Natriuretic peptides increase in heart failure. C-type natriuretic peptide (CNP) generates cyclic 3’,5’ guanosine monophosphate (cGMP) by activating the NPR-B receptor in cardiomyocytes. There are studies showing CNP-induced negative inotropic and positive lusitropic responses in normal hearts, but less is known about the effects of CNP in failing hearts.2+ removal from cytosol and thus less Ca2+ available for the myofilaments during contraction. Cyclic GMP levels, contraction and relaxation, Ca2+ transients, troponin I (TnI) and phospholamban (PLB) phosphorylation were measured in left ventricular muscle strips or cardiomyocytes from Wistar rats with heart failure 6 weeks after myocardial infarction. CNP increased cGMP levels and evoked negative inotropic and positive lusitropic responses concentration-dependently. TnI and PLB phosphorylation also increased in the presence of CNP. The functional responses to CNP were reduced in the presence of a PKG-blocker/cGMP-analogue (Rp-8-Br-Pet-cGMPs), demonstrating activation of the PKG pathway. In the presence of CNP, Ca2+ transient amplitude and Ca2+ extrusion rate were increased. CNP elicited both negative inotropic and positive lusitropic responses in the presence of the L-type Ca2+ channel activator BAY K 8644, whereas in the presence of full activation of the cAMP system by isoproterenol these responses were not seen. This indicates that the downstream targets causing functional responses to CNP were already activated in the presence of isoproterenol. All these results could be explained by an increased SERCA activity and a reduced myofibrillar sensitivity to Ca2+ in the presence of CNP, due to increased phosphorylation of PLB and TnI, respectively. An obligatory role of SERCA activation was revealed in a mouse model with cardiomyocyte-specific excision of the SR Ca2+-ATPase gene (SERCA-KO). The functional responses to CNP were abolished in 8-week SERCA-KO mice compared to 4-week SERCA-KO mice still possessing some SERCA activity.We investigated the functional effects of CNP in heart failure and suggested that increased activation of SERCA is the main mechanism of CNP-induced negative inotropic and positive lusitropic responses. Increased SERCA activity could cause a faster CaThe functional responses to CNP are mediated through the PKG pathway. Activation of SERCA thus seems to be the major and indispensable mechanism of CNP-induced functional responses in failing hearts.


The family of classical transient receptor potential channels (TRPC) has been implicated in both the receptor/second messenger as well as in store-operated Ca2+ entry pathway, and represents an attractive target for therapeutic intervention.Calcium governs a wide range of cellular processes. Specifically, control of gene transcription involves Ca2+ entry into RBL-2H3 mast cells and HEK293 cells overexpressing TRPC3.We tested a series of pyrazol compound structurally related to Pyr3 , a recen2+ entry inhibitors, which are able to discriminate between the two tightly related pathways of receptor/second messenger-activated and store-operated calcium entry. These compounds appear suitable for selective modulation of Ca2+-dependent gene transcription in a variety of mammalian cells.We identified novel Ca


Like other cells, red blood cells (RBCs) rely on Ca2+ dependent signalling during differentiation from precursor cells. Intracellular Ca2+ levels in the circulating human RBCs take part not only in controlling biophysical properties such as membrane composition, volume and rheological properties, but also physiological parameters such as metabolic activity, redox state and cell clearance. Extremely low basal permeability of the human RBC membrane to Ca2+ and a powerful Ca2+ pump maintains intracellular free Ca2+ levels between 30 and 60 nM, whereas blood plasma Ca2+ is approximately 1.8 mM. Thus, activation of Ca2+ uptake has an impressive impact on multiple processes in the cells rendering Ca2+ a master regulator in RBCs. Malfunction of Ca2+ transporters in human RBCs leads to excessive accumulation of Ca2+ within the cells. This is associated with a number of pathological states including sickle cell disease, thalassemia, phosphofructokinase deficiency and other forms of hereditary anaemia. Continuous progress in unravelling the molecular nature of Ca2+ transport pathways allows harnessing Ca2+ uptake, avoiding premature RBC clearance and thrombotic complications. This review summarizes our current knowledge of Ca2+ signalling in RBCs emphasizing the importance of this inorganic cation in RBC function and survival.Ca When bound and buffered Ca2+ are included, total intracellular Ca2+ in red blood cells (RBCs) reaches 5.7 μM [2+ concentration in RBCs of healthy human beings under physiological conditions is estimated to be in the range of 30 to 60 nM [2+ concentration reaches up to 1.8 mM is maintained due to particularly low permeability of membranes to Ca2+ (~50 μmol/(lcells h)) and efficient extrusion of Ca2+ from the cells by the plasma membrane Ca2+ pump (PMCA) [6 ions per second over several milliseconds may result in >10-fold changes in free Ca2+ levels in the sub-membrane space, causing acute changes in the activity of multiple Ca2+ sensitive proteins involved in structural, signalling, metabolic and transport functions. Such a huge Ca2+ influx seems to exceed the RBC’s Ca2+-buffering abilities. Long-term increases in the Ca2+ permeability result in serious dysregulation of multiple cellular functions. The subsequent onset of proteolysis, oxidation, irreversible shrinkage and phosphatydylserine (PS) exposure to the extracellular membrane leaflet facilitate clearance of Ca2+ overloaded cells in case the latter have not been haemolysed when passing through capillaries.Camolecule ,2, reguls 5.7 μM . Basal fto 60 nM . A tremep (PMCA) . This gr2+ levels, Ca2+ dependent processes and the potential role that Ca2+ might play in the development of RBC pathologies. For obvious reasons this review makes no claim to be complete, but wants to draw attention to open questions and mechanisms to be resolved in the field of Ca2+ signalling in RBCs.This review summarizes the current knowledge on the regulation of RBC Ca2+ increase [2+ influx remained unknown for several decades, the extrusion mechanism was realized to be mediated by a plasma membrane Ca2+ pump (PMCA). For human RBC membrane the presence of the B-splice isoform of the PMCA1 was shown [N- and C-terminals. The latter contains a Ca2+ calmodulin binding domain, phosphorylation sites and a PDZ-binding domain serving as a docking terminal for a number of proteins [2+ extrusion rate in RBCs can vary within RBC populations of a single donor between >60 mmol/(lcells h) and <4 mmol/(lcells h) [Vmax in cells from the dense fraction was referred to as a hallmark of RBC senescence [2+ turnover rate of PMCA in the RBC membrane of healthy humans not loaded with Ca2+ is most likely never reached. The apparent Vmax for PMCA measured in such cells is ~50 μmol/(lcells h), the value equal to the passive Ca2+ uptake. The enzyme half-activation constant for Ca2+ was reported to be 4 μM, far above the actual free cytosolic Ca2+ levels in human RBCs [It was realised very early on that the inhibition of the energy supply in RBCs leads to a Caincrease . Althougas shown . This P-proteins . The maxcells h) . The maxcells h) . This renescence . The maxman RBCs .2+ are sensed by the PMCA and occur in response to the interaction of the Ca2+ calmodulin complex with the C-terminus of the enzyme. In Ca2+-loaded RBCs the limiting factor of the PMCA transport capacity is ATP availability. The pump is fuelled preferentially by a pre-membrane ATP pool [Kd (ATP) of 145 μM [2+ leak, activation of PMCA results in rapid ATP depletion.Increases in the intracellular free CaATP pool ,12,13 anRBC-derived plasma membrane vesicles contain factors activating PMCA, such as arachidonic acid, ceramide and acidic phospholipids, whereas sphingosine suppresses its function –17. The 2+ influx through the plasma membrane of healthy human RBCs is extremely slow but increasing up-to 5-fold in cells of patients with sickle cell disease (SCD) and several other forms of hereditary haemolytic anaemia [2+. Significant progress in identification of ion channels in control of Ca2+ uptake has recently been reviewed [Ca anaemia . Severalreviewed .2+ permeable [Voltage-activated non-selective cation channels –22 were ermeable . These iermeable  and furtermeable –27. Howe2+ channel was pharmacologically identified [V2.1 channel by Western blot analysis [Furthermore, a P-type Caentified  and showanalysis . This chanalysis . Howeveranalysis .A recent report provided evidence for the presence of a transient receptor potential (TRP) channel of subtype C6 in the RBC membrane . HoweverN-methyl d-aspartate (NMDA), homocysteine, homocysteic acid, glycine and d-serine [In addition, the expression of an NMDA receptor channel was initially reported for rat  and lated-serine .Recently, the protein PIEZO1 was reported as being mutated in RBCs in hereditary xerocytosis  without Furthermore there is evidence for an AMPA receptor related channel activity in RBCs .sickle [All the channels mentioned above were reported to be present in human RBCs from healthy donors. However, some currents were only shown to be present in cells of patients. An example is an increase in non-selective cation conductance on RBC of SCD patients mediating or contributing to Psickle ,40, an isickle . Howeversickle .2+ activates numerous Ca2+ dependent proteins. Each of them has its own activation threshold. Thus, gradual increase in Ca2+ levels is associated with gradual activation of various groups of Ca2+-sensitive proteins involved in physiological and pathophysiological processes in RBCs. In 2+ sensitive proteins is by far not comprehensive and can hardly be covered within one review. Despite a large number of such proteins and diversity of their functions, only few of them are “true” Ca2+ sensors interacting directly with calcium ions [2+ binding domains enriched with carboxyl and carbonyl groups  interconnected with a flexible linker  include, e.g., elements of the cytoskeletal network, the Na+/H+ exchanger NHE1, PMCA and the endothelial NO synthase (eNOS). Cytoplasmic CaM becomes active when recruited to the plasma membrane where its action is often coupled to that of phosphatidylinositol 4,5-bisphosphate (PIP2) localised at the inner leaflet of the membrane. An example of such coupling is a competitive binding of both co-regulators to the intracellular domain of NHE1 . UpoNHE1 see .2+ sensors (~650 proteins included) contain Ca2+ binding C2 domains interacting with 2–3 Ca2+ [2+ to the C2 domains, triggers translocation of these proteins to the specific areas within plasma membrane containing their substrates [Another class of “true” Ca2–3 Ca2+ ,46. In R2+ activated protein kinases, the conventional protein kinase C (cPKC) [2+ binding, PKCα translocates to the plasma membrane, where it phosphorylates its target proteins. The kinase domain of PKCs lacks specificity [Changes in phosphorylation are among the most important modulations