to definitively establish the value of rabbits as a model for vaccine efficacy in pneumonic tularemia.The current work suggests that rabbits also constitute a valuable model as they reproduce the key pathophysiological aspects of the human disease. In addition to showing similar gross pathology to humans infected with virulent Young female New Zealand White (NZW) rabbits were obtained from Myrtle's Rabbitry  and housed in the University of Pittsburgh Regional Biocontainment Laboratory (RBL) at ABSL-3 for the duration of the studies. Prior to exposure, IPTT-300 temperature/ID chips  were implanted subcutaneously. Temperature was read using a DAS-7000 reader (BioMedic Data Systems). Food and water intake was monitored and recorded twice daily. All studies were approved by the University of Pittsburgh's Institutional Animal Care and Use Committee, protocol number 1004196. The University of Pittsburgh is an AALAC-approved facility.F. tularensis strain SCHU S4 was used in these studies. A stock was prepared by one in vitro passage of an isolate obtained from Dynport Vaccine Company . This stock was washed one time and frozen at −80°C in Brain Heart Infusion broth (BHI) containing 20% glycerol. For aerosol exposures, SCHU S4 was streaked on cysteine heart agar (CHA) containing 1% hemoglobin and incubated for 2 days at 37°C in 5% CO2. Individual colonies were picked off the plate and resuspended in PBS to an OD600 reading of 0.1; 0.5 ml of diluted SCHU S4 was added to a baffled polycarbonate vented 125 ml flask containing 24.5 ml of BHI. The flask was placed in a shaking incubator and incubated overnight at 37°C. At approximately 18 h after the culture was started, it was removed from the shaker and the OD read at 600 nm. Using a growth curve from prior studies, the concentration was adjusted in BHI to the concentration desired for aerosol exposures. During preparation and throughout the exposures, F. tularensis was kept on ice. After the exposures were completed, samples of the nebulizer and all-glass impinger (AGI) contents were diluted 10-fold in PBS, spread on quad CHA plates in duplicate and incubated at 37°C 5% CO2 for 2 days prior to counting.Virulent Aerosols were conducted inside a class III biological safety cabinet  located inside the aerobiology suite of the University of Pittsburgh Regional Biocontainment Laboratory (RBL). Animals are transported from their holding room to the aerobiology suite via mobile transfer carts (Baker). Rabbits were exposed two at a time for 10 minutes in a nose-only exposure chamber  while plethysmography data was collected in real-time using Buxco XA software  during the exposure. Aerosols were created with a vertical discharge 3-jet Collison nebulizer controlled by the AeroMP aerosol management platform . Aerosol samples were collected using an all-glass impinger (AGI) containing 10 ml of BHI and 0.001% Antifoam A  operating at 6 lpm for the duration of the exposure. At the conclusion of the aerosol, the system was purged with clean air for 5 minutes after which the rabbits were transported back to their holding room. Aerosol concentration was determined was described previously: the bacterial count measured in the AGI was multiplied by the volume of liquid in the AGI which was then divided by the product of the flow of air through the AGI and the duration of the exposure Rabbits were anesthetized first by subcutaneous injection of ketamine (80 mg/kg) and xylazine (8 mg/kg). Isoflurane (dose) was used to maintain anesthesia while the rabbit was bled via the ear vein. Approximately 2 ml of blood was collected per procedure; a portion was used for clinical chemistries, for bacteremia, and for erythrocyte sedimentation rate, and for measurement of antibody titers. Once phlebotomy was completed the rabbit was laid prone and lateral and chest digital x-rays were taken (Kodak). The xylazine was reversed by i.m. injection of dose (mg/kg) yohimibine. The rabbit was returned to its cage and monitored until it recovered from anesthesia.Rabbits were monitored twice daily after exposure. Body weight was recorded once in the morning and body temperature was