Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites.Egress of 2+ homeostasis on the egress programme.Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER) Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell.A steady increase in cytoplasmic free Ca2+ for egress initiation, vacuole swelling, and host cell cytoskeleton digestion. The evidence that parasitophorous vacuole swelling, a stage of unaffected egress, is dependent upon a rise in intracellular Ca2+ suggests a mechanism for ionophore-inducible egress and a new target for Ca2+ in the programme liberating parasites from the host cell. A regulatory pathway for egress that depends upon increases in intracellular free Ca2+ is proposed.The parasite egress programme requires intracellular free Ca To avoid cell photodamage, the time of observation was extended but the interval between frames was increased.To test the hypothesis that a quasi-static Ca2+ probe Fura Red AM. The use of a ratio-metric probe mitigates measurement concerns that arise from volume variability, probe incorporation and hydrolysis of AM ester groups, and photobleaching bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]] bis[(acetyloxy)methyl] ester; cGMP: Cyclic guanosine monophosphate; CPA: Cyclopiazonic acid; EDTA: Ethylene diamine tetraacidic acid; EGTA: Ethylene glycol tetraacidic acid; EMCD: Electron multiplying charge coupled device; ER: Endoplasmic reticulum; Kd: Dissociation constant; LSM: Laser scanning microscope; NA: Numeric aperture; NIH: National Institutes of Health; PfATP6: The authors declare that they have no competing interests.JZ, SG and VL conceived and designed the experiments. SG and VL performed the experiments. SG, VL, PSB, KM, GH and JZ analysed the data. JZ, SG, PSB, VL, GH and KM wrote the paper. All authors read and approved the final manuscript.Free calcium kinetics in schizonts undergoing parasite egress. The data provided show the variability in free calcium kinetics observed in schizonts undergoing parasite egress. Cells were labeled with Fluo-4 AM (5 μM), and monitored at 37°C in full medium. A sharp increase in fluorescence reflects an influx of free calcium from the medium into the cell through pores formed in the erythrocyte membrane. Egress occurred in the frame marked by an arrow. Different kinetic behaviors are observed: stable  or steadily increasing  fluorescence prior to membrane poration and parasite egress; sharp  or steady (B) drops in fluorescence to the background level reflecting leakage of Fluo-4 from erythrocytes following membrane poration but before membrane rupture.Click here for fileChelation of internal but not external Ca2+inhibits parasite egress. The data provided show control experiments that tested the effect of external and internal free calcium chelation on parasite egress. A. Parasite egress proceeds normally in a Ca2+ free isotonic salt solution (PBS) supplemented with glucose and AlbuMax II. In some experiments EDTA (3 mM) was added to reduce trace amounts of free calcium in the AlbuMax II solution. Cultures were treated 30–60 min at 37°C in chambers. Control cultures were maintained in complete medium . B. Inhibition of parasite egress by BAPTA AM does not depend on ATP-depletion in erythrocytes. Cells were pretreated 30 min at 37°C in media with 30 μM BAPTA AM and different concentrations of Na-pyruvate and then incubated in chambers for 90 min at 37°C. Control cultures were incubated in medium without BAPTA AM and Na-pyruvate. An individual experiment, mean of four measurements. C. Hydrolysis of the AM ester in cells labeled with calcein AM does not affect parasite egress. Cultures were pretreated 30 min at 37°C in the presence of calcein AM or BAPTA AM and then incubated 30 additional minutes at 37°C in the chamber . Bars: BAPTA AM, black; calcein AM, grey. DIC (upper image), calcein fluorescence (green) and merged images of calcein-labeled infected and normal erythrocytes. Bar = 5 μm. D. Chelation of intracellular calcium by BAPTA within the last 45–60 min of the parasite cycle inhibits parasite egress. Cultures were pretreated 30 min at 37°C in the presence of 30 μm BAPTA AM and then incubated 15 or 30 additional minutes in the chamber .Click here for fileDepletion of intracellular calcium blocks cycle progression upstream of the morphological transformations of infected erythrocytes that precede parasite egress. The data provided show light microscopy images of BAPTA AM treated cells. BAPTA AM treatment blocks progression of schizont into the schizont “flower” form characterized by a swelled parasitophorous vacuole and reduced erythrocyte cytoplasm volume. Mature schizonts were pretreated with 60 μM BAPTA AM (30 min at 37°C) and analysed using light microscopy. Randomly selected schizonts do not demonstrate the expected cycle progression towards parasite egress over relatively long observation times (up to 37 minutes of observation). Bar = 5 μm.Click here for fileEffect of calcium ionophore A23187 on parasite egress, cell morphology and erythrocyte membrane of infected cells. The data provided show additional experimental results on the effect of A23187 on parasite egress and morphology of treated cells. A. Activation of parasite egress upon short-time treatment of schizonts with calcium ionophore A23187 is dose-dependent. Culture medium was supplemented with different concentrations of A23187 and cells were then placed in the chamber for 30 min incubation at 37°C. Parasite egress in treated and control cultures was assessed as described in the Methods . B. Marked differential morphological changes in ionophore-treated cells. Normal erythrocytes were crenated (lower left cell), mature schizont was accelerated to egress and has blebbed erythrocyte membrane (upper left cell) and trophozoite appeared ballooned due to the swelling of the parasitophorous vacuole (cell on the right). Bar = 5 μm. C. Ionophore-induced shading and blebbing of erythrocyte membrane in immature schizont. Blebbed erythrocyte membrane (white arrowhead) shaded from the immature schizont (black arrowhead) damaged by ionophore treatment suggesting that Ca2+ fluxes activated calpain and cytoskeleton digestion in this cell. Green colour - erythrocyte actin cytoskeleton labeled with fluorescent phalloidin-Alexa 488. Bar = 5 μm.Click here for fileSuggested Ca2+-dependent steps in parasite egress programme. A table-style presentation of experimental data on the involvement of Ca2+ in the parasite egress programme.Click here for file