of protein activity in RBCs. Among the kinases there is a group of CaC (cPKC) . Among cC (cPKC) . Upon Cacificity ,68 and tcificity , cytoskecificity ,71 and scificity  and posscificity ,30,73 ar2+ levels in the vicinity of the plasma membrane. The latter, most likely serves as a signal to mediate rapid reversible changes in cytoskeletal flexibility.Opening of cation channels in response to mechanical stress and the presence of activators, such as amino acids, proinflammatory cytokines and others, result in local transient increase in CaThe calcium-calmodulin complex (Ca-CaM) plays a key role in regulation of cytoskeletal stability. Selected elements of the cytoskeletal architecture interacting with Ca-CaM are schematically shown in 2 resulting in activation of this ion transporter and dysregulation of cell volume and cytosolic alkalosis [It is shown in lkalosis ,77.2+ concentration required for half-activation of calmodulin is 100 nM [2+ [The Cas 100 nM –920 nM [s 100 nM , Figure 0 nM [2+ . Phospho0 nM [2+ .2+ dependent phosphorylation is a second mode of action of Ca2+ on cytoskeletal proteins. Phosphorylation of the 4.1R protein at serine 312 and serine 331 by PKC was reported [2+ uptake is known to be triggered by mechanical deformations [2+ is permanently upregulated, on the contrary compromises mechanical stability of the RBC membrane [Careported . These treported . As Ca2+rmations , controlmembrane . In RBCsmembrane . One mormembrane , which r2+ entry with a rapid release of its inhibitory GTP and activation resulting in formation of Nɛ (γ-glutaminyl)lysine cross-linking  of 4.7 μM, +, Cl− and water resulting in cell shrinkage. Although the physiological function of the Gardos channel is not completely elucidated, there are two complementary concepts: (i) Openings of RBC Ca2+ channels by platelet released substances [2+ permeability with secondary activation of the Gardos channels [RBC volume regulation is a complex process with contributions of numerous molecular players including, e.g., band 3 protein . A marke in RBCs . The cor in RBCs ,89. Thisly 20 pS , a selecout 15:1  and an Ebstances ,93 initichannels . This wachannels .2+ activated [2+ sensitivity is mediated by an EF hand motif [2+ [Scramblase is a protein responsible for bidirectional transmembrane movement of phospholipids  leading ctivated ,52. Its nd motif . The scrnd motif . This protif [2+ .2+ in turn was suggested to promote band 3 tyrosine phosphorylation [Numerous reports emphasize the possible role of the Ca-CaM system in regulation of activity of glycolytic enzymes including pyruvate kinase ,99. Howerylation . Phosphorylation . A similrylation .2+ concentration and the haemoglobin oxygen saturation. In cells of healthy individuals, passive Ca2+ uptake was unaffected by deoxygenation, but the Vmax of the PMCA was reduced by 18%–32% [2+ levels was mainly attributed to changes in haemoglobin protonation, increases in protonation of deoxyhaemoglobin and a shift in the intracellular pH towards more alkaline values [2+ buffering capacity of haemoglobin. In the cytosol of deoxygenated RBCs release of Ca2+ ions from protein binding sites and lowering of extrusion capacity of the PMCA contribute to both an increase in the ionised Ca2+ fraction by 34%–74% even in the absence of Ca2+ influx from the extracellular medium [In RBCs there is a direct link between the intracellular free Ca 18%–32% . This is 18%–32% . An incre values ,105. Alor medium .2+ were recently linked to a lower oxygen affinity of haemoglobin promoting the release of oxygen [2+ from intracellular buffers may promote further deoxygenation enhancing O2 dissociation from haemoglobin. Molecular mechanisms of this phenomenon remain to be investigated.Increases in the free Caf oxygen . Deoxyge2+ is also involved in the regulation of the RBC’s redox state. Ca-CaM complexes are co-activators of endothelial NO synthase (eNOS) activity [2+ uptake during shear stress [l-arginine eNOS itself gets uncoupled and is capable of generating superoxide anions, which is turned into H2O2 in a reaction catalysed by SOD [Aside to the control of haemoglobin oxygenation, Caactivity ,107. Recactivity ,109 and r stress ,110. Nitr stress . Howeverd by SOD . Pro-oxid by SOD .2+ dependent cysteine protease μ-calpain  was mainly detected in human RBCs, m-calpain  was found to be virtually absent [2+ with a half-activation concentration in the range of 3–50 μM [2+ concentration found in both human RBCs from healthy individuals (30–60 nM) and values of 100–300 nM reported in RBCs from patients with hereditary forms of anaemia [2+ sensitive, shifts Ca2+ concentrations required for half-maximal activation from 40–50 μM down to 0.2 μM [2+ levels were raised when compared to those from healthy donors. [While the Cay absent . μ-Calpa 3–50 μM ,115. The 3–50 μM . This is anaemia . Howevero 0.2 μM . In humao 0.2 μM ,117. Acto 0.2 μM  was used2+ levels beyond physiological concentrations, namely in the range 50–150 μM [Targets of activated calpain are mainly transmembrane or membrane-associated proteins including PMCA, and bands 1, 2, 2.1, 3, 4.1, 4.2 proteins, but also calpain itself . Autolys0–150 μM . Limited0–150 μM .2+ loaded normal RBCs but not in the KO RBCs [Recently, RBCs of a μ-calpain knock-out (KO) mouse displayed an improved deformability . Further KO RBCs .2+ gradients [Cleavage of the PMCA by μ-calpain was associated with an activation of the pump, rapid ATP depletion, inactivation of the pump and gradual loss of the transmembrane Caradients .2+ sensitive. Half-maximal activation of calpastatin occurs at 40 μM Ca2+ [Calpastatin, an endogenous inhibitor of μ-calpain, is a natural regulator of the enzyme activity in RBCs. Both major and minor components of calpastatin, calpastatin H and L were detected in human RBCs . Interac μM Ca2+ , in bovi μM Ca2+ ,124.2+ levels, an altered rheology was observed [Due to the protein activity and lipid remodelling described above one would expect changes in rheological properties of RBC suspensions and indeed in numerous studies provoking increased cytosolic Caobserved –127. Theobserved ,129. How2+ channels in blood clot formation was proposed previously [2+ mediated aggregation of RBCs [2+ concentration at which aggregation occurs. Additionally the modulations of the aggregation properties under in vivo conditions need to be elucidated.A participation of RBC Caeviously ,93 folloeviously –132. Fur of RBCs –136 and  of RBCs –139. Fur2+ uptake is of key importance for promoting differentiation and proliferation of erythroid precursors at the stages of burst-forming units erythroid (BFU-E) colony-forming units erythroid (CFU-E) [2+ is an integral part of the signalling pathway activated by binding of erythropoietin to its receptor [2+-free medium, Ca2+ uptake is absent and differentiation and survival of erythroid precursors is compromised [2+ uptake by erythroid precursor cells cultured from mononuclear cells by the NMDA receptor antagonist MK-801 resulted in 45.5% ± 12.8% mortality of cells at the stage of basophilic and polychromatic erythroblasts suggesting that these receptors are actively contributing to erythropoiesis [2+ levels in reticulocytes are higher than in mature RBCs comes from secondary Ca2+-dependent processes such as phosphorylation [Ca (CFU-E) ,141. Increceptor ,143. In promised ,141. Inhopoiesis . Additiorylation . Proteinrylation .2+ in mature RBCs was obscure and was believed to be limited to the involvement in RBCs aging and clearance [2+ in RBC regulating a broad range of processes including O2 transport [2+ homeostasis in RBCs results in development of severe life-threatening systemic pathologies.For a long time, the physiological function of Calearance ,146,147.ransport , rheologransport , clottinransport ,136 and 2+ associated mechanism was reported. Here, rises in the intracellular Ca2+ appear to promote the ability of RBCs to deliver oxygen [Very recently, additional evidence in favour of a physiologically important Car oxygen .2+ levels exceed those in reticulocytes and young RBCs [2+ levels largely depend on the age markers employed. Typical age markers include glycosylated haemoglobin HbA1c, band 4.1a/b ratio, cell density, de-sialation and changes in CD47 abundance at the membrane surface, PS exposure, and several others [At present it is suggested that in senescent RBCs the intracellular Caung RBCs . Howeverl others –151.2+ accumulation only when coupled to the unchanged or increasing activity in Ca2+-transporting ion channels. However, according to recent findings, this is not necessarily the case. Young rat and human RBCs contain higher number of NMDA receptors, that upon stimulation with plasma glycine and glutamate can cause significant Ca2+ influx [2+ levels [2+ overload, which may well trigger RBC clearance, but the relation of this mechanism to other Ca2+ independent clearance mechanisms and to in vivo relevance is still obscure.Activity of the PMCA in RBCs was shown to decrease with progressing HbA1c accumulation . However+ influx ,33. Youn+ influx ,153–155.+ levels . Thus, b2+ levels [2+ that could not be compensated for by the activation of PMCA was reported for patients with SCD [2+ seem to be sequestered in vesicles or bound to cytosolic proteins) [2+ transport in diseased RBCs was so far obtained for SCD patients. In these cells relatively high rates of Ca2+ uptake in RBCs are partially compensated for by sequestration of Ca2+ into intracellular inside-out vesicles, in which Ca2+ is pumped actively by PMCA [2+ taken into the cells is immobilised by the intracellular proteins. Sickle cell transformation associated with polymerisation of deoxygenated mutated S-haemoglobin is amplified by 20–40-fold by dehydration [2+ uptake and release of ionised Ca2+ from the intracellular proteins (reduction in buffering capacity) [2+ RBCs causes opening of the Ca2+ sensitive Gardos channel and anion channels [Independent of its origin, hereditary haemolytic anaemia is often associated with an increase in the intracellular Ca+ levels . “Leakinwith SCD –159, betroteins) –162, phoroteins) . Most of by PMCA . Apparen by PMCA . Furtherydration . Deoxygeapacity) ,105,167.channels –170.2+ comprise activation of μ-calpain [Downstream events triggered by augmentation of free intracellular Ca-calpain  and acti-calpain . In RBCs-calpain .Cross-linked polymers have been observed in RBCs of patients with SCD suggesting hyperactivation of transglutaminase .Psickle) mediating Ca2+ uptake by RBCs remains unknown (compare Section 2.2).Oxidation of membrane proteins and impaired NO production by eNOS ,174, inc2+ overload is involved in dramatic reduction of the life span of stored RBCs used for transfusion [2+ free glucose-containing preservation citrate buffers at low temperatures. These storage conditions favour Ca2+ depletion of the cytosol, oxidative stress and ATP deprivation [2+ transport and evoke acute Ca2+ overload when re-exposed to the plasma of patients receiving transfusions. The resulting processes include dehydration, rigidity, fragmentation of cytoskeletal proteins and oxidative stress and increased adherence of RBCs to the endothelium