recorded twice daily. A clinical scoresheet was used to record body temperature and changes in weight, behavior and appearance. Scores were recorded for four criteria at least once daily. Temperature: >34°C and <40°C = 1; >40 = 2; >41 = 3; >42 = 4; >32 & <34 = 5; <32 = euthanize promptly. Weight Loss: up to 10% = 1; 11–15% = 2; 16–20% = 3; 21–25% = 4; >25% euthanize promptly. Appearance: Normal = 1; reduced grooming = 2; ruffled fur = 3; hunched = 4; respiratory distress = 5. Behavior: Normal = 1; less peer interaction = 2; huddled = 3; moving only when prodded = 4; no response = 5. Monitoring was increased to every six hours if an animal's score was 12 or higher and if the score reached 15 or higher the rabbit was considered moribund and was euthanized promptly. Food and water intake was recorded separately. When rabbits reached a score at which they were considered to be moribund, they were promptly euthanized.Rabbits were euthanized by intravenous injection of sodium pentobarbital at 100 mg/kg. Once death was confirmed rabbits were necropsied. Pictures were taken of organs removed from the rabbits and samples of the liver, spleen and lung were taken for determining bacterial loads.Rabbit whole blood collected in EDTA was pipetted using a glass Pasteur pipet into glass Wintrobe tubes. After one hour, the degree of sedimentation was recorded in mm for each rabbit.Rabbit whole blood collected in EDTA was analyzed on a VetScan HM2  to determine the complete blood count and on a VetScan VS2 (Abaxis) to measure blood chemistry, using a profile optimized for mammalian liver function.Data was collected and organized using spreadsheets in Microsoft Excel 2007; analysis was conducted using a two-tailed Student's t-test available in the Analysis Toolpak add-in.


Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR).Rate-dependent effects on the Ca2+ channel ; and the enhancement in SERCA pump activity via phosphorylation of PLB.Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type β-adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current  and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR.Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated ICa,L as the key mechanisms underlying the aforementioned positive FFR.Our model provides mechanistic biophysically based explanations for the rate-dependence of CICR, generating useful and testable hypotheses. Although rat ventricular myocytes exhibit a positive peak FFR in the presence/absence of beta-adrenergic stimulation, they show a characteristic increase in the positive slope in FFR due to the presence of Norepinephrine or Isoproterenol. Our study identifies cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of  Ca2+ concentration[ Ca2+] myo. Sarcolemmal (SL) membrane depolarization triggers Ca2+ influx via the dihydropyridine (DHP)-sensitive L-type Ca2+ channels. Following diffusion across a small sub-membrane dyadic space, this influx activates ryanodine receptors (RyRs) controlling ryanodine-sensitive Ca2+ release channels in the junctional portion of the sarcoplasmic reticulum (jSR). Fabiato and Fabiato  myo is returned to resting levels by combination of: (a) Ca2+ buffering in the dyadic space and myoplasm; (b) sequestration of Ca2+ by sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-type calcium pumps lining the longitudinal portion of the sarcoplasmic reticulum (LSR); and (c) Ca2+ extrusion from the myoplasm by Na+/ Ca2+ exchangers and Ca2+-ATPase pumps on the sarcolemmal membrane.Cardiac muscle contraction is a result of a transient increase in intracellular  Fabiato  named thCa2+ is an extremely important and highly versatile second messenger in cardiac cells, which plays a crucial role not only in excitation-contraction (E-C) coupling but also in excitation-transcription coupling  myo is small due to a low SR load. We utilize a 2-state model, as shown in Figure In CDI,  channel , modulatthe pore ,30. CDI Ca2+-dependent facilitation (CDF) is a Ca2+/CaM-mediated enhancement in ICa,L via activation of CaMKII  present ; (c) the large Q10 values used by Rice et al. , we refrain from modeling this effect as