Acute myeloid leukemia (AML) is nearly always a fatal malignancy. For the past 40 years, the standard of care remains a combination of cytarabine and an anthracycline known as 7 + 3. This treatment regimen is troubled by both low survival rates (10% at 5 years) and deaths due to toxicity. Substantial new laboratory findings over the past decade have identified many cellular pathways that contribute to leukemogenesis. These studies have led to the development of novel agents designed to target these pathways. Here we discuss the molecular underpinnings and clinical benefits of these novel treatment strategies. Most importantly these studies demonstrate that clinical response is best achieved by stratifying each patient based on a detailed understanding of their molecular abnormalities. The greatest strides in treating myeloid malignancies have come from the development of targeted agents. These include all location . Similarlocation .Other than these few therapeutic successes, there have been few clinical advances in the treatment of myeloid malignancies, and in particular AML. The standard treatment option for the majority of young, fit patients with AML remains continuous infusion of cytarabine for days 1 to 7 and an anthracycline, daunorubicin or idarubicin, as a daily bolus for days 1 to 3 (7 + 3), a long-lived standard combination in use for at least the last 40 years  that an Many elderly patients, who comprise more than half of those with AML, are unfit for any induction chemotherapy or likely not to benefit due to poor cytogenetic risk . For theNovel targeted therapies are needed to improve survival for more patients with AML. Numerous agents are in development with the aim of targeting both well established and more recently identified leukemogenic pathways. This review begins with a discussion of some promising novel targeted agents recently tested in AML patients and, within this context, the clinical hurdles to drug development in AML are highlighted. Non-targeted, conventional chemotherapeutic agents and combinations of these agents remain outside the scope of this review.While the development of novel therapies has been slow, the knowledge of mutations associated with AML has increased dramatically in recent years. There are known translocations e.g., t, t8;21), inv 16, t which are associated with a favorable prognosis; e.g., t, t, inv 16, t, the so-called core binding factor (CBF) leukemias ;21, inv . It is aMechanisms of leukemogenesis have been attributed to many of the known mutations, but most have not been the subject of a targeted therapeutic plan. This may be due to: (1) a lack of identified therapies to target the mutations; (2) the genetic complexity of AML which is often characterized by several co-existing mutations that all contribute to leukemogenesis; and, (3) low numbers of patients with any given mutation, which makes it difficult to devise a clinical trial with a reasonable accrual rate. For example, c-kit mutations have been described in CBF leukemias ,15. HoweThe most promising target for AML therapy in this past decade has been the FLT3 (FMS-like tyrosine kinase 3) protein. FLT3 is a transmembrane receptor tyrosine kinase that belongs to the same family as FMS, kit, and PDGFRa/b. It is mutated in about 30% of all AML . The mutIn AML, there is constitutive activation of FLT3 either due to interference with the negative regulatory function of the juxtamembrane region with ITD mutations, or changes in the activation loop with TKD mutations . As a reGiven these data and the availability of several TKIs with activity against FLT3-mutated leukemia , severalin vivo [in vitro studies indicate that samples with high FLT3-ITD allele burden are more sensitive to the cytotoxic effect of FLT3 inhibitors compared to samples with a low allele burden [in vivo following exposure to lestaurtinib [There have been several reasons identified for the limited success of FLT3 inhibitors. The problem of bioavailability has been highlighted by a randomized Phase III trial of induction chemotherapy combined with lestaurtinib in relapsed AML with FLT3-ITD, where there was no improvement in survival over chemotherapy alone. This was attributed to decreasing plasma levels of lestaurtinib with time, and increasing alpha-1 acid glycoprotein, which binds lestaurtinib in vivo . In addie burden ,37. FLT3aurtinib . Finallyaurtinib . Nonetheetc. In many cases these fusion proteins contribute to the development of leukemia partly by disrupting the modification of chromatin, through recruitment of chromatin-modifying coregulators [Anomalous epigenetic changes, including DNA hypermethylation and histone acetylation/deacetylation occur frequently in acute leukemia. The recurring chromosomal translocations seen in AML lead to the generation of chimeric fusion oncoproteins e.g., TEL-AML, AML-ETO, PML-RARa, gulators . In AML,gulators ,41. Unligulators . Incorpogulators ,44,45, ap = 0.037). A reason for the modest benefit was that patients with high percentage blast count and high white counts were included in this study. This was in contrast to the patients enrolled in the AZA-001 study, who had low white counts and blast counts not exceeding 30% [Clinically, 5-azacytidine treatment leads to improved overall survival in AML patients with low blast counts, as demonstrated in a sub-analysis of the AZA-001 study of high risk myelodysplastic syndrome, in which approximately one third of patients had low blast count AML . When deding 30% . Finallyding 30% . HDACi hding 30% . Combinading 30% ,49 or arMessenger RNA (mRNA) processing and mRNA translation are important steps in the regulation of protein levels in cells and these processes are coupled to essential cellular events such as growth, proliferation, differentiation and apoptosis. In normal hematopoietic cells, translation of transcripts into protein is tightly regulated at the initiation phase by signal transduction pathways including PI3K/AKT pathways that can ultimately affect eukaryotic translation initiation factor 4E (eIF4E). Most AMLs are characterized by elevated PI3K and Akt activity ,51. Furtex vivo [The only direct approach to target eIF4E in AML patients in the clinic thus far has been with ribavirin ,32,60,62ex vivo  and servex vivo .ex vivo [ex vivo [Most AML cells show activated PI3K/AKT/mTOR pathways ,51. For ex vivo ,65. Akt ex vivo , in prec[ex vivo . There iex vivo [Downstream of PI3K and AKT is the mTOR pathway. mTOR activation results in the phosphorylation of both eIF4E binding protein BP1 and ribosomal S6 protein. In AML, S6 phosphorylation is a robust marker (see below). Early studies with rapamycin, an mTORC1 inhibitor, failed to show responses in clinical trial as a single agent . Howeverex vivo . This heex vivo .Tosedostat is an inhibitor of aminopeptidase and therefore acts by depleting amino acids. In the HL-60 leukemia cell line, gene array studies have shown that this amino acid depletion resembles