and to each other [Cansfusion . Storagerivation ,178. Thech other ,180.2+ entry into RBC may cause side effects of drugs involved in therapies unrelated to RBCs. An example is photodynamic therapy, where the oxidative stress produced by the photosensitizer leads to the activation of cation channels in the RBC membrane and the consecutive Ca2+ entry triggers the mechanism described above, which is the major cause of an increased formation of blood aggregates as well as haemolysis [Additionally, unwanted modulations of the Caemolysis . Thus, Remolysis  and can emolysis .2+ in RBCs physiology and pathophysiology cannot be overestimated. Many links between Ca2+ and RBC related diseases still need to be explored [2+ within the cytosol are essential to predict the onset of changes related to the abnormally high Ca2+ levels, which are particularly important in patients with haematological disorders [2+ binding entities within the cells will need further attention and research. Following the broad variety of Ca2+ mediated processes mentioned here, monitoring the following parameters may be used to indirectly predict abnormally increased intracellular free Ca2+ levels in RBCs: (i) changes in cell volume and morphology ; (ii) congenital haemolytic anaemia associated with stomatocytosis, reticulocytosis, and shortened RBC survival; (iii) decrease in the intracellular K+ levels, pseudohyperkalemia; (iv) loss of RBC deformability, changes in osmotic resistance, an increase when dehydration has occurred but cytoskeletal stability is still maintained, or a decrease when cytoskeleton is partially disassembled; (v) appearance of calpain-induced band 3 cleavage fragments; (vi) oxidative stress or unusually high NO production ; (vii) ATP depletion due to hyperactivation of PMCA; (viii) increase in inter-RBC aggregability; and (ix) increase in PS exposure.The role of Caexplored . Methodoexplored . This isisorders ,186. How2+ that occur in other cells and may involve several organelles—have been rediscovered in a modified and/or simplified form in RBCs.For many years the RBC was the cell of choice for membrane transport investigations. In the age of genomics, interest in RBC research decreased, but numerous signalling cascades—also in respect to the second messenger Ca


Relapse after chemotherapy is inevitable in the majority of patients with acute myeloid leukemia (AML). Thus, it is necessary to develop novel therapies that have different antileukemic mechanisms. Recent advances in immunology and identification of promising leukemia-associated antigens open the possibilities for eradicating minimal residual diseases by antigen-specific immunotherapy after chemotherapy. Several methods have been pursued as immunotherapies for AML: peptide vaccines, granulocyte-macrophage colony-stimulating factor-secreting tumor vaccines, dendritic cell vaccines, and adoptive T cell therapy. Whereas immunogenicity and clinical outcomes are improving in these trials, severe adverse events were observed in highly avid engineered T cell therapies, indicating the importance of the balance between effectiveness and side effects in advanced immunotherapy. Such progress in inducing antitumor immune responses, together with strategies to attenuate immunosuppressive factors, will establish immunotherapy as an important armament to combat AML. The immune system has an exquisite ability to specifically kill cells that express particular antigens. This specificity is the heart of immunotherapies that eliminate tumor cells without damaging normal cells. Recent advances in immunology research have revealed many facets in the immune system that are important to develop tumor immunotherapy. At the same time, recent studies have identified many promising acute-myeloid-leukemia- (AML-) associated antigens that can be targeted by immunotherapy. The combination of such advancement may enable antigen-specific immunotherapies to be established as a viable choice of therapy for AML.Here we review recent advance in antigen-specific autologous immunotherapy for AML and raise several issues to overcome in order to improve clinical efficacy in the future. This review excludes graft-versus-leukemia (GVL) effects exploited in allogeneic hematopoietic stem cell transplantation, which are mainly allogeneic immune reactions.Cytotoxic chemotherapy and allogeneic transplantation are practically the only two modalities to treat AML. Chemotherapy has inevitable limitations of effectiveness due to chemoresistance in the majority of AML patients, except for a small fraction of patients with favorable karyotypes. Allogeneic transplantation is inherently accompanied by a variety of life-threatening complications related to graft-versus-host disease (GVHD), which limits candidates to younger and fit patients. Given such situations, immunotherapy may potentially play an important part in the treatment for AML, mainly from the following two standpoints. Although it is possible to achieve complete remission by a series of initial chemotherapies in about 80% of AML patients, recurrence is inevitable in the majority of the patients without allogeneic transplantation. It has been reported that leukemia stem cells are resistant to chemotherapy and that it may be an important reason why it is difficult to eradicate AML cells in the majority of patients . This neAML most often occurs in elderly people. It has been reported that 2-year overall survival rate of elderly patients with AML is only 6% [Recent studies have identified several promising AML antigens suitable for targets of immunotherapy.∗2402- [∗0201- [Wilms' tumor 1 (WT1) is one of the most promising AML-associated antigens. It was originally reported that HLA-A∗2402-  and HLA- [∗0201- , and thu [∗0201- –10, indi∗0201-restricted proteinase 3 peptides induce CTLs that preferentially kill myeloid leukemia cells compared to normal marrow cells [Proteinase 3 is a myeloid cell-restricted serine protease abundantly expressed in azurophilic granules and is another promising myeloid leukemia-associated antigen. It was originally reported that HLA-Aow cells . Proteinow cells , 9, 12. Other than WT1 and proteinase 3, the receptor for hyaluronic-acid-mediated motility RHAMM/CD168) , human t8 , humanAntigen-specific immunotherapies can be largely divided into two categories: active immunization and adoptive T cell therapy. In active immunization, tumor antigens are injected in order to provoke antigen-specific immune responses in vivo. To do so, there are mainly the following three ways reported for AML: peptide vaccines, granulocyte-macrophage colony-stimulating-factor- (GM-CSF) secreting tumor vaccines, and dendritic cell (DC) vaccines. ∗2402-restricted WT1 peptide vaccination for malignancies including 12 AML patients [Peptides in combination with an appropriate adjuvant are injected to stimulate CD8+ CTLs specific to the MHC-class-I-restricted peptides. WT1 peptide vaccines have been actively pursued. Oka et al. have first reported a clinical trial of HLA-Apatients . Among 8patients . This im∗0201-restricted WT1 peptide vaccination for 19 AML or MDS patients, most of whom had large tumor burden [Keilholz et al. reported a clinical trial of HLA-Ar burden . They ob∗0201-restricted WT1 and proteinase 3 peptides to 8 patients with myeloid malignancies [Rezvani et al. reported a clinical trial of combined administration of HLA-Agnancies . Immune ∗0201-restricted heteroclitic peptide that has higher affinity to the HLA class I molecule than a native peptide, and three are long peptides that bind to multiple HLA-DRB1 haplotypes [∗0201-restricted heteroclitic peptide in it. The combination of peptides has three potential advantages. First, the heteroclitic peptide is expected to stimulate low avidity tumor-specific CD8+ T cells that preferentially remain in cancer patients. Second, the MHC-class-II-binding peptides can exploit CD4+ T cell help that is required to induce a robust memory CD8+ T cell response. Third, it has been reported that a long peptide containing an MHC-class-I-restricted peptide can be preferentially targeted to professional antigen-presenting DCs that are capable of presenting the embedded MHC-class-I-restricted peptide for a long period [Maslak et al. reported a clinical trial of a novel combination of WT1 peptide vaccination for 9 AML patients . They usplotypes . Interesg period . This mag period . It is dSchmitt et al. reported a clinical trial of the HLA-A2-restricted RHAMM peptide vaccination in patients with AML, MDS, and multiple myeloma . In 7 ofCollectively, these clinical trials of peptide vaccines have established safety and immunogenicity of this modality. Efforts to improve clinical efficacy by combining with superior adjuvants or with other therapeutic modalities will increase the potential of peptide vaccines for AML.Random mutations in tumor cells are expected to generate many individually specific antigens that may induce multivalent antitumor immune responses of both CD4+ and CD8+ T cells. Thus, the whole autologous tumor cell vaccination is a viable option as long as a sufficient number of tumor cells are harvested in advance.A mixture of killed autologous leukemia cells and a GM-CSF gene-transduced K562 leukemia cell line was used for vaccination in combination with primed T cells after autologous stem cell transplantation for 54 patients with AML . LeukemiAutologous leukemia cells transduced with GM-CSF were administered after allogeneic transplantation to 28 patients with AML or high-risk MDS . VaccinaDCs generated ex vivo from monocytes or CD34+ progenitor cells are modified to present tumor antigens and are injected. It has also been reported that AML cells can be differentiated into DCs and they can be injected.In the first DC vaccination for AML, Fujii et al. used CD34+ progenitor cell-derived DCs pulsed with autologous leukemic cells in combination with primed T cells for 4 relapsed patients after allogeneic stem cell transplantation . This meClinical trials of DC-based immunotherapy for AML using leukemic cell-derived DCs have also been reported , 32. HowLee et al. reported the first study of MoDC-based immunotherapy for 2 AML patients with relapse after autologous peripheral blood stem cell transplantation . Althoug∗2402-positive patient, indicating cross-priming in vivo. In another study, we administered MoDCs pulsed with an HLA-A∗2402-restricted modified WT1 peptide that has higher affinity to the HLA molecule than the natural peptide to 3 patients [We recently reported two clinical studies of MoDC-based immunotherapy for AML at morphologic complete remission in elderly patients. In one study, we administered MoDCs that engulfed autologous apoptotic leukemic cells to 4 patients . We obsepatients . We obseCollectively, these studies indicate that MoDC-based immunotherapy is immunogenic even in elderly patients with AML after remission-inducing chemotherapy and warrant further study of this strategy.Active immunization relies on immune competence of cancer patients. However, tumor antigen-specific T cells may be nonfunctional or deleted in the presence of tumor cells in vivo in cancer patients . In addiTumor-specific adoptive T cell therapy was initially developed by expanding tumor-infiltrating lymphocytes from melanoma lesions in vitro . However∗0201-restricted MART-1, gp100, NY-ESO-1, and p53 as targeted antigens [The first clinical trial of TCR-transduced T cell transfer was performed to advanced melanoma patients by Rosenberg's group, using HLA-Aantigens . The tra∗0201-restricted MART-1 and gp100 peptides [∗0201+ patients with metastatic colorectal cancer [Subsequently, the same group reported a clinical trial using high avidity TCR against HLA-Apeptides . Objectil cancer . All patThese promising results of adoptive transfer of TCR-transduced T cells for solid tumors pave the way for its application to hematological malignancies. Two groups reported mouse models of adoptive transfer of T cells with WT1-specific TCR genes , 45. BotIn the T cells transduced with a new TCR gene, their original TCRs are still functional, and thus mispairing of endogenous and introduced TCR chains occurs. This decreases the expression level of introduced TCR, resulting in reduced antitumor activity . In addiA CAR contains an extracellular antigen-binding domain, a transmembrane region, and a signaling endodomain. The extracellular domain is typically a single chain variable fragment (scFv) derived from a tumor-specific monoclonal antibody. There are two advantages of using an antibody-derived domain for antigen recognition. First, antibodies are not dependent on MHC presentation. Second, antibodies bind antigens with much greater affinity than TCRs, permitting the formation of a more stable immunological synapse.