the molecular mechanisms involved remain unresolved.Our model for cardiac contractile mechanics is based on the approximate model of cooperative activation and crossbridge cycling reported by Rice et al.  with theoponin I ,56 at Ser et al. , the unpCa2+ concentration. We now extend our voltage clamp studies to address the subject of frequency-dependent characteristics of CICR and begin with the study of frequency dependence of the DCU, one of the most important components of the model. All the frequency-dependent behavior discussed here is at steady state (150 cycles) unless otherwise specified. The first task is to examine the frequency dependence of the ICa,L trigger current, followed by the RyR Ca2+ channel, highlighting the rate-dependent CaM-mediated signalling involved. This analysis is followed by examining the SR Ca2+ content and the various factors controlling it in a rate-dependent manner. A quantitative study of the overall cellular Ca2+ balance is performed to highlight its rate-dependent feature. Emphasis is placed on relative roles played by the longitudinal sarcoplasmic reticulum (LSR) membrane SERCA pump and the plasma membrane Na+/ Ca2+ exchanger, as two principal Ca2+ transport routes in the maintenance of Ca2+ homeostasis. We finally examine the myoplasmic Ca2+ transient as a function of frequency with a particular interest in the rate dependence of the force-frequency response (FFR) generated by the coupled electromechanical model. This is subsequently followed by an investigation of the rate-dependent influence of cAMP-mediated β-adrenergic stimulation on the cardiac contractile response.From our modeling standpoint, the dyadic coupling unit (DCU) as defined by Krishna et al.  is a funICa,L channel activity is the rate-dependent change in the average level of activated-CaMKII . As stimulation frequency is increased from 0.5 Hz to 8 Hz, the increase in peak ICa,L rates, the lack of a substantial rate-dependent increase in its average level . This small modulation of peak current  versus [ Ca2+] RyR constructed from model-generated data corresponding to different stimulation rates with the inset showing the peak open probability attained by each of the phase loops. As the stimulation frequency is increased from 0.5 Hz to 4 Hz, a marginal increase (< 1%) in peak RyR open probability occurs , due to increased trigger current and the subsequent increase in [ Ca2+] myo combined with an increase in [ Na+] myo  a direct CaMKII-mediated enhancement in RyR release. As the stimulation frequency is increased from 4 Hz to 8 Hz, despite a moderate increase in both the trigger current and SR Ca2+ content, a small decrease in RyR open probability occurs as a result of an increase in post-release [ Ca2+] jSR  frequencies can be seen in Figure Ca2+ released into the dyad) mirrors that of the peak RyR open probability. The SR Ca2+ release from the Ry-sensitive receptor exhibits strong frequency-dependent behavior as shown in Figure Ca2+ content , despite decreasing time available for uptake, increasing pre-release SR Ca2+ content is achieved only by increased rate of SR filling. This increased rate of SR filling translates into a faster decline in inhibition via the luminal sensor  increase in the rate of RyR channel inactivation. The RyR channel experiences a frequency-dependent modulation by both the trigger current and the amount of activated-CaMKII on its dyadic side. On its luminal side, it experiences modulation by the luminal sensor . Hence,receptor ,43,45-48a et al. ) controlCa2+ level in the SR is a result of several factors namely: (a) available [ Ca2+] myo for sequestration; (b) the rate-dependent behavior of the SERCA pump; (c) the frequency dependence of the release characteristics of Ry-sensitive Ca2+ channel; and (d) the cumulative transmembrane Ca2+ transport via ICa,L and NCX. The combined effect of these factors results in the biphasic relationship  enhancement of maximal uptake rate of the SERCA pump due to increase in PLB phosphorylation  increase in half-activation constant for the backward (SR to myoplasm) operation of the SERCA pump  stimulation frequencies is a result of the following: (a) a decrease in maximal uptake rate of the SERCA pump  a decrease in Ca2+ entry into the cell via the trigger current ICa,L  Figure B capablet Figure B linked Ca2+ when compared with the combined influence of Na+/ Ca2+ exchange (INaCa) as well as Ca2+ transport via the plasma membrane Ca2+ ATPase pump,(IPMCA). As the frequency of stimulation is increased from 0.5 Hz to 4.0 Hz, increasing activated-CaMKII-dependent enhancement in SERCA activity assisted by a decrease in Ca2+ extrusion via NCX due to increasing [ Na+] myo  frequency-dependent, the SERCA pump has a primary role in resequestration of myoplasmic o Figure B result Ca2+] myo at increasing frequencies showing an increase in the peak myoplasmic Ca2+ transient during systole with increasing stimulation frequency in the range 0.5 Hz to 8.0 Hz. A ’primary phase’ negative peak FFR (a decrease in peak contractile force with increase in stimulation frequency observed at low (< 1.0 Hz) stimulation rates) reported in studies on rat ventricular myocytes [rise) and decay (R decay) of the [ Ca2+] myo transient increases with the frequency of stimulation from 0.5 Hz to 8.0 Hz. The increase in rate of rise is a result of an increase in peak trigger current ICa,L , which parallels a strong increase in pre-release diastolic [ Ca2+] jSR  is comparable to that reported by Kentish et al. β-adrenergic stimulation bears a very crucial physiological relevance in normal functioning of the heart. We have investigated the effect of β-adrenergic stimulation on [ Ca2+] myo under voltage clamp conditions. The degree of CaMKII mediated ICa,L facilitation is kept identical to the control case discussed earlier. Besides a moderate CaMKII-mediated  myo  versus maximum rate of contraction (R act) for increasing stimulation frequency with and without β-adrenergic stimulation (shows significant overlap). This linear relationship highlights contraction-relaxation coupling, and represents a key intrinsic property of the contractile myofilaments [β-adrenergic stimulation further assists  a CaMKII-mediated rate-dependent increase in peak [ Ca2+] myo, CaMKII is the key factor responsible for this behavior (a 2-fold increase in peak [ Ca2+] myo from 4 Hz to 8 Hz both in the presence/absence of cAMP-mediated effects). In the presence of β-adrenergic stimulation, Figure Ca2+] myo  is maximum at the stimulation rate of 5 Hz  stimulation frequencies  of ICa,L-71. The encies > .0 Hz as encies > .0 Hz as encies > .0 Hz as  Figures B), β-adrilaments . As showo Figure B supportz Figure C and decz Figure D on stimz Figure C showingβ-adrenergic stimulation on Ca2+ cycling and their effect on the force-frequency response. In particular, we have modeled their two crucial signalling mechanisms: the rate-dependent upregulation of the DHP-sensitive ICa,L channel : calcium ion concentration; [Ca2+]jSR: luminal Ca2+ concentration in the jSR; [Ca2+]myo: myoplasmic Ca2+ concentration; [Ca2+]o: extracellular Ca2+ concentration; [Ca2+]ryr: Ca2+ concentration at the “mouth” of the RyR channel on the dyadic side; CaM: calmodulin; CaMKII: Ca2+/calmodulin-dependent protein kinase II; CaMKIIact: activated Ca2+/calmodulin-dependent protein kinase II; cAMP: cyclic adenosine monophosphate; CaN: calcineurin; CaNact: activated calcineurin; CDF: calcium-dependent facilitation; CDI: calcium-dependent inactivation; CICR: calcium-induced calcium-release; CRCP: contraction-relaxation coupling point; DCU: dyadic coupling unit; DHP: dihydropyridine; DHPR: dihydropyridine receptor; E-C: excitation contraction; EC50bwd: affinity of backward Ca2+ flux from LSR to myoplasm; : ; ICa,L: L-type Ca2+ current; Icyt,serca: Ca2+ uptake current directed from the myoplasm to the SERCA; INa,b: background sodium current; INaCa: sodium calcium exchanger current; INaCs: sodium cesium pump current; IPMCA: plasma membrane Ca2+ ATPase pump current; Iryr: Ca2+ current due to CICR from an individual jSR; Iserca,sr: Ca2+ uptake current directed from the SERCA to the LSR; jSR: junctional portion of the sarcoplasmic reticulum; LCC: L-type DHP-sensitive Ca2+ channel; L-type: long lasting type; LSR: longitudinal portion of the sarcoplasmic reticulum; mM: milli molar; mV: milli volt; [Na+]myo: myoplasmic Na+ concentration; NCX: Na+/Ca2+ exchanger; nM: nano molar; NO: Nitric oxide; pC: pico coulomb; Po: Open probability; PKA: protein kinase A; PLB: phospholamban; : ; PLBp: phosphorylated phospholamban; PSR: phospholamban to SERCA ratio; Ry: ryanodine; RyR: ryanodine receptor; SERCA: sarcoplasmic reticulum Ca2+ ATPase; SL: sarcolemma; SR: sarcoplasmic reticulum; VC: voltage clamp; VDI: voltage-dependent inactivation.The authors declare that they have no competing interests.AK developed the coupled electromechanical model of the rat ventricular myocyte, carried out the voltage clamp modeling studies, and drafted the manuscript. MV made substantial intellectual contributions to the study and in drafting of the manuscript. PTP made intellectual contributions to the manuscript as well as significant contributions to the drafting of the manuscript. JWC made key contributions to the conception of the study, design, analysis and interpretation of results, and drafting of the manuscript. All authors read and approved the final manuscript.


Here we show that exogenously expressed Mcu is mitochondrially localized and increases mitochondrial Ca2+ levels following NMDA receptor activation, leading to increased mitochondrial membrane depolarization and excitotoxic cell death. Knockdown of endogenous Mcu expression reduces NMDA-induced increases in mitochondrial Ca2+, resulting in lower levels of mitochondrial depolarization and resistance to excitotoxicity. Mcu is subject to dynamic regulation as part of an activity-dependent adaptive mechanism that limits mitochondrial Ca2+ overload when cytoplasmic Ca2+ levels are high. Specifically, synaptic activity transcriptionally represses Mcu, via a mechanism involving the nuclear Ca2+ and CaM kinase-mediated induction of Npas4, resulting in the inhibition of NMDA receptor-induced mitochondrial Ca2+ uptake and preventing excitotoxic death. This establishes Mcu and the pathways regulating its expression as important determinants of excitotoxicity, which may represent therapeutic targets for excitotoxic disorders.The recent identification of the mitochondrial Ca Calcium uptake by the mitochondrial calcium uniporter is implicated in excitotoxicity. This study shows that the uniporter gene product mediates mitochondrial calcium uptake and depolarisation in neurons during excitotoxicity, and is transcriptionally repressed by neuroprotective nuclear calcium signals. Early attempts to interfere with mitochondrial Ca2+ uptake in neurons indirectly involved the use of protonophores in order to depolarize the mitochondria 2+ uptake, with protective consequencesMany important studies into the responses of mitochondria to NMDA receptor (NMDAR) activity suggest that mitochondrial Ca2+ uptake in excitotoxic cell death.Pharmacological agents based on the hexavalent cation ruthenium red have also been utilized. Ruthenium red itself is able to selectively block the uniporter in isolated mitochondria, but has non-selective effects on certain ion channels in intact cells and is unable to cross the plasma membrane of many cell types151516Mcu) was identified as the ubiquitously expressed gene previously known as Ccdc109a182+ uptake20Mcu expression and knockdown to be employed to determine the role of mitochondrial Ca2+ uptake in all aspects of cellular physiology and pathology.In two recent papers, the gene product encoding the uniporter channel  Liposome-mediated transfection of an Mcu expression vector or Mcu-directed siRNA in mouse cortical neurons, and (b) Recombinant adeno-associated virus (rAAV)-mediated transduction of expression vectors encoding Mcu (tagged with GFP or tDimer) or Mcu-directed shRNA in mouse hippocampal neurons. The reason for the liposome-mediated transfection approach was to achieve low efficiency, enabling a direct comparison between transfected and juxtaposed untransfected cells, while the viral transduction technique was chosen for high efficiency of infection (80–90%), which facilitates ease of data analysis, particularly cell death. The two neuronal types used served to broaden the applicability of our findings.2+, loss of mitochondrial membrane potential and cell death. In all Ca2+ imaging experiments we performed experiments with control and 'treated' conditions using sister cultures interleaved on the same day to control for any culture-to-culture variation in NMDA-induced Ca2+ influx. Thus, the only 'control' data shown in any particular figure are those data collected on the same day on the same culture as when the ‘treated condition’ data  were collected. As an important additional control, we verified that Mcu overexpression did not have any effect on NMDA-evoked whole-cell currents  that was barely detectable over background (data not shown). Using this technique, we also found that Mcu overexpression strongly increased both basal [Ca2+]mit and NMDA-induced increases in [Ca2+]mit, compared with control-transfected neurons treated in parallel experiments  and an rAAV containing an expression cassette for the FRET-based mitochondrial Ca2+ indicator 4mtD3cpv4mtD3cpv. The rAAV-mediated infection resulted in high efficiency (80–90%) expression of Mcu–tDimer in mouse hippocampal neurons (data not shown). Compared with parallel experiments performed on control neurons Mcu–tDimer overexpression led to enhanced elevation of [Ca2+]mit, following NMDAR activation  is one of the earliest observable events in response to excitotoxic insults, and mitochondrial CaMcu–GFP: the expression of Mcu–GFP led to increased basal cell death rates in neurons and elevated levels of NMDA-induced cell death  with control or Mcu-encoding vectors plus an eGFP marker were taken before and 24 h after a 1-h exposure to an excitotoxic dose of NMDA, and their viability scored. We found that Mcu overexpression increased the level of basal toxicity in neurons . In addill death . For botll death .2+, loss of mitochondrial membrane potential and cell death. Two independent approaches were taken: liposome-mediated transfection of Mcu-targeted pre-made siRNA, and rAAV-mediated expression of Mcu-directed small hairpin RNA (shRNA). The efficacy of the pre-made siRNA was investigated using a high-efficiency transfection approach (nucleofection), which revealed a partial (50%) knockdown at protein and mRNA levels , as employed previously32It has long been known that episodes of synaptic activity can promote neuroprotection against excitotoxic insults2329302+ uptake following subsequent NMDA treatment. Indeed, this was found to be the case: mitochondrial Ca2+ uptake during NMDA treatment was substantially reduced in BiC/4-AP pre-treated cortical neurons . We studied mRNA stability using the standard method of inhibiting transcription with Actinomycin D and harvesting RNA at multiple time points thereafter. By comparing the level of Mcu mRNA with a relatively stable mRNA (18S), one can gain a measure of mRNA degradation, and assess the influence of prior synaptic activity on this stability. An episode of prior synaptic activity had no significant difference in the decay kinetics of Mcu mRNA levels  represses Mcu expression.We then investigated the mechanism of this transcriptional repression. We found that activity-dependent repression of Mcu was blocked by cycloheximide, indicative that 2+ signaling2+-induced neuroprotective genes, five of which were found to inhibit NMDA-induced mitochondrial depolarization2+ and CaM kinase-dependent induction of Npas4, which in turn mediates the transcriptional repression of Mcu. These data establish Npas4 as a potentially central regulator of Mcu activity in neurons.Our laboratories have previously described the neuroprotective effects of nuclear CaMcu gene product in the control of mitochondrial Ca2+ uptake in neurons following an excitotoxic insult. Moreover, we established that Mcu is an important mediator of death signal-induced loss of mitochondrial membrane potential and relays NMDA receptor stimulation to excitotoxic cell loss. Intriguingly, we also find that Mcu expression and mitochondrial Ca2+ uptake are subject to dynamic regulation by neuroprotective firing activity.In this study, we used gain- and loss-of-function experiments to demonstrate the central role of the 2+ uptake will tell us what role this process has in several key pro-death cascades. For example, mitochondrial Ca2+ uptake is implicated in the activation of PARP-1 via mitochondrial ROS generation, which combine with NO to trigger ONOO−-mediated DNA damage, the trigger for PARP-1 activation11412+ uptake and ROS production have also been implicated in JNK activation2+ uptake in mitochondrial membrane depolarization. The coupling of the GluN2B C-terminal domain to downstream cell death pathways, including nitric oxide production, contributes to excitotoxicity344452+ uptake has been reported to involve nitric oxide102+-dependent depolarization+/Ca2+ exchanger. PARP-1 has also been shown to contribute to excitotoxic mitochondrial depolarization, likely via depletion of NADH levelsSeveral key pro-death pathways are activated in parallel in response to an excitotoxic insult, including nitric oxide and ROS production, oxidative stress, JNK and PARP-1 activation, and CREB shut-off33738392+ uptake contributes to the differential effects of synaptic versus extrasynaptic NMDAR activity282+ influx triggered by bath activation of NMDARs is more strongly coupled to mitochondrial Ca2+ uptake than influx through other routes2+-carrying capacity of the uniporter2+), Ca2+ uptake may be substantially enhanced if mitochondria are positioned extremely near the site of Ca2+ entry where local Ca2+ levels are high. Synaptic NMDARs may be somewhat shielded from close proximity to mitochondria by the post-synaptic density, and more significantly by the fact that dendritic spines rarely contain mitochondria2+ transporters such as Letm1 or uncoupling proteins contribute in any way in neurons492+]mit  awaits further investigation. Given that the MCU gene is also subject to Ca2+-mediated transcriptional repression in human ESC-derived neurons , a search for phylogenetically conserved elements within the Mcu promoter may yield the sequences responsible for mediating Ca2+ sensitivity.Protection against excitotoxic insults by CaMcu both as an effector of excitotoxic cell death and a target of neuroprotective signals. Exploiting these facts may lead to new interventions aimed at ameliorating neuronal dysfunction and death in disorders associated with aberrant NMDAR activity.To conclude, we have established Hippocampal and cortical neurons from newborn C57Bl/6 mice (sex not determined) were cultured and maintained as described previously33−1 total) and/or siRNA (100 nM) using Lipofectamine 2000. Nucleofection was performed using the Amaxa rat Neuron Nucleofector Kit (Lonza). See Neurons were transfected in trophic transfection mediumGel electrophoresis and western blotting were performed using the Xcell Surelock system (Invitrogen) using precast gradient gels (4–20%) as describedMcu and Npas4.The vector containing the mouse CaMKIIα promoter used to construct and package rAAV has been described previously11 rAAV particles per μl at DIV 4. Infection efficiencies were determined at DIV 10 by analyzing the fluorescence of eGFP or mCherry; they ranged from 80 to 90% of the viable neurons. rAAV expression vectors for Npas4, CaMBP4 and mCherry have been described previously35Neurons were infected with 10NMDA-evoked whole-cell steady-state currents  were measured 48 h after transfection as described2+ imaging experiments, we performed experiments with control and 'treated' conditions using sister cultures interleaved on the same day to control for any culture-to-culture variation in NMDA-induced Ca2+ influx. Thus, the only 'control' data shown in any particular figure are those data collected on the same day on the same culture as when the 'treated condition' data  were collected. Several imaging approaches were used: (a) Rhod-2 imaging: this was performed as described2+ were expressed as (F−Fmin)/(Fmax−F) according to the equation [Ca2+]=Kd × (F−Fmin)/(Fmax−F). Fmax was obtained when cells were treated with the cell-permeable Ca2+ ionophore ionomycin which both inserts into the plasma membrane and passes into the cell, inserting into mitochondrial membranes2+). Fmin was obtained under the same conditions except in zero Ca2+ medium. The linear relationship between [Ca2+] and (F−Fmin)/(Fmax−F) was confirmed by calibrating the indicator as expressed in neurons, exposing them to ionomycin in the presence of sequentially different solutions of precise [Ca2+], obtained by mixing K2EGTA and CaEGTA solutions  at different ratios (data not shown). (c) 4mtD3cpv imaging was performed as described2+ indicator and an mCherry or tDimer tag were chosen for analysis. Ca2+ concentration changes were quantified using the crosstalk- and bleaching-corrected FRET ratio2+ responses to NMDA were expressed as (%Rfret/(ΔRmax−%Rfret))1/n according to the equation [Ca2+]=K′d × (%Rfret/(ΔRmax−%Rfret))1/nfret represents the percent of the maximum response obtained in SGG medium containing 10 μM ionomycin and 10 mM Ca2+. The values for DRmax and n  were derived from published calibrations2+] and (%Rfret/(DRmax−%Rfret))1/n was confirmed in neurons exposed to ionomycin in the presence of sequentially different solutions of precise [Ca2+], obtained by mixing K2EGTA and CaEGTA solutions  at different ratios (data not shown). Control values for mitochondrial [Ca2+] obtained using 4mtD3cpv were subject to some culture-to-culture variation, which is likely to be due to the fact that it is operating closer to it's maximum than GCaMP2-mt is. As a result of this, we have normalized all 4mtD3cpv [Ca2+] data to the average NMDA-induced [Ca2+] obtained in control cells on that day of recording. This allows us to directly assess the effect of Mcu expression, Mcu knockdown, or BiC pre-treatment on NMDA-induced mitochondrial [Ca2+], expressed as a percentage of the NMDA-induced mitochondrial [Ca2+] level obtained in that exact day of recording.In all Ca−1 or 26 μM) in SGG medium for 10 min followed by extensive washing with SGG. Rh123 partitions into polarized mitochondria where it self-quenches at the concentration used. When mitochondria depolarize, Rh123 leaks out of the mitochondria into the cytoplasm where it dequenches and fluoresces strongly. Maximum Rh123 signal  was obtained by completely eliminating the mitochondrial potential by exposing the neurons to the mitochondrial uncoupler FCCP . For analyzing the effect of transfecting Mcu expression vectors or Mcu-directed siRNA, transfected cells were identified by co-transfecting mCherry expression plasmid. The NMDA-induced increase in Rh123 signal of each transfected cell (as a % of the FCCP-induced level for that cell) was compared with that observed in surrounding untransfected cells in a 100-μm radius and subsequently expressed as a percentage of that observed in those untransfected cells. The NMDA-induced increase in Rh123 signal in Mcu-1 shRNA-infected hippocampal neurons was compared with that observed in Scr shRNA-infected cultures analyzed in parallel.Mitochondrial membrane potential was analyzed as described10Excitotoxicity experiments performed on Lipofectamine-transfected neurons were performed as described45Mcu silencing by the pre-made Mcu-directed siRNA, and study activity-dependent Mcu regulation in cortical neurons, QRT–PCR was performed using an Mx3000P QPCR System as describedMcu-directed rAAV–shRNA, and study activity-dependent Mcu regulation in hippocampal neurons, QRT–PCR was performed using real-time TaqMan technology with a sequence detection system model 7300 Real Time PCR System . For further details including primer sequences see To determine t-test, as appropriate. For studies employing multiple testing, we used a one-way ANOVA followed by Bonferroni post hoc test. All data were presented as mean±s.e.m.Statistical testing involved a two-tailed paired or unpaired Student’s G.E.H and H.B. conceived the project and designed the research. G.E.H., H.B., P.A.S. and D.J.A.W. supervised the research. J.Q., Y-W.T., A.M.H., M.M., N.K. and G.E.H. performed the experiments. G.E.H. and H.B. wrote the manuscript.How to cite this article: Qiu, J. et al. Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals. Nat. Commun. 4:2034 doi: 10.1038/ncomms3034 (2013).Supplementary Figures S1-S5 and Supplementary Methods