an amino acid deprivation response (AADR) . In the In a phase II study of tosedostat as a single agent in elderly patients with relapsed AML, median age 72, and half with refractory disease prior to starting therapy, 12% had a complete response and 10% a partial response. Some of these responses occurred among patients having failed hypomethylating agents and patients who had MDS previously. The median survival among patients with a CR was 323 days, 195 days among patients with partial response, and 162 days in those with stable disease . Based ovs. 0.8%) and no improvement in complete remission rates, relapse free survival, post-consolidation disease free survival, or overall survival. Untreated AML patients under the age of 61 were randomized to cytarabine and daunorubicin with or without GO (6 mg/m2) [Monoclonal antibody therapy has proven extremely successful in other malignancies. In AML, gemtuzumab ozogomycin (GO) is an antibody-drug conjugate where a humanized monoclonal IgG4 antibody directed against CD33 is linked to a derivative of calicheamicin, a potent DNA-binding cytotoxic antibiotic . CD33 an6 mg/m2) . In June6 mg/m2) .AML15 compared the addition of GO to one of three induction schedules and/or one of two consolidation schedules . A total2. GO was given on day 1, 4 and 7 in order to deliver a cumulative dose of 9 mg/m2 and to reduce liver toxicity. Event free survival at 2 years showed a significant advantage at 41.4% in patients treated with GO in comparison to 15.6% for the chemotherapy alone arm. Similarly, there was an overall survival benefit of 53.2% versus 41.9% as well as a benefit in relapse free survival of 50.3% versus 22.7% in the GO-containing regimen, and no increase in treatment related death [Finally, a phase III study by Castaigne and colleagues performed in 26 centers in France, evaluated the benefit of low dose GO when added to 7 + 3 in newly diagnosed AML patients between the ages of 50 and 70 . A totaled death . The finNew drug development relies heavily on the results of Phase II trials, which determine whether a therapy will be tested in large, Phase III trials. Phase II clinical trial design includes not only the method by which patients will be enrolled and the sample size, but also the dose and schedule for single and combination drugs, patient selection, determination of primary and secondary endpoints, and response assessments. This section reviews some of these elements and current difficulties.Most phase II trials in AML are single arm, uncontrolled trials with small sample sizes  mainly bIn order to deal with the small sample sizes and lack of comparator arm, the “Pick-a-Winner” design has been proposed . In thisin vitro with the novel agent. A recent example includes the combination of salvage chemotherapy and plerixafor, an inhibitor of CXCR4, in relapsed and refractory AML [Single agent studies are obviously limited by both low response rates and remission duration. An obvious way to circumvent these problems is to combine them with standard chemotherapeutic drugs known to have activity in AML and, ideally, synergy tory AML . CXCR4 itory AML ,86. In Atory AML . In moustory AML . On the tory AML .In combining targeted agents with chemotherapeutic drugs, toxicity becomes a concern. In the SWOG study of GO and induction chemotherapy, there was excess death in the GO arm attributed to the dose and schedule of GO, whose toxicity may have been enhanced by chemotherapy . When thin vitro. In clinical trials, the fixed ratio is maintained [One sophisticated approach to ensuring greater predictability in drug delivery is exemplified by the development of CPX-351, a liposomal preparation of cytarabine and daunorubicin, which maintains a fixed ratio of 5:1 cytarabine:daunorubicin. This preparation has shown promise in a small randomized Phase II trial of older patients with newly diagnosed AML. By maintaining a fixed ratio of drugs, CPX-351 maximizes synergy and minimizes antagonism intained , and in intained . How thiAn important consideration in the development of new therapies for AML is the selection of patients for Phase I and II trials. For the majority of trials, these are patients with relapsed AML or elderly patients for whom induction chemotherapy is not feasible. These are important populations to target given the dearth of therapeutic options. However, these patients are often more frail, less able to tolerate chemotherapy and have a more resistant and more genetically heterogeneous disease than young, newly diagnosed patients.From our experience with Phase I and II trials in AML, the early attrition rate due to rapid disease progression, infection, or overall clinical deterioration approaches 33%. Such a high rate of early attrition could result in a therapy being falsely determined to be ineffective if all patients having taken at least one dose of treatment are considered evaluable for response.p-glycoprotein and other mechanisms) that affect not only standard agents, but also the novel therapeutics. For instance, if drug transporters are downregulated as a response to one therapy, all therapies requiring this drug transporter, despite being based on different intracellular pathways, would be expected to fail. This leads to a conundrum in using heavily pretreated patients in these studies as drugs effective as first line agents may fail in this setting.Further, difficulties in testing new drugs in refractory and relapsed AML patients include the development of drug resistance mechanisms (including Another common issue in clinical trials of agents without a rapid rate of response, which is the case for many novel agents, has been that patients with a high peripheral blood blast count, or rapidly doubling white count are less likely to respond . Some grAs with other cancers, a detailed understanding of the molecular profile of a particular AML is needed to appropriately place individuals on suitable clinical trials. Mutational testing is a first step in identifying potential targets and in selecting therapies, but, as can be seen from the FLT 3 inhibitor data, targeting a single mutation is likely insufficient. In addition, other molecular predictors of response characterize certain AMLs, including eIF4E overexpression in AML FAB subtypes M4 and M5. In the proof-of-principle trial, responses to ribavirin were seen irrespective of FLT3 mutation status . As discSome of the most relevant pathways and mutations in AML have been outlined, as well as the therapies being developed to target these pathways. No single molecular abnormality accounts for leukemogenesis in AML and given this complexity, it is not surprising that targeted agents have not led to widely applicable therapies. However, some of these agents, even as monotherapies have been associated with activity including some complete responses, when the most apt patient cohorts are considered . How bes