ζ chain alone, which transmits a signal inadequate to fully activate T cells. In second-generation CARs, the CD28 intracellular domain is inserted proximal to the CD3ζ endodomain to enhance the stimulatory effects of the CAR. This encouraged further addition of other signaling sequences from costimulatory molecules such as 4-1BB and OX40 in third-generation CARs. A complete response was observed in a patient with follicular lymphoma who received T cells transduced with a second-generation anti-CD19 CAR [CARs can be grouped into three generations with progressively increasing costimulatory activity. These differ primarily in the structure of the signaling endodomain. First-generation CARs contain a single signaling unit derived from the CD3CD19 CAR . HoweverCD19 CAR  and anotCD19 CAR . AlthougY is expressed in about 50% of multiple myeloma and AML cases. LewisY CAR-transduced T cells delayed growth of myeloma xenografts in NOD/SCID mice [Y CAR T cell transfer is a promising therapy for myeloma and AML.The carbohydrate antigen LewisCID mice . This paAdvances in immunology and identification of promising leukemia-associated antigens are making it possible to develop truly effective immunotherapies for AML. Fortunately, AML is relatively more chemosensitive than most solid tumors, and thus it is possible to reduce a tumor burden by chemotherapy in the majority of patients. Thereafter, immunotherapy will play a complementary role in eradicating minimal residual diseases, which contain chemoresistant leukemia stem cells. Thus, leukemia-associated antigens expressed in leukemia stem cells will be important to achieve cure. Recent concerns are immunosuppressive factors expressed by tumor cells or built in the immune system, which curtail antitumor immunity. Universal immunosuppressive factors built in the immune system, such as CTLA-4 , 56, PD-Furthermore, epigenetic therapies with DNA methyltransferase and histone deacetylase inhibitors are prevailing as novel therapies for myeloid malignancies. Notably, recent studies have shown that epigenetic modification upregulates the expression of cancer testis antigens in AML and induces CTL responses , 63. Thi


It is also occasionally used to activate eggs in the clinics practicing in vitro fertilization. However, neither the precise molecular action of ionomycin nor its secondary effects on the eggs' structure and function is well known. In this communication we have studied the effects of ionomycin on starfish oocytes and zygotes. By use of confocal microscopy, calcium imaging, as well as light and transmission electron microscopy, we have demonstrated that immature oocytes exposed to ionomycin instantly increase intracellular Ca2+ levels and undergo structural changes in the cortex. Surprisingly, when microinjected into the cells, ionomycin produced no Ca2+ increase. The ionomycin-induced Ca2+ rise was followed by fast alteration of the actin cytoskeleton displaying conspicuous depolymerization at the oocyte surface and in microvilli with concomitant polymerization in the cytoplasm. In addition, cortical granules were disrupted or fused with white vesicles few minutes after the addition of ionomycin. These structural changes prevented cortical maturation of the eggs despite the normal progression of nuclear envelope breakdown. At fertilization, the ionomycin-pretreated eggs displayed reduced Ca2+ response, no elevation of the fertilization envelope, and the lack of orderly centripetal translocation of actin fibers. These alterations led to difficulties in cell cleavage in the monospermic zygotes and eventually to a higher rate of abnormal development. In conclusion, ionomycin has various deleterious impacts on egg activation and the subsequent embryonic development in starfish. Although direct comparison is difficult to make between our findings and the use of the ionophore in the in vitro fertilization clinics, our results call for more defining investigations on the issue of a potential risk in artificial egg activation.Ionomycin is a Ca It has been demonstrated that Ca2+-linked second messengers such as InsP3 , cADPr (cyclic ADP ribose) and NAADP (nicotinic acid adenine dinucleotide phosphate) are the mediators of the intracellular Ca2+ release in response to various stimuli 2+ waves inside fertilized eggs 2+ levels can also be conveniently elevated by use of ionophores. Ionomycin is a widely used Ca2+-selective ionophore that has been isolated from the bacterium Streptomyces conglobatus2+ in one-to-one stoichiometry and promotes mostly electrically neutral exchange of Ca2+ for 2H+ or other divalent cations such as Mg2+, and thereby transports Ca2+ ions across the vesicle membranes or through the water-lipid interface 2+-selective ionophore, ionomycin is known to be more specific and potent than A23187 2+ levels inside an integral living cell is not fully understood and still remains controversial 2+ influx, or act on the intracellular organelles to release Ca2+. Alternatively, it may take an indirect route either to potentiate the existing Ca2+-mobilizing mechanisms or to activate other enzymes such as phospholipase C (PLC) that in turn produces the Ca2+-mobilizing second messenger InsP3. The alternative actions of ionomycin might depend on its concentration, as ionomycin has predominantly ionophoretic effects at >1 µM In physiological conditions, intracellular Ca2+ from internal stores 2+ mobilization has not been fully established because artificial elevation of intracellular Ca2+ levels with A23187 did not induce maturation by itself 2+ signals with calcium chelators blocked GV breakdown and the meiotic progress in starfish oocytes 2+ signaling that cannot be mimicked by the Ca2+ ionophore, as was exemplified in the Ca2+-dependent cytoskeletal changes in neuronal growth cones 2+ increase by ionophores might have caused other structural and functional changes in cells.As an experimental model, starfish provide a synchronous population of oocytes that are arrested at the prophase of the first meiotic division. These immature oocytes characteristically containing a large nucleus termed ‘germinal vesicle (GV)’ can be induced to reenter the cell cycle (meiotic maturation) by exposure to the maturation hormone, 1-methyladenine (1-MA). At the onset of meiotic maturation, starfish oocytes readily (<2 min after 1-MA addition) releases Ca2+ levels with ionomycin immediately led to drastic alteration of the actin cytoskeleton in the immature oocytes, which exhibited depolymerization of the actin meshwork in the subplasmalemmal regions along with highly enhanced polymerization and bundling of the actin filaments in the inner cytoplasm. These cytoskeletal changes were accompanied by retraction of the microvilli at the oocyte surface and the loss of cortical granules that were either engulfed by other vesicles or exocytosed to the perivitelline space, which mimicked the changes at egg activation. Ionomycin neither provoked nor inhibited breakdown of the nuclear envelope, which is the hallmark of the meiotic maturation. However, the oocytes' history of being briefly exposed to ionomycin prevented completion of the other processes of meiotic maturation: the structural changes in the ectoplasm of the oocytes 3 with no elevation of the fertilization envelope. The monospermic zygotes derived from these eggs tended to have problems in cell cleavage and showed a higher rate of abnormal development. The detrimental effect of ionomycin on the early stage of development was also confirmed in the monospermic zygotes briefly exposed to ionomycin.In this communication, we have studied the effects of ionomycin on egg activation and subsequent development in starfish. Since ionomycin by itself activates mature eggs, we have used the GV stage oocytes briefly exposed to the ionophore, and followed the consequences in meiotic maturation, fertilization, and early development. We observed that artificial elevation of intracellular CaAstropecten aranciacus were microinjected with Calcium Green and Rhodamine Red and examined with the CCD camera to monitor the changes of cytosolic Ca2+ levels in response to 5 µM ionomycin. In the artificial seawater containing 10 mM Ca2+ (ASW), oocytes promptly started to increase the intracellular Ca2+ level after the addition of ionomycin. The Ca2+ signal surpassed half the maximal level within 90 sec and arrived at the plateau by 5 min  at the initial stage of the Ca2+ rise (10.5±2.9 sec) (2+-free seawater (CaFSW), however, the Ca2+ response came out with a significantly longer latent period after the addition of ionomycin  in comparison with the oocytes in ASW , as shown in 2+ influx from outside 2+, the ionomycin-exposed oocytes released Ca2+ from the internal stores, but the amplitude of the Ca2+ peak was significantly lower  than that in the Ca2+-containing seawater . In addition, the Ca2+ level inside the oocytes exposed to 5 µM ionomycin in CaFSW fell to the basal level within 15 min  , arrow. n 15 min , whereasd levels . Hence, We noted that the starfish oocytes at the GV stage often responded to ionomycin with slight elevation of the vitelline layer , as was 2+ rise, the actin cytoskeleton underwent highly accelerated rearrangement. Specifically, in the inner cytoplasm, the actin cytoskeleton became extensively polymerized, forming much longer and thicker microfilament bundles and patches. In the subplasmalemmal region, however, the typical actin filaments that are intimately associated with the plasma membrane . As shown in 2+ increase. The failure of the second dose of ionomycin to evoke a Ca2+ response is not due to the potential acidification of the eggs and the reported inefficacy of ionomycin at low pH; unlike ionomycin that has no Ca2+-complexing activity below pH 7.0, A23187 maintains its Ca2+ ionophore activity at pH 5 to 10 2+ increase in control eggs, the ionomycin-pretreated eggs at the GV stage did not mobilize Ca2+ . Thus, the mobilized Ca2+ had mostly returned or was at least en route to its original stores. Although not responding to the second exposure to ionomycin nor A23187 with Ca2+ release  was consistently lower (by 43%) than that of the control mature eggs . Thus, the alteration of the egg cortex, e.g. retraction of microvilli and disruption of cortical granules  and the ionomycin-pretreated eggs   in comparison with the control eggs in the same batch of experiment . Similar to the results obtained with uncaged InsP3, the peak amplitude of the sperm-induced Ca2+ transient in the eggs that had been exposed to ionomycin at the GV stage was about 35.9% lower  than that of the control eggs   was not significantly different from that in the control , implying that the intracellular mechanism that sustain the propagation of the Ca2+ wave remained intact in these eggs (p<0.05) (p = 0.0005)  . It is nll cases . In addiof cases . Even wh<0.0001) . Again, ese eggs . Another(p<0.05) . The amp 0.0005) . Hence, 3 and fertilizing sperm  using the same method produced high level of Ca2+ response as was expected (2+ even with 10 times higher dose (500 µM in the injection pipette), and the same negative result was observed with the immature oocytes (not shown).Ionomycin-pretreated eggs responding to InsPng sperm  and 7 bung sperm  and 5 raexpected . Microinp = 0.7614). To test the effect of the ionomycin-pretreatment on early development, we followed the fate of the monospermic zygotes. Firstly, all the zygotes from the ionomycin-pretreated eggs underwent their developmental process without being shielded by the thick fertilization envelope that is normally seen in the control  (p<0.05)  p<0.001) . In the p<0.001) . After 3(p<0.05) .2+ levels in cell biology laboratories. On more rare occasions, it has also been used as a part of the standard protocol to activate the eggs that do not respond to ICSI in the in vitro fertilization clinics 2+ in the oocytes. Owing to the large cell size, we were able to visualize the spatiotemporal changes of the intracellular Ca2+ levels following ionomycin treatment. With the removal of the external Ca2+ in the media, the peak of the Ca2+ response by the immature oocytes were merely 53.4% of the level in the Ca2+-containing seawater, implying a significant contribution of the influx to the Ca2+ rise  of that in the ASW  containing 10 mM Ca2+. Hence, with meiotic maturation, mobilization of Ca2+ from internal stores seems to make more contribution to the net intracellular Ca2+ increase by ionomycin. We have then shown that the rapid increase of intracellular Ca2+ was accompanied by accelerated rearrangement of the actin cytoskeleton, which exhibited depolymerization near the plasma membrane with concomitant massive polymerization and bundling in the inner cytoplasm  2+ ion, once bound and buried in the groove of F-actin, is virtually inaccessible for exchange 2+ increase at least in part 2+ upheaval. On the other hand, depolymerization of actin in the subplasmalemmal region might be involved in the regulation of store-operated Ca2+ entry that was reported to take place in ionomycin-exposed cells Since its first discovery in the 1970s, ionomycin has been widely used to increase intracellular Caa2+ rise . This diytoplasm . This is2+ increase and the drastic reorganization of the actin cytoskeleton are accompanied by the formation of the large white vesicles, which are often vested with actin filaments (Pisaster ochraceus) undergoing atresia to give room to other growing oocytes Arbacia punctulata2+ and polyphosphate. While the large white vesicles in the ionomycin-pretreated oocytes might be indicative of a cell stress after cytoskeletal rearrangement and the prolonged elevation of intracellular Ca2+ levels, yet to be resolved is the question about the physiological role of the ‘small’ white vesicles seen in the control oocytes.We have also shown that the ionomycin-induced massive Cailaments . This tyilaments  found in2+ rise, cortical granules are either exocytosed or engulfed by large white vesicles (2+ increase. Our result is in line with the early studies in which Xenopus oocytes did not produce Ca2+-dependent Cl− currents in response to the second dose of ionomycin if the oocytes had been previously exposed to the suprathreshold (1 µM) level of ionomycin Xenopus oocytes could be overcome by 50 min of washing, but our starfish eggs did not show any sign of recovery from the refractory phase 70 min after the removal of ionomycin in the media. Thus, we were not able to establish whether the refractory phase of ionomycin is reversible in starfish eggs. In contrast to the apparent loss of the responsiveness to ionomycin, the eggs of the same treatment (5 min exposure to ionomycin at the GV stage) still responded to InsP3 and fertilizing sperm with substantial increase of intracellular Ca2+ , our result might suggest that, to transport Ca2+ ions across the membrane, ionomycin should be added to the side of higher Ca2+ concentration. Alternatively, Ca2+ is transported into the cell from outside by ionomycin and this priming Ca2+ increase may ignite further Ca2+ release in an indirect mechanism either by inducing calcium-induced calcium release (CICR) or activating Ca2+-dependent enzymes such as phospholipase C. However, we have seen no evidence of PIP2 hydrolysis after ionomycin-induced Ca2+ increase inside starfish oocytes in an assay using PH-GFP (data not shown), which is at variance with the results obtained with smaller cells such as fibroblasts Our results in this communication raise a question about the way how the Calization , might bthe eggs . Being p2+ ionophore ionomycin might have a detrimental effect on both egg activation and the early embryonic development. Starfish eggs that had been briefly exposed to ionomycin at the GV stage may still undergo meiotic maturation process at least in the nucleus, but the resulting eggs displayed several problems at fertilization. First, these eggs had difficulties in producing effective sperm interaction at fertilization. While generation of the initial Ca2+ spot underneath the egg plasma membrane is considered as one of the indices for a meaningful sperm-egg interaction at fertilization 2+ waves in the ionomycin-pretreated eggs required more than three times longer latent period than in the control  were maintained in circulating cold seawater (16°C). The female gonads were dissected from the central dorsal area near the arms and transferred to the filter-sterilized cold seawater. Free individual oocytes were isolated by repeatedly passing through gauze and rinsing in cold filtered seawater. Oocytes were collected by low speed  sedimentation in each step. Immature oocytes obtained in this way were marked by the presence of the large nucleus . Ionomycin and InsP3 were purchased from Invitrogen. All other chemicals were purchased from Sigma-Aldrich unless stated otherwise, and all solutions were prepared following the manufacturers' instruction. Certain experiments were performed in artificial seawater  or Ca2+-free seawater . Hoechst 33342 was purchased from Sigma and the final concentration of 5 µM was used to stain sperm for 1 min before insemination. The number of egg-incorporated sperm was counted in epifluorescence microscopy.Captured starfish (3 (Molecular Probes), microinjected eggs were irradiated with 330 nm UV light by using the computer-controlled shutter system Lambda 10-2 . Alexa Fluor 488-phalloidin (Molecular Probes) was injected as described previously 2+ imaging work in this study was done by use of epifluorescence microscopy. Cytosolic Ca2+ changes were detected using a cooled CCD camera  mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 20x/0.50 objective. The quantified Ca2+ signal at a given time point was normalized to the baseline fluorescence (F0) following the formula Frel = [F−F0]/F0, where F represents the average fluorescence level of the entire oocyte. Fluorescence of Ca2+ images were analyzed with the MetaMorph Imaging System software .Microinjection of the oocytes was performed with an air-pressure Transjector (Eppendorf), following the procedure that was previously described in detail The numerical MetaMorph data were compiled and analyzed with Excel of Microsoft Office 2003. The average and variation of the data were reported as ‘mean ± standard deviation (SD)’ in all cases. The paired t-test and the one-way ANOVA were performed by use of Prism 3.0 , and the P-values smaller than 0.05 (P<0.05) were considered statistically significant.Starfish oocytes and eggs to be analyzed following the ionomycin and 1-MA treatment were fixed together with proper control samples in filtered seawater containing 1% glutaraldehyde (pH 8.0) for 1 h at room temperature and then post-fixed with 1% osmium tetroxide for 1 h. Specimens were dehydrated in increasing concentrations of alcohol and embedded in EPON 812. The polymerized resin containing the fixed material was sectioned in two ways. Semi-thin (1 micron) sections were stained with toluidine blue and examined by light microscopy with a Zeiss Axiovert 200 microscope, and the images were captured by the CCD camera. For TEM, ultrathin sections were stained with 2% uranyl acetate and 0.2% lead citrate, and examined with a LEO 912 AB energy filter transmission electron microscope.GV stage oocytes and eggs microinjected with Alexa Fluor 488–conjugated phalloidin were viewed with a Zeiss LSM 510 META Laser Scanning Confocal Microscope  with excitation at 488 nm and emission at 500/555 nm. A Plan-Neofluar 25x/0.80 objective water lens was used to produce optical slices from the specimens. The images were captured as digital computer files and examination of the fluorescence was performed using MetaMorph image analysis software.Data S1Translocation of white vesicles during egg maturation and activation. (A) Light microscope images of immature oocytes of A. aranciacus show that white vesicles are mainly located in the cortex but not tightly packed underneath the plasma membrane (white arrows). In sharp contrast, white vesicles in the mature eggs are closely associated with plasma membrane just like cortical granules. Note that the white vesicles are largely eliminated in the activated egg that underwent massive exocytosis and the elevation of the vitelline layer. (B) TEM image of white vesicles containing remnants of fibrillary contents in the normal eggs of Astropecten aranciacus at fertilization. As a result of cortical granules (black arrow) exocytosis and microvilli (gray arrow) elongation, the fertilization envelope was fully elevated . PS, perivitelline space. The white vesicle fusing with the plasma membrane is marked by a white arrow. Scale bar 1 µm.(PDF)Click here for additional data file.


Low-Ca2+ fresh water stimulated Ca2+ influx and expressions of epithelial calcium channel (ecac), vitamin D-25-hydroxylase (cyp2r1), vitamin D receptor a (vdra), and vdrb in zebrafish. Exogenous vitamin D increased Ca2+ influx and expressions of ecac and 3-24-hydroxylase (cyp24a1)25-hydroxyvitamin D, but downregulated 1α-OHase (cyp27b1) with no effects on other Ca2+ transporters. Morpholino oligonucleotide knockdown of VDRa, but not VDRb, was found as a consequence of calcium uptake inhibition by knockdown of ecac, and ossification of vertebrae is impaired. Taken together, vitamin D-VDRa signaling may stimulate Ca2+ uptake by upregulating ECaC in zebrafish, thereby clarifying the Ca2+-handling function of only a VDR in teleosts. Zebrafish may be useful as a model to explore the function of vitamin D-VDR signaling in Ca2+ homeostasis and the related physiological processes in vertebrates.The purpose of the present study was to use zebrafish as a model to investigate how vitamin D and its receptors interact to control Ca Decrease of renal Ca2+ reabsorption and intestinal Ca2+ absorption is an important factor causing osteoporosis 2+ uptake is vital to sustain life and enable bone formation 2+ uptake is highly essential to vertebrate life. The major source of Ca2+ in terrestrial vertebrates is from food. Fish, unlike terrestrial vertebrates, continually face ambient water with variable Ca2+ levels and absorb Ca2+ from the surrounding water. In adult fish, the predominant route of Ca2+ entry from the environment is across the gill epithelium, while in larvae, the body skin is the major route of Ca2+ uptake before full development of the gills occurs 2+ uptake in specific cells and organs. According to the current model in mammals and teleosts, active transcellular Ca2+ transport is carried out through the operation of apical epithelial Ca2+ channels (ECaC), and the basolateral plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+ exchanger (NCX) In vertebrates, one of physiological roles of Ca2+ uptake in mammals. A vitamin D precursor is initially synthesized in the skin. Through a series of reactions, vitamin D-25 hydroxylase (CYP2R1) converts the vitamin-D precursor into 25-hydroxyvitamin D3 (25(OH)D3), which is then converted to 1α,25-dihydroxytamin D3 2D3), the active form of vitamin D, by renal 1α-OHase (CYP27B1) 2D3 level is modulated by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1). CYP24A1, a mitochondrial enzyme in target cells, functions to degrade 1α,25(OH)2D32D3 in the kidneys and degradation of this hormone at peripheral sites are associated with the homeostasis of 1α,25(OH)2D3 in mammals. Although 1α,25(OH)2D3 was also detected in the lamprey, one of the earliest vertebrate lacking a calcified skeleton and teeth, it was found to play a non-calcemic role there 3 may initially have evolved a Ca2+ regulatory function in bony vertebrates. From evolutionary and physiological points of view, teleosts have been an important model to explore the hypothesis of whether vitamin D also has a calcemic function in bony vertebrates because Ca2+ uptake mechanisms of teleosts were demonstrated to be similar to those of mammals as described above. In teleosts 1α,25(OH)2D3 was also demonstrated to be produced by renal tissues and the liver 2+ level in carp and cod 2+ turnover 2D3 concentrations and expressions of vitamin D receptor (VDR) were noted in Atlantic salmon undergoing smoltification and migrating from fresh water (with low Ca2+ concentrations) to seawater (with high Ca2+ concentrations), suggesting that regulation of the synthesis of 1α,25(OH)2D3 and VDR is dependent upon ambient Ca2+ concentrations 2+ uptake function in teleosts are still largely unclear.Vitamin D is well documented as vital endocrine regulating Ca3-VDR complex upon binding with vitamin D. This complex could upregulate mammalian intestinal ecac translation by binding the vitamin D3-responsive element (VDRE) in the promoter region of ecac2+ absorption, which is an important pathway for controlling Ca2+ homeostasis in mammals Takifugu rubripes), putative VDREs were also identified by a bioinformatics analysis 2D32D3 and induced different transcripts of VDRE-containing expression constructs in cell lines 2D3, and the 2 paralogous VDRs may undergo functional divergence in teleosts; however, these notions lack in vivo molecular/physiological evidence to support them.The vitamin D receptor (VDR), a ligand-activated transcription factor, forms a vitamin D2+ uptake function 2+ uptake mechanisms. The purpose of the present study was to use zebrafish to clarify the molecular physiological mechanisms of vitamin D control of epithelial Ca2+ transport in teleosts. Experiments were designed to address 2 specific questions: (1) does the vitamin D system control zebrafish Ca2+ uptake function by regulating the Ca2+ transporters, ecac, ncx1b, and/or pmca2? and (2) does vitamin D control zebrafish Ca2+ uptake function through 1 or both of the paralogous VDRs, VDRa and VDRb? Hence, the effects of environmental Ca2+ levels and exogenous vitamin D on Ca2+ contents and influx, and the mRNA expressions of Ca2+ transporters  and vitamin D-related genes  were investigated. Moreover, the effects of knockdown of VDRa or VDRb on Ca2+ contents/influx and the expression of Ca2+ transporters and Ca2+-regulatory endocrines in zebrafish embryos were also examined.Zebrafish with a well-established genomic database and advantages of morpholino gene knockdown technique is a competent model for research on ion regulation and related endocrine controls Danio rerio) were kept in local tap water ([Ca2+] of 0.2 mM) at 28.5°C under a 14∶10-h light-dark photoperiod at the Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan. Experimental protocols were approved by the Academia Sinica Institutional Animal Care and Utilization Committee .The wild-type AB strain of zebrafish (2+ (0.02 mM) levels was prepared with double-deionized water  supplemented with adequate CaSO4·2H2O, MgSO4·7H2O, NaCl, K2HPO4, and KH2PO4. Ca2+ concentrations  of high- and low-Ca2+ media were 2 and 0.02 mM, respectively, but the other ion concentrations of the 3 media were the same  as those in local tap water. Variations in ion concentrations were maintained within 10% of the predicted values. Fertilized zebrafish eggs were transferred to high- and low- Ca2+ media, respectively, and incubated thereafter until sampling at 3 or 5 d post-fertilization (dpf).Artificial fresh water with high- (2 mM) or low-Ca2D3  was dissolved in 95% ethanol at 0 (control) and 20 µg/l (0.48 nM). Zebrafish embryos were incubated in 1α,25(OH)2D3-containing media immediately after fertilization, and were sampled at 3 dpf for subsequent analysis. Incubation media were changed with a new 1α,25(OH)2D3 solution every day to maintain constant levels of 1α,25(OH)2D3. During incubation, neither significant mortality nor abnormal behavior was found.1α,25(OH)3 (13.1 N) was added to samples for digestion at 60°C overnight. Digested solutions were diluted with double-deionized water, and the total calcium content was measured with a Z-8000 atomic absorption spectrophotometer . Standard solutions  were used to make the standard curves.Three-dpf zebrafish embryos were anesthetized with 0.2% buffered MS-222 (Sigma) and then briefly rinsed in deionized water. Thirty individuals were pooled as 1 sample. HNO45Ca2+ -containing medium for a subsequent 4-h incubation. After incubation, embryos were washed 4 times in fresh water without isotope. Six embryos were pooled into 1 vial, anesthetized with 0.2% buffered MS-222, and digested with tissue solubilizer  at 60°C for 8 h. The digested solutions were supplemented with counting solution , and the radioactivities of the solutions were counted with a liquid scintillation beta counter . The Ca2+ influx was calculated using the following formula: Jin = Qembryo Xout−1t−1; where Jin is the influx (pmol·h −1), Qembryo is the radioactivity of the embryo  at the end of incubation, Xout is the specific activity of the incubation medium (cpm/pmol), t is the incubation time (h), The influx was expressed in pmol·mg−1·h−1 by dividing Jin by the embryo wet weight (mg). Both the data of Qembryo and W were from pooled samples, and the averaged values were used during calculation.By following previously described methods g for 30 min, supernatants were obtained. Samples were mixed with an equal volume of isopropanol. Pellets were precipitated by centrifugation at 4°C and 12,000×g for 30 min, washed with 70% alcohol, and stored at −20°C until use.After anesthetization with 0.03% MS222, appropriate amounts of zebrafish tissues or embryos were collected and homogenized in 1 ml Trizol reagent , then mixed with 0.2 ml chloroform, and thoroughly shaken. After centrifugation at 4°C and 12,000×20, 250 ng random primers, 5 mM dithiothreitol, 40 units RNase inhibitor, and 200 units Superscript RT (Invitrogen) for 1 h at 50°C followed a 70°C incubation for 15 min. For PCR amplification, 2 µl cDNA was used as a template in a 50-µL final reaction volume containing 0.25 mM dNTPs, 2.5 units Taq polymerase , and 0.2 µM of each primer (For complementary (c)DNA synthesis, 1∼5 µg of total RNA was reverse-transcribed in a final volume 20 µl containing 0.5 mM dNTPs, 2.5 µM oligo (dT)h primer . Thirty A qPCR was performed with a Light Cycler real-time PCR system  in a final volume of 10 µl, containing 5 µl 2x SYBR Green I Master Mix (Roche), 300 nM of the primer pairs, and 20∼30 ng cDNA. The standard curve for each gene was checked in a linear range with β-actin as an internal control. The primer sets for the qPCR are shown in Danio rerio) VDRa (NP_570994), zebrafish VDRb (NP_001153457), flounder  VDRa (BAA95016), flounder VDRb (BAA95015), frog (Xenopus laevis) VDR (NP_001079288), chicken  VDR (NP_990429), human (Homo sapiens) VDR (BAH02291), medaka (Oryzias latipes) VDRα (NP_001121988), medaka VDRβ (NP_001121989), Nile tilapia (Oreochromis niloticus) VDRa (XP_003449167), Nile tilapia VDRb (XP_003441588), Atlantic salmon VDR (NP_001117029), stickleback (Gasterosteus aculeatus) VDRa (ENSGACT00000006308), stickleback VDRb (ENSGACT00000010601), seabass (Dicentrarchus labrax) VDRb (CBN80914), mouse (Mus musculus) VDR (NP_033530), lizard (Anolis carolinensis) VDR (ENSACAT00000013576), and lamprey (Petromyzon marinus) VDR (AAP05810). Alignment of the amino-acid sequences was conducted using ClustalW via the SDSC Biology Workbench (http://workbench.sdsc.edu). Phylogenetic analyses were carried out using the Neighbor-joining method. Six hundred bootstrap replicate analyses were carried out with MEGA5.0 software.All protein sequences were obtained from the Ensembl and NCBI databases. The accession number of the sequence were as follows: zebrafish  or vdra  fragments were obtained by a PCR and inserted into the pGEM-T easy vector . The inserted fragments were amplified with the T7 and SP6 primers by a PCR, and the products were used as templates for in vitro transcription with T7 and SP6 RNA polymerase (Roche) in the presence of digoxigenin (DIG)-UTP (Roche) to synthesize sense and antisense probes, respectively. Zebrafish embryos were anesthetized on ice and fixed with 4% paraformaldehyde (PFA) in a phosphate-buffered saline solution  at 4°C overnight. To perform the in situ hybridization, we followed a previous description ecac-expressing cells in 12 randomly-selected areas (100×100 µm each) on the yolk-sac surface of an embryo were counted.Zebrafish 5′- AACGGCACTATTTTCCGTAAGCATC-3′) and VDRb MO (5′- AACGTTCCGGTCGAACTCATCTGGC-3′) were prepared with 1x Danieau solution 2, and 5.0 mM HEPES; pH 7.6). A standard control MO (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was used as the control. To analyze the physiological function of VDRa/b under normal development, we chose to inject 4 ng/embryo of the MO into embryos in the following experiments. At this dose, neither significant mortality nor abnormal behavior was found. The MOs (4 ng/embryo) were injected into embryos at the 1∼2-cell stage using an IM-300 microinjector system . MO-injected embryos at 3 dpf were sampled for subsequent analyses.The zebrafish VDRa MO (Thirty embryos were pooled as 1 sample and homogenized. Protein at 50 µg/well was loaded onto 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V for 2 h. After separation, proteins were transferred onto a polyvinylidene difluoride membrane  at 100 V for 2 h. After being blocked for 1.5 h in blocking buffer, blots were incubated with zebrafish VDRa or VDRb polyclonal antibodies overnight at 4°C, diluted 1∶1000 with alkaline-phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgG)  for another 2 h. Blots were developed with 5-bromo-4-chloro-3-indolylphosphate/nitro-blue tetrazolium.Zebrafish embryos were incubated in the staining solution  for 10 min. After incubation, embryos were washed with fresh water, and then euthanized in MS-222. Observations were carried out using a microscope with a green-fluorescence filter set.Fresh zebrafish gills were fixed with 4% PFA at 4°C for 3 h and then immersed in PBS containing 5%, 10%, and 20% sucrose for 15 min each at room temperature. Finally, gills were soaked in a mixed PBS solution (OCT compound: 20% sucrose at 1∶2) overnight and embedded with OCT compound embedding medium  at 20°C. Cryosections at 6 µm were made with a cryostat , and these were placed onto poly-L-lysine-coated slides .Prepared slides were rinsed in PBS and blocked with 3% bovine serum albumin (BSA) for 30 min. Afterward, slides were first incubated with an α5 monoclonal antibody against the α-subunit of avian Na,K-ATPase (NKA)  overnight at 4°C. Slides were washed twice with PBS and incubated with an Alexa Fluor 568 goat anti-mouse IgG antibody  for 2 h at room temperature. Images were acquired with a Leica TCS-NT confocal laser scanning microscope (Leica) or an Axioplan 2 imaging microscope.p>0.05).Data are presented as the mean ± SD and were analyzed by one-way analysis of variance (ANOVA) and Student’s t-test.Group data sets were confirmed to be normal distribution by Anderson Darling Normality Test 2D3 at the 1∼2-cell stage and lasted 3 d. Compared to the control group (0 µg/l), 1α,25(OH)2D3-treated groups (20 µg/l) showed significant increases in Ca2+ contents and influxes in 3-dpf embryos 2D3 treatment 2D3 in 3-dpf embryos 2D3 significantly inhibited the mRNA expression of cyp27b1, but significantly stimulated cyp24a1expression  and zVDRb (NP_001153457) were identified from NCBI. The 2 VDRs have 453 (zVDRa) and 422 (zVDRb) amino acids , and calvdra and vdrb mRNA expressions were first detected by an RT-PCR at 0 h post-fertilization (hpf) and throughout development  in all tissues except the testes and ovaries by the qPCR analysis  after injecting the MOs. Compared to the control group 2D3), both groups of the control MO with 1α,25(OH)2D3 and the VDRb MO with 1α,25(OH)2D3 exhibited significantly higher Ca2+ influxes at 3 dpf, but VDRa MO-injected embryos with 1α,25(OH)2D3 showed no difference 2D3 and the VDRb MO with 1α,25(OH)2D3 was significantly stimulated, while that of the VDRa MO-injected embryos was not affected by 1α,25(OH)2D3 fference . Similar5(OH)2D3 .2+ medium is known to stimulate Ca2+ influx and ecac expression in zebrafish 2+ influx and ecac expression were upregulated by low-Ca2+ medium as mediated by VDRa or VDRb was further clarified in the following experiments. Embryos at the 1∼2-cell stage were respectively injected with the control MO, VDRa MO, and VDRb MO, and then incubated in 2.0 mM (high) or 0.02 mM (low) Ca2+ medium. Compared to the control MO in low-Ca2+ medium, VDRa morphants in low-Ca2+ medium had significantly lower Ca2+ influx and ecac mRNA expression at 3 dpf, but VDRb morphants in low-Ca2+ medium had similar values to the control group  cells, HR (H+-ATPase-rich) cells, and NCC (Na+/Cl− cotransporter expressing) cells in zebrafish gill/skin ionocytes 2+ uptake function in the skin of developing embryos and gills of adults 2+ uptake function, we further tested the hypothesis of whether VDRa is expressed in NaR ionocytes by double in situ hybridization/immunocytochemistry for vdra mRNA and Na,K-ATPase (a marker of NaR cells) in zebrafish gills. As shown in vdra mRNA signals were colocalized in NaR ionocytes that were labeled with Na,K-ATPase.There are at least three subtypes of ionocytes, NaR 2D3 regulates expressions of cyp27b1 and cyp24a1 through mediation by the VDR in zebrafish as in mammals 2D3. The VDRb MO could not neutralize effects of exogenous 1α,25(OH)2D3 on mRNA expressions of cyp27b1 and cyp24a1 in 3-dpf zebrafish embryos  in freshwater teleosts must be strictly regulated to cope with a fluctuating environment. Vitamin D increased plasma calcium levels in cod 2+ handling in Atlantic salmon because 1α,25(OH)2D3 concentrations and VDR mRNA expressions changed in salmon undergoing smoltification and migrating from fresh water  to seawater  2+ uptake function by stimulating ECaC expression during acclimation to low-Ca2+ fresh water 2+ fresh water stimulated mRNA expressions of ecac, vdra, vdrb, and vitamin D-25hydroxylase (cyp2r1) in 3- and/or 5-dpf zebrafish embryos, implying a possible role of vitamin D in the functional control of Ca2+ uptake in zebrafish. To test this hypothesis, we treated zebrafish with exogenous 1α,25(OH)2D3, which was found to stimulate the mRNA expression of ecac and Ca2+ influx and result in increased total calcium contents in the whole body. The following experiments from the molecular to the physiological level demonstrated the calciotropic effects of vitamin D in zebrafish.Besides the source from food and drinking, teleostean fish actively absorb Ca2D3 stimulated branchial ecac mRNA in trout, but they did not attempt to examine other Ca2+ transporters (NCX or PMCA) 2+ uptake function 2+ uptake mechanisms. The present study first reports that 1α,25 (OH)2D3 only regulates the expression of ecac but not that of ncx1b or pmca2. These results support a previous notion that ECaC is the major regulatory player in the epithelial Ca2+ uptake pathway in fish 2+ influx by regulating the expression of ecac, but neither affected that of ncx1b or pmca2 in zebrafish ecac appears to be the major regulatory target transporter gene not only in response to environmental Ca2+ but also in control pathways of related hormones in zebrafish.Qiu et al., (2007) showed that 1α,25(OH)2D3 treatment was found to stimulate transcription of VDRE-containing expression constructs in a mammalian cell line that overexpressed the teleost VDR 2D3 treatment 2+-uptake function through only one of the paralogous receptor genes, VDRa, in zebrafish based on the loss-of-function experiments of the 2 VDRs.Most physiological functions of vitamin D signaling are mediated by the VDR, which is a ligand-activated transcription factor 2D3 treatment in a cell line experiment ecac promoter region of fugu and zebrafish ecac in fish. In the present study, paralogous VDRs of zebrafish were differentially activated by vitamin D in the control pathways of Ca2+ uptake and ecac expression, implying some divergences in the functions between these two paralogous VDRs in zebrafish, as what was previously reported using in vitro experiments on the 2 medaka VDRs 2D3, but a stronger and more-specific response was observed in the VDRβ chimera 2D3, and this may have resulted from the difference of amino-acid sequence in the LBD. Comparison of amino-acid compositions between zebrafish VDRa and VDRb showed a higher degree of similarity in the DBD (∼97%) than LBD (∼92%), demonstrating that slight changes in the amino-acid composition may be associated with a significant difference in transactivation and thus physiological functions.The teleost VDR simulated the transcript level of the VDRE-containing construct with 1,25(OH)cyp24a1 transcript and inhibit the cyp27b1 transcript through its binding to the VDR in experiments on mammalian cell lines 2D3) suppressed the mRNA expression of cyp27b1 and simultaneously stimulated that of cyp24a1 in 3-dpf zebrafish embryos, reflecting a feedback mechanism in homeostasis of vitamin D levels as found in mammals. In experiments on mammals or mammalian cell lines, exogenous 1α,25(OH)2D3 caused negative feedback which directly suppressed gene expression and activity of CYP27B1 2D3 caused positive feedback to directly stimulate gene expression of CYP24A1 2D3. Accordingly, stimulation of cyp24a1 and inhibition of cyp27b1 by exogenous 1,25(OH)2D3 treatment may provide feedback to control 1α,25(OH)2D3 levels in zebrafish. Taken together, 1α,25(OH)2D3 may regulate the function of the Ca2+ uptake mechanism in fish through feedback pathways, in which expressions of cyp27b1 and cyp24a1 are differentially modulated. Subsequent loss-of-function experiments further indicated that this feedback control by the differential regulation of cyp27b1 and cyp24a1 expressions appears to be mediated by only one of the paralogous receptors, VDRa, supporting results of previous experiments (see the preceding paragraph). Loss-of-function of VDRa was found to modulate exogenous 1α,25(OH)2D3-induced changes in cyp24a1 and cyp27b1expressions in zebrafish, while that was not the case in VDRb morphants. Notably, a continuous feedback effect of 1α,25(OH)2D3 appeared to cause an inhibitory effect on the Ca2+ uptake capacity through downregulation of ecac expression in 5-dpf embryos to prevent excess Ca2+ uptake (unpublished data).Vitamin D was found to directly stimulate the 2+ uptake by upregulating ECaC in zebrafish through feedback pathway associated with the differential regulation of CYP27B1and CYP24A1. Compared to VDRa, VDRb seems to be nonfunctional in calcemic regulation of zebrafish. The present study for the first time clarifies the divergent physiological functions of paralogous VDRs in a teleost after a gene-duplication event. Similar actions of vitamin D on Ca2+ homeostasis evolved in zebrafish as in mammals, and thus zebrafish may serve as a model to explore the function of vitamin D-VDR signaling in Ca2+ homeostasis and related physiological processes in vertebrates.In summary, vitamin D-VDRa signaling was demonstrated to stimulate CaFigure S1Specificity and effectiveness of vitamin D receptor (VDR)a and VDRb morpholino oligonucleotides (MOs). VDRa and VDRb MOs were respectively injected into 1- or 2-cell embryos. To clarify the MO specificity and effectiveness, Western blotting was used to detect VDRa and VDRb protein expressions in wild-type (WT) and MO-injected embryos at 3 d post-fertilization (dpf). An asterisk (*) indicates the position of VDRa or VDRb expression.(TIF)Click here for additional data file.Figure S2Effect of vitamin D receptor (VDR)a and VDRb morpholino oligonucleotides (MOs) on ossification of vertebrae of 5-d post-fertilization (dpf) zebrafish embryos. Control (A), VDRa (B), and VDRb (C) MO. Ossification of vertebrae was observed by calcein staining. Scale bar: 100 µm.(TIF)Click here for additional data file.Figure S3Colocalization of vdra mRNA with Na,K-ATPase rich (NaR) cells in zebrafish gill cryosections. (A) In situ hybridization of vdra mRNA; (B) immunocytochemical staining of Na,K-ATPase. The arrow indicates colocalization of vdra mRNA and Na,K-ATPase protein signals in the same cells. Scale bar: 5 µm.(TIF)Click here for additional data file.Table S1Primers for the RT-PCR and qPCR analyses.(DOCX)Click here for additional data file.Table S2Identities (%) of amino-acid sequences among the DNA-binding domain (DBD) and ligand-binding domain (LBD) of vitamin D receptors (VDRs) in different species.(DOCX)Click here for additional data file.


Cell length and ratiometric (fura2) Ca2+fluorescence were monitored by video microscopy. At 0.5 Hz stimulation, theextent of shortening was increased ~40% and maximal rate of shorteningwas increased ~80% in cardiomyocytes overexpressing Rrm1+Rrm2 ascompared to non-transduced cardiomyocytes. The maximal rate of relaxation wasalso increased ~150% with Rrm1+Rrm2 over expression, resultingin decreased time to 50% relaxation over non-transduced cardiomyocytes.These differences were even more dramatic when compared to cardiomyocytesexpressing GFP-only. Interestingly, Rrm1+Rrm2 over expression had noeffect on minimal or maximal intracellular[Ca2+] (Fura2 fluorescence), indicatingincreased contractility is primarily due to increased myofilament activitywithout altering Ca2+ release from the sarcoplasmicreticulum. Additionally, functional potentiation was maintained withRrm1+Rrm2 over expression as stimulation frequency was increased (1 Hzand 2 Hz). HPLC analysis indicated cellular [dATP] was increasedby approximately 10-fold following transduction, becoming ~1.5% of theadenine nucleotide pool. Furthermore, 2% dATP was sufficient tosignificantly increase crossbridge binding and contractile force duringsub-maximal Ca2+ activation in demembranated cardiac muscle.These experiments demonstrate the feasibility of directly targeting theactin-myosin chemomechanical crossbridge cycle to enhance cardiac contractilityand relaxation without affecting minimal or maximal Ca2+.We have previously demonstrated that substitution of ATP with 2 deoxy-ATP(dATP) increased the magnitude and rate of force production at all levels ofCa Thus, replacement of ATP with dATP offers the potential toimprove contraction independent of changes in[Ca2+]i or adrenergicsignaling.We have previously shown that replacing ATP with 2 deoxy-ATP (dATP) as thesubstrate for contraction of demembranated cardiac muscle increased isometric forceand the rate of force development and shortening at all levels ofCapCa 4.0) –9. The pin situ isunknown. Cellular production of dATP occurs in the cytoplasm of mammalian cells byribonucleotide reductase (Rrm), which removes a hydroxyl moiety from the 2-positionon the ribose ring of ADP to produce dADP. dADP is then rapidly converted to dATP.Rrm consists of two subunit proteins, a catalytic activator (Rrm1) and free radicalcontaining (Rrm2) subunit and is regulated by nucleoside triphosphate allostericeffectors, rate and extent of shortening, and rate ofrelaxation, with minimal effects on Ca2+ transients, at 0.5 Hz, 1Hz and 2 Hz stimulation. Additionally, the [dATP] found intransduced cells (1–2% of adenine nucleotide content) was sufficientto increase sub-maximal Ca2+ activated force in skinned cardiactrabeculae. These experiments suggest that increases in cardiac intracellular Rrmand/or the dATP pool can significantly alter the actin-myosin crossbridge cycle toenhance cardiac contractility without impairing diastolic function or cardiomyocyteCa2+ handling.In the current study, we produced adenoviral vectors expressingcytomegalovirus (CMV) promoter driven Rrm1 or Rrm2, each along with greenfluorescent protein (GFP) as a transduction reporter. Cultured adult ratcardiomyocytes were transduced with these vectors, and the rate and extent ofmyocyte contraction and relaxation and CaGreater details of plasmid design and vector production, cell culture,contractile assessment, nucleotide binding affinity, and western blot analysis areprovided in These studies were approved by the University of Washington (UW) AnimalCare Committee and conducted in accordance with federal guidelines. Animals werecared for in accordance with US NIH Policy on Humane Care and Use of LaboratoryAnimals in the Department of Comparative Medicine at UW. Adult rat (Fischer 344)cardiomyocytes (ARCs) were isolated from heart using aortic retrograde perfusionfor enzymatic (collagenase/protease) dispersion of cells. NeonataHEK293 cells were used to generate adenoviral vectors expressi2. The rateconstant for ATP-induced actin-S1 dissociation (kobs) was determinedfrom Rapid kinetic measurements of nucleotide binding and actin-myosindissociation were taken at 10°C and 20°C (Hi-Tech ScientificSF-61 DX2 stopped-flow system) as previously described using py2+ activation as previously described or[GFP] for 2 days. Successful gene transfer, grossly indicated bygreen fluorescence with microscopy, indicated nearly 100% transductionefficiency . This is2+transients (Fura2 fluorescence), for non-transduced (black), GFP-only (green),and Rrm1+Rrm2 (red) transduced cardiomyocytes. The data for allmeasurements at 0.5 Hz is summarized in 2+ transient. ransient, Table 2t 0.5 Hz  stimulat2+ transients was also assessed,and is summarized in 2+ (3a), maximal Ca2+ (3b) and timeto 50% (3c) and 90% (3d) Ca2+ decay. As with contraction, there was nodifference in Ca2+ transient behavior with increasedstimulation frequency between non-transducer and Rrm1+Rrm2 transducermyocytes. GFP-only transducer cardiomyocytes had a slight increase in minimalCa2+ at 2 Hz, and an increase in maximalCa2+ at 1 Hz and 2 Hz, as compared to non-transducermyocytes, but the times to 50% and 90% decay were similar. As at0.5 Hz, the times to 50% and 90% decay were decreased (fasterdecay) in Rrm1+Rrm2 transducer myocytes at both 1 and 2 Hz. Mostimportantly, although higher stimulating frequencies increased relaxationparameters in all groups, the relative increase in relaxation kinetics wasmaintained with Rrm1+Rrm2 over expression, such that relaxation wasimproved, not impaired. Results for 1 Hz and 2 Hz stimulation are summarized inIt is important to determine whether Rrm1+Rrm2 over expressionaffects normal cellular response to increased stimulation frequency, as changesin heart rate are a normal physiological adaptation to systemic demand. 2+ transients werefaster than at 22–24°C, but were similarly increased incardiomyocytes transducer with Rrm1+Rrm2 vs. GFP-only transducer andnon-transducer cells. Similarly, the rates of Ca2+ releaseand re-uptake were also increased at 37°C vs. room temperature, but withRrm1+Rrm2 over expression resulting in faster Ca2+transient decay as was observed at ambient temperature.We chose to perform these experiments at room temperature(22–24° C) to compare with the predominant number of reports forcultured cardiomyocytes in the literature –29. Howe2+, changes in contractility can best be explained by achange in myofilament responsiveness to activating Ca2+. Thisis illustrated in 2+). Cardiomyocytes expressingRrm1+Rrm2 had significantly higher contractile response thannon-transducer or GFP transducer cardiomyocytes at all stimulation frequencies.There was no difference in contractile response between GFP only ornon-transducer myocytes except at 2 Hz, which can be primarily be attributed toincreased maximal Ca2+ in GFP only myocytes with no increasein fractional shortening, reducing response.Since there was little difference between groups in minimal and maximalCaTo verify increased Rrm mRNA, Rrm protein, and dATP production inRrm1+Rrm2 transducer cells, neonatal rat cardiomyocytes were collectedand processed for RT-PCR, western blotting and HPLC analysis of intracellular[ATP] and [dATP]. Neonatal cardiomyocytes wereused to achieve high enough cell density for accurate nucleotide contentanalysis, as intracellular [dATP] is known to be in the pMrange. Although there are structural differences between neonatal and adultcardiomyocytes, it is important to note that Rrm1+Rrm2 over-expressionincreased contractility to a similar extent in both cell types .Interesobs) for ATP vs. dATP. obs at 10°C and 20°C formouse cardiac  myosin. There was no difference in kobs betweenATP and dATP at any [NTP] at either temperature. This was alsotrue for fast and slow skeletal S1 myosin . The example force trace in 2+ (pica 4.0) activation. Similarly, crossbridgebinding, assessed by high frequency sinusoidal stiffness measurements, increased16.0 ± 0.03% (p<0.05), which indicates that the increasedforce with 2% dATP, 98% ATP is due to increased crossbridgebinding. Thus the data demonstrate that a relatively small increase in cellular[dATP] (1–2% of adenine nucleotide) issufficient to significantly increase the contractile strength of intactcardiomyocytes by increasing the number of strong crossbridges.To determine if small amounts of dATP increase force production, weactivated contraction of demembranated rat cardiac trabeculae at pica 5.6 and pica 4.0  in solutions containing 5 mM NTP, composedof either 100% ATP or 2% dATP, 98% ATP. The sub-maximalCa2+ transient properties.The main objective of this study was to determine if over expression of ribnucleotide reeducates (Rrm1+Rrm2) increases cellular [dATP]and, in turn, increases contractility in intact cardiomyocytes without adverselyaffecting cardiomyocyte relaxation. Over expression of Rrm1+Rrm2 resulted inincreased cellular [dATP] to ~1.5% of the total adeninenucleotide pool, and this dramatically increased the extent and rate of myocyteshortening and rate of myocyte relaxation, while having no apparent effectCa2+ activation, including saturating[Ca2+] (pica 4.0), but these studies wereperformed with 100% replacement of 5 mM ATP with 5 mM dATP in bathingsolutions. These resultswarrant progression to animal studies to determine its potential to improve globalcardiac function in normal and diseased hearts.In summary, dATP provides a dual benefit of positive inotropy and lusitropyin cultured rat cardiomyocytes, with little alteration of Ca01