While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca2+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent ISOC, activated in response to Ca2+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated ICRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca2+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd3+, removal of extracellular Ca2+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca2+-containing, but not Ca2+-free, medium. Consistent with this, ICRAC is activated in cells pretreated with thapsigargin in Ca2+-free medium while ISOC is activated in cells pretreated in Ca2+-containing medium. Significantly, TRPC1 function is required for sustained KCa activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca2+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca2+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.Store-operated Ca 2+ entry is present in all cell types and determines sustained cytosolic [Ca2+] increases that are critical for regulating a wide variety of physiological functions. This Ca2+ entry mechanism is activated in response to depletion of Ca2+ in the endoplasmic reticulum (ER). When ER [Ca2+] is decreased, the Ca2+-sensor protein STIM1 aggregates in the ER membrane and moves to regions in the periphery of the cells where it interacts with and activates two major types of channels that contribute to store-operated Ca2+ entry: CRAC and SOC. While gating of Orai1 by STIM1 is sufficient for CRAC channel activity, both Orai1 and transient receptor potential channel 1 (TRPC1) contribute to SOC channel function. The molecular composition of SOC channels and the critical role of Orai1 in activation of TRPC1 have not yet been established. In this study, we demonstrate that TRPC1 and Orai1 are components of distinct channels, both of which are regulated by STIM1. Importantly, we show that Orai1-mediated Ca2+ entry triggers plasma membrane insertion of TRPC1 which is then gated by STIM1. Ca2+ entry via functional TRPC1-STIM1 channels provides additional increase in cytosolic [Ca2+] that is required for regulation of specific cell functions such as KCa activation. Together, our findings elucidate the critical role of Orai1 in TRPC1 channel function. We suggest that the regulation of TRPC1 trafficking provides a mechanism for rapidly modulating cytosolic [Ca2+] following Ca2+ store depletion.Store-operated Ca SOCE generates local and global [Ca2+]i signals that regulate a wide variety of cellular functions 2+ channel to be characterized, the Ca2+ release-activated Ca2+ (CRAC) channel, has a high selectivity for Ca2+ versus Na+ and displays a typical inwardly rectifying current-voltage relationship. CRAC channel accounts for the SOCE in lymphocytes and mast cells 2+ binding protein that has been established as the primary regulator of SOCE Store-operated Ca2+-permeable cation channels, usually referred to as SOC channels, that have been associated with SOCE in several other cell types Ca activation and, consequently, salivary fluid secretion 2+ store depletion 684KK685) and TRPC1(639DD640) Store depletion also leads to activation of relatively non-selective Ca2+ store depletion. Functional Orai1 is also required since knockdown of Orai1 or expression of functionally deficit Orai1 mutants prevents TRPC1 activation 2+ store depletion and determined the contributions of TRPC1 and Orai1 to SOCE. We report that TRPC1 and Orai1 constitute two distinct channels that contribute to SOCE in HSG cells. Suppression of TRPC1 function unmasks the underlying CRAC channel function. Further, in response to store depletion, STIM1 mediates association of Orai1 and TRPC1 within ER/PM junctional domains. Ca2+ entry via Orai1/STIM1-CRAC channel triggers plasma membrane insertion of TRPC1 and gating is achieved by interaction with STIM1(684KK685) residues. Remarkably, while both Orai1 and TRPC1 contribute to [Ca2+]i increase following store depletion, they impact different cellular functions. Ca2+ entry mediated by TRPC1 is the primary regulator of KCa channel and partially contributes to NFκB activation while Orai1-mediated Ca2+ entry alone is sufficient for maximal NFAT activation and partial NFκB activation. Together these findings reveal the molecular events that determine activation of TRPC1 channels following store depletion. We suggest that local Ca2+ entry mediated by Orai1 determines plasma membrane insertion of TRPC1 while gating by STIM1 controls its activation. Thus, Orai1 and STIM1 not only determine Ca2+ signals generated by CRAC channels but by regulating TRPC1 channel activity rapidly modulate [Ca2+]i and thus significantly impact various cell functions.An intriguing finding is that STIM1 alone is not sufficient for activation of TRPC1-SOC channels following Ca2+ influx by >90%, >80%, or >60%, respectively . Silencing of Orai1 expression blocked generation of ISOC while knockdown of TRPC1 by shRNA significantly reduced the amplitude of the inward current but induced more pronounced loss of the outward current. Thus the residual current detected in 6/10 shTRPC1 treated cells was more inwardly rectifying, i.e. more like ICRAC  The contribution of TRPC1 and Orai1 to SOCE in HSG cells was further examined by using whole cell patch clamp technique epletion . Consist684KK685) mediate gating of TRPC1 via electrostatic interaction with TRPC1(639DD640) residues 684EE685), induced suppression of SOCE in HSG cells while expression of WT-STIM1 resulted in a small increase in function (639KK640), induced a similar suppression of endogenous SOCE (684EE685) in HEK293 cells (684EE685) stimulated Orai1 similar to WT-STIM1 (684EE685) resulted in generation of ICRAC in response to Tg-induced Ca2+ store depletion in >70% of HSG cells displaying currents. Together the data in SOC in HSG cells is composed of a small Orai1-mediated ICRAC and a larger TRPC1-mediated non-selective current (note that we have not yet measured an isolated TRPC1+STIM1 current).The two C-terminal residues of STIM1 2+ entry (CRAC was seen in these cells (2+ entry in SOAR-expressing cells was significantly reduced by knockdown of TRPC1 (2+ entry reflects spontaneous Orai1-dependent Ca2+ influx). In aggregate, these data provide strong evidence that endogenous Orai1 mediates ICRAC without any contribution from TRPC1 while SOCE and ISOC display significant contribution from TRPC1. Importantly, the function of TRPC1 requires Orai1.To conclusively demonstrate that endogenous Orai1 mediates I2+ entry  and sponse cells . SOAR-inse cells . In cont2+ store depletion on the surface expression of TRPC1. In resting cells the surface expression of TRPC1 (i.e. in the biotinylated fraction) was relatively low. Tg treatment of cells . The role of Orai1-mediated Ca2+ entry was more directly assessed by using Orai1-E106D, an Orai1 mutant that is permeable to Ca2+ in Ca2+-containing medium, but unlike the wild type channel, it is permeable to Na+ in nominally Ca2+-free medium. Tg treatment of cells expressing this mutant induced surface expression of endogenous TRPC1 in Ca2+-containing medium but not in Ca2+-free medium (684EE685), which display ICRAC in response to Ca2+ store depletion   . TRPC1 c2+]i increase regulates plasma membrane insertion of TRPC1. 2+]i increase was completely suppressed (2+]i increase in cells loaded with low [BAPTA-AM]). In addition, Tg-stimulated ISOC was not altered by replacing EGTA in the pipette solution with 10 mM BAPTA . Note that 1 µM Ca2+ induces >90% activation of TRPC4 and TRPC5 2+-dependent cation channels to SOCE We next examined whether relatively global or local i elevation due to intracellular Ca2+ release is insufficient for triggering TRPC1 insertion. Further when cells were treated with Tg in a Ca2+ free medium for 5 min, there was no increase in TRPC1 expression in the plasma membrane until Ca2+ was added to the external solution  did not change the level of TRPC1 in the surface membrane. Functional consequences of these treatments are shown in 2+-free medium prior to whole cell current measurements in DVF medium. Typical inwardly rectifying ICRAC with rapid inactivation was detected in these cells  in HSG cells induced a slow, much diminished (>80% reduction), and transiently activated KCa current compared to that in control cells. As shown above  mutant, which does not gate TRPC1, facilitates detection of ICRAC. Further, SOAR-activated ICRAC required Orai1 but not TRPC1. Thus Orai1 and TRPC1 are components of two distinct channels. These findings provide strong argument against the possibility that TRPC1 and Orai1 contribute to the same channel pore or that Orai1 is a regulatory, non-conducting, subunit of TRPC channels The findings described herein address several important and as-yet unresolved questions regarding the molecular components of TRPC1-SOC channel, the mechanism involved in regulation of the channel in response to store depletion, and its contribution to SOCE. We report that the previously described I2+ entry triggers plasma membrane recruitment of TRPC1. These data reveal a novel function for Orai1 that can explain its critical requirement in the activation of TRPC1 channels following Ca2+ store depletion. We show that Ca2+ store depletion leads to enhanced surface expression of TRPC1, which is blocked when Ca2+ is removed from the external medium or SOCE is inhibited by addition of Gd3+. Knockdown of endogenous Orai1 expression or expression of non-functional Orai1 mutants (Orai1-E106Q) also lead to loss of TRPC1 in the plasma membrane. Notably, in cells expressing Orai1-E106D, TRPC1 trafficking is supported in Ca2+-containing medium but not Ca2+-free medium. Together, these findings provide strong evidence that surface expression of TRPC1 is determined by the Ca2+ permeability of Orai1 and that TRPC1 is gated by STIM1 and not directly by [Ca2+]i increase. Presently we cannot conclusively rule out the involvement of possible downstream signaling pathway(s) activated by Orai1-mediated Ca2+ entry.We also report that Orai1-mediated Ca684KK685 residues of STIM1 are involved in gating the channel, the ERM domain 2+ entry to locally regulate plasma membrane insertion of TRPC1. However, our data show that internalization of TRPC1 is apparently not dependent on [Ca2+]i  Ca2+ store depletion leads to translocation of STIM1 to ER/PM junctional domains and recruitment of Orai1  and TRPC1 , (ii) Orai1 is activated by STIM1 and Ca2+ entry via Orai1 triggers exocytosis of TRPC1, and finally (iii) STIM1 gates plasma membrane TRPC1  and transfected as required; experiments were done 48 h post-transfection. Cells were loaded with 5 µM Fura-2 (Invitrogen) for 30 min at 37°C. Fluorescence was recorded using a Till Photonics-Polychrome V spectrofluorimeter and MetaFluor imaging software (Molecular Devices). Each fluorescence trace (340/380 nm ratio) represents an average from at least 50–150 cells from >6 individual experiments. Student's 2+ containing standard external solution (Ca2+-SES) with the following composition (in mM): NaCl, 145; KCl, 5; MgCl2, 1; CaCl2, 1; Hepes, 10; glucose, 10; pH 7.4 (NaOH). The patch pipette had resistances between 3 and 5 milliohms after filling with the standard intracellular solution that contained the following (in mM): cesium methane sulfonate, 145; NaCl, 8; MgCl2, 10; Hepes, 10; EGTA, 10; pH 7.2 (CsOH). For KCa measurements, pipette solution contained 150 mM KCl, 2 mM MgCl2, 1 mM Mg-ATP, 5 mM Hepes, 0.1 mM EGTA, and pH 7.2, potassium hydroxide. Osmolarity for all the solutions was adjusted with mannose to 300±5 mosM using a vapor pressure Osmometer . All electrophysiological experiments were performed in the tight-seal whole cell configuration at room temperature (22–25°C) using an Axopatch 200B amplifier (Molecular Devices). Development of the current was assessed by measuring the current amplitudes at a potential of −80 mV, taken from high resolution currents in response to voltage ramps ranging from −90 to 90 mV over a period of 100 ms imposed every 2 s  and digitized at a rate of 1 kHz. Liquid-junction potentials were less than 8 mV and were not corrected. Capacitative currents and series resistance were determined and minimized. For analysis, the current recorded during the first ramp was used for leak subtraction of the subsequent current records. Thapsigargin (Tg 1 µM), dissolved in the bath solution, was used to stimulate the cells. DVF solution contains (mM): NaCl 165; CsCl 5; EDTA 10; HEPES 10; glucose 10; pH 7.4 (NaOH). Cells were pretreated with 1 µM Tg for 10 min either in Ca2+ containing or Ca2+ free medium before whole cell configuration was achieved. Cells were switched to DVF 1 min after achieving whole cell configuration in Ca2+ free external medium.Coverslips with HSG cells were transferred to the recording chamber and perfused with CaTransfected HSG cells were washed with phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) protein extraction buffer , 0.5% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, 1 mM dithiothreitol (DTT), pH 7.4) supplemented with Complete Protease Inhibitor Cocktail tablets (Roche Diagnostics). Where indicated, cells were first stimulated for 5 min with 1 µM Thapsigargin (Tg), lysates was then centrifuged at 12,000 x g for 30 min at 4°C, and the supernatant was collected and analyzed by SDS-PAGE and Western blotting (50 µg of protein were loaded per lane). Protein concentrations in the lysate was adjusted to 2 mg/ml and incubated with 10 µg/ml IP antibody. Immunoprecipitates were released by incubating in SDS-sample buffer and resolved in 4%–12% NuPAGE gels (Invitrogen) followed by Western blotting. Anti-STIM1 , anti-Orai1 , anti-GAPDH , and Anti-TRPC1 antibody 2+) or absence (−Ca2+) of extracellular calcium, and incubation time was 5 min or otherwise as indicated. The reaction was stopped by adding ice-cold quenching solution. In BAPTA-AM loading experiments, cells were pretreated with 200 µM BAPTA-AM (Invitrogen) in SES containing 100 µM extracellular Ca2+ for 30 min at 37°C. Treated cells were then incubated for 20 min with 1.5 mg/ml Sulfo-NHS-LC-Biotin (Pierce) in 1XPBS (pH 8.0) on ice. Following biotin labeling, cells were washed and harvested in RIPA buffer using the same protocol as described above. Biotinylated proteins were pulled down with NeutrAvidin-linked beads (Pierce) and detected by Western blotting. Band intensities of surface proteins were obtained using Image J software.Cells were transfected with vector or scrambled control as required. For stimulation experiments, cells were pretreated with 1 µM Thapsigargin in the presence  driven by the TK promoter was used to control for cell number and transfection efficiency. Transfected cells were stimulated as described in An Olympus IX81 motorized inverted microscope (Olympus) was used as described previously Balb/c mice were anesthetized and injected subcutaneously with either saline (Resting) or 0.5 mg of pilocarpine/kg (Stimulated). After the saliva secretion was observed in stimulated mice, the animals were perfusion fixed with 10% buffered formalin and immediately euthanized. Salivary glands were excised and embedded in paraffin for histologic processing. Slides of paraffin sections were deparaffinized and rehydrated. Sections were unmasked by microwaving samples for 10 min in a microwave pressure cooker (NordicWare) in 1 mM EDTA, pH 8.0, containing 0.05% Tween 20. After cooling, sections were blocked either with 0.5% BSA in PBS (for direct conjugates) or with 10% donkey serum in PBS (for samples using secondary antibodies). After blocking for 30 min at room temperature, primary antibodies were applied and incubated at 4°C overnight. For samples using two or more rabbit host primary antibodies, direct-conjugation with a fluorescent tag using Invitrogen's Zenon labeling kit was used. For antibodies requiring secondary antibody labeling, donkey anti-rabbit Alexa conjugates were used (Invitrogen). A negative control using normal rabbit IgG at the same concentration as specific primaries was included for both methods. After labeling with primary antibodies only, samples were washed extensively and incubated with secondary antibodies for 1 h at room temperature, washed, and mounted with VectaShield mounting medium containing DAPI. Zenon conjugated samples were washed extensively and mounted with cover slips as above. Images were collected by using a Leica Confocal microscope and MetaMorph software .t test. Experimental values are expressed as means ± SD or SEM. Differences in the mean values were considered to be significant at p<0.01.Data analysis was performed using Origin 8 (OriginLab). Statistical comparisons were made using student's Figure S13+ and 20 µM 2APB on endogenous SOCE as well as in cells expressing TRPC1, STIM1, and Orai1 (as indicated in the figure). 1 µM Gd3+ or 20 µM 2APB were added immediately before Ca2+. (G) SOCE measured under the different conditions. ** indicates values that are significantly different from that in control cells . Ca2+ entry under all the different conditions is blocked by 1 µM Gd3+ or 20 µM 2APB.(A) Western blot showing knockdown of TRPC1 in HSG cells treated with shTRPC1 for 48 h. 5 µg of the construct was used for transfection in all the experiments shown in the article. (B–G) Effect of 1 µM Gd(TIF)Click here for additional data file.Figure S22+ entry stimulated by maximal and sub-maximal stimulation of HSG cells with CCh. Cells were transfected with shTRPC1, siOrai1, or siSTIM1 for 48 h. Cells were treated with CCh; 100 µM  or 1 µM  in Ca2+ free medium for 5 min (release component not shown) prior to addition of Ca2+. Average values for Ca2+ entry are shown in (C). ** indicates cells significantly different from the control conditions . The relative contributions of the three proteins in the two conditions of stimulation are similar.Contribution of TRPC1, STIM1, and Orai1 to Ca(TIF)Click here for additional data file.Figure S3684EE685) activates Orai1 but not TRPC1-mediated SOCE. (A) HEK293 cells were transfected with TRPC1, STIM1, STIM1(684EE685), or STIM1(684EE685) + TRPC1(639KK640). (B) HEK293 cells were transfected with Orai1, STIM1, or STIM1(684EE685). Additions of Tg or Ca2+ are indicated. (C) Average data showing statistical analysis. ** indicates values significantly different from the control value in each case .STIM1((TIF)Click here for additional data file.Figure S42+ store depletion.Co-localization of STIM1 and TRPC1 is stimulated by store depletion. (A) Left: TIRFM image of HSG cells expressing CFP-STIM1 and YFP-TRPC1 . Right, upper panel shows five cells used to evaluate the increase in fluorescence intensity of the two proteins; 5 was a control ROI shown in the graph given below. Accumulation of both proteins increased at the same rate (each has been expressed relative to their respective maximum fluorescence). (B) Similar experiment using cells in which Orai1 had been knocked down by siOrai1 . Other details are similar to that given for (A). Knockdown of Orai1 does not affect STIM1-TRPC1 clustering stimulated by Ca(TIF)Click here for additional data file.Figure S52+ buffers in the pipette solution on Tg-stimulated currents. (A) [Ca2+]i was clamped in the pipette solution at the levels indicated. ISOC was not activated by increasing the [Ca2+] in the pipette solution to 1 µM but was activated following Tg stimulation of cells with pipette solution buffered to 100 nM Ca2+.  Inclusion of 10 mM BAPTA instead of 10 mM EGTA in the pipette solution did not change the activation and properties of Tg-stimulated ISOC . (D) KCa channel activity measured in Tg-stimulated HSG cells with 10 mM BAPTA in the pipette solution, and Ca2+ in external solution was increased from 1 mM to 10 mM (third panel in C).Effect of including Ca(TIF)Click here for additional data file.Figure S6Localization of Orai1, TRPC1, STIM1, and AQP5 in resting and pilocarpine stimulated submandibular glands. Salivary gland sections were obtained from mice (treated as described) and used for immunofluorescence Click here for additional data file.


Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca2+ imaging and Ca2+ selective microelectrodes we found that changes in e-c coupling, SR Ca2+content and resting [Ca2+] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca2+ regulation than Jct/CASQ association.Triadin (Tdn) and Junctin (Jct) are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ) and ryanodine receptor (RyRs) at the junctional sarcoplasmic reticulum (jSR). However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c) coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null), Jct (Jct-null) or both (Tdn/Jct-null), we identify Tdn as the main component of periodically located  The SR has two clearly delimited domains with distinct function, structure and composition: the free SR (fSR) rich in sarco-endoplasmic reticulum Ca2+ ATPase (SERCA1) important for Ca2+ re-uptake and the junctional SR (jSR), containing among other proteins the ryanodine receptor Ca2+ release channels (RyRs) and the Ca2+ binding protein calsequestrin (CASQ). The jSR functionally communicates with invaginations of the surface membrane  where RyR1 interacts with several protein components forming functional multi-protein complexes defined as the Calcium Release Unit (CRU).The sarcoplasmic reticulum (SR) of skeletal muscle is a differentiated domain of the endoplasmic reticulum 2+binding protein 2+ storage capacity 2+, at physiological concentrations In adult skeletal muscle CRUs are in the form of triads with two jSR cisternae, also called lateral sacs, facing a central T-tubule. In the junctional face membrane of the jSR, RyR1 interacts with Tdn, Jct and CASQ forming a macromolecular complex thought to regulate RyR1 activity Junctin 2+ homeostasis in skeletal muscle 2+ homeostasis comes primarily from studies in cardiomyocytes where either overexpression 2+ release and contractility. Studies in C2C12 myotubes 2+ store size and mediating the signaling between CASQ1 and RyR1.In addition to being involved in facilitating the cross-communication between CASQ and RyR1 −/−, −/−, anchoring of the CASQ polymer to the jSR membrane and a synergistic effect of both Tdn and Jct (but with a predominance of Tdn) in stabilizing the CASQ polymer within the jSR vesicles. Consistent with their corresponding effects on jSR CASQ retention Tdn-null cells showed a reduced e-c coupling efficiency, but the lack of Jct had very limited, if any, functional effects.Taking advantage of the currently existing Jct-null Jct−/−,  and Tdn-V1.1, Tdn, FKBP12, SERCA-1, CASQ, Junctophilin-1 [JP-1] and Histidine-rich Ca2+ binding protein, HRC)  . Tdn-nulin, HRC) , consistV1.1 and HRC expression in double null muscles. Altogether, these results suggest that the absence of Jct alone had a minimal effect on the expression profile of its other CRU partners. However, in the absence of Tdn the lack of Jct had a dramatic effect on expression of CASQ.The expression profile of CRU proteins of Tdn/Jct-null muscles closely resembles that of the Tdn-null muscle showing a similar up-regulation of RyR1 (∼55%), SERCA-1 (∼28%) and FKBP12 (∼320%) and down-regulation of JP-1 (∼10%) . InteresIn order to relate the structural changes to specific fiber types, we used three types of muscles. Mouse EDL contains a majority of fast twitch type IIB fibers The ultrastructure of a skeletal muscle triad consists of a central T-tubule profile (TT) flanked by two jSR cisternae that are joined to it by two SR feet (RyRs) on either side  A. In a anchors, . The line is of the same general appearance as those constituting the randomly disposed linear CASQ polymers. Periodic anchors and lines were observed in all muscles from WT mouse analyzed in this study.A structural detail that has been poorly emphasized in the past is the presence of noticeable periodically disposed electron densities, Since the expression level of either Tdn or Jct is not affected by the absence of the other, the single and double null mutants mice offered the unique opportunity of clearly distinguishing the specific structural functions of the two proteins. The loss of Tdn, Jct and both proteins did not affect either the overall appearance of the junctional gap between the membranes of SR and T-tubules or the frequency and disposition of feet within it, but differentially and sometimes profoundly affected the architecture of the jSR lumen.anchors, the fine line connecting the anchors to the CASQ network and the network itself are still present  on jSR structure is more profound than that of deletion of Tdn alone. In this case jSR profiles show no evidence of any internal substructure, although they show a slight diffuse density, and they are quite narrow  and caffeine-induced Ca2+ release, as well as their ability to modulate total SR Ca2+ content and myoplasmic resting free Ca2+ concentration. In spite of obvious structural and functional differences between cultured myotubes and adult fibers, myotubes were chosen for the current work based on our previous studies in the Tdn-null model. Those studies showed that the behavior of cultured myotubes closely resembled the behavior of adult muscle fibers in terms of e-c coupling efficiency, caffeine-induced Ca2+ release, SR Ca2+ content and cytoplasmic resting calcium concentrations 2+ transients as a result of using BTS to prevent contraction, we chose myotubes and not adult muscles to make physiologic measurements in the current study.To correlate the extent of the jSR alterations of each phenotype with its corresponding effect on Ca2+ transients and the sensitivity to K+  with no evident change in K+ sensitivity . Tdn/Jct double-null cells, on the other hand, displayed a small but statistically significant reduction (p<0.01) in the rate of Mn2+ quench when compared to WT myotubes. These results suggest that structural alterations of the jSR induced by lack of Tdn and Jct expression had only a minor effect on ECCE. The fact that peak amplitudes of K+-induced Ca2+ transients of Tdn-null and Tdn/Jct-null myotubes are not statistically different strongly suggests that the reduction in ECCE observed in Tdn/Jct-null cells had a negligible effect on the global Ca2+ signal induced by depolarization.To evaluate the potential role of extracellular Ca2+ release we compared the caffeine-induced Ca2+ release in Fura-4F loaded myotubes from each phenotype. Jct-null cells displayed average caffeine dose responses curves that closely resembled that of WT myotubes (WT 5.0±0.3 mM vs EC50Jct 4.7±0.3 mM (p>0.05). By comparison, Tdn-null cells showed both a significant reduction in peak Ca2+ release amplitude  and a noticeable rightward shift in caffeine sensitivity . Tdn/Jct double-null myotubes displayed an even greater reduction in peak caffeine induced Ca2+ transient amplitude . Caffeine EC50 was shifted to the right compared to WT but was unchanged relative to Tdn-null .To assess the direct effect of absence of either protein on RyR1-mediated Camyotubes  A, with 2+ content of cultured myotubes was estimated from the Ca2+ signal obtained by emptying SR stores with the SERCA pump inhibitor cyclopiazonic acid (CPA). 2+ release traces of Fura-2 loaded myotubes challenged with 10 µM CPA. Average peak 340/380 ratios values for WT, Jct-null, Tdn-null and Tdn/Jct-null myotubes (2+ load of 6% (p>0.05), 17% (p<0.01) and 38% (p<0.001). These data support the hypothesis that there is a significant reduction of SR Ca2+content in Tdn-null and Tdn/Jct-null but not Jct-null myotubes, and seems consistent with the caffeine-induced Ca2+ release data and the relative CASQ expression levels observed in each phenotype.SR Camyotubes  C are; 12+ concentrations for each phenotype were determined in cultured myotubes by direct measurement with Ca2+ selective microelectrodes (2+]rest than WT myotubes (188±12 nM vs 118±4 nM for Tdn-null and WT respectively). By comparison, Jct-null myotubes had a modest, although significant, increase in [Ca2+]rest to 136±7 nM while [Ca2+]rest in double null myotubes (255±8 nM) was significantly more elevated than Tdn-null (p<0.001).Resting Caectrodes . As prevanchors). The disappearance of the anchors in Tdn-null muscles, while Jct and CASQ are still present, is a direct indication that Tdn is the protein responsible for CASQ anchorage. Identification of Tdn and not CASQ as the major component of the anchor is quite consistent with the fact that anchors are present in the junctional face membrane of CASQ1-null fast twitch fibers CASQ polymer within the jSR cisternae is anchored to the RyR-bearing jSR membrane by periodically disposed electron opaque densities (anchors and whether their periodic positioning is consistent with known Tdn/RyR interactions anchors (∼4 nm) is consistent with two alternate models of the protein et al., 1995 may constitute the visible anchors. This is depicted in the model  upon depletion of Ca2+ stores Decrease in jSR cisternae size, the loss of visible content and expression levels of CASQ follow the same trend: all are hardly changed in Jct-null; significantly reduced in Tdn-null and greatly reduced in Tdn/Jct-null. This ties CASQ expression levels (an indirect indication of the protein stability) to its retention within the jSR and establishes a correlation between volume of the jSR cisternae, visibility of the CASQ mass in the electron microscope as a structured coil of protein and the content of polymerized CASQ. Extending the observation of monomer to polymer transition in the movement of CASQ from the rough ER to the jSR In skeletal muscles Tdn is the prevalent CASQ retaining protein and in our view anchors are to be considered initiation sites for its polymerization while Jct has a less critical role, but both on their own can retain a portion of CASQ within the jSR. The very reduced size of jSR cisternae and the presence of large SR sacs containing what appears to be polymerized CASQ in fibers of the double null muscles suggests that CASQ protein may not be retained within the jSR at all when both proteins are missing.2+ load and [Ca2+]rest) were equivalent in myotubes and adult muscle fibers 2+ load parallels the reduction in CASQ content and jSR volume. Importantly, we show that in Tdn-null the well-defined phenotype is accompanied by a seemingly total loss of CASQ achoring to the jSR. The effects of Tdn ablation on e-c coupling and SR Ca2+ content have been associated with hyperactivation of RyR1 as a result of disruption of the FKBP12/RyR1 interaction 2+ homeostasis in these cells.Although cultured myotubes do not exactly replicate adult muscle physiology in our previous studies in Tdn-null mice we showed that the same functional features analyzed in this study , humbug and junctate 2+ cycling regulation of cardiac or skeletal muscles is unknown but because of this nonspecific effects on [Ca2+]rest as a result of Jct ablation can not be ruled out.All nulls show elevated [Ca2+ homeostasis. Thus, despite the similarities between the two proteins, triadin and junctin in skeletal muscle are not structurally and functionally equivalent.Overall, our study indicates that in skeletal muscle Tdn plays a more critical role than Jct in defining the structural architecture of the jSR and identifies Tdn as the preferred anchor points for CASQ. The loss of anchor points with ablation of Tdn and Tdn/Jct coincided with a loss of polymerized CASQ that ultimately determined the size and shape of the jSR cisternae. Importantly, the severity of the anchor's disruption was mirrored by its functional effects on intracellular CaAll experiments on animals from creation of null and double mice to establishment of their structural and physiological phenotypes were conducted using protocols approved by the institutional animal care and use committees at the Harvard Medical School.Triadin-null (Tdn-null) and Junctin-null (Jct-null) mice were generated as described previously 1S , Junctin  Junctophilin-1 and HRC  and GAPDH . Membranes were then incubated with either goat anti-mouse or goat anti-rabbit horseradish-peroxidase-conjugated, secondary antibody and developed with SuperSignal ultra chemiluminescent substrate  and the intensity of the signal collected using a Kodak Imaging Station 4000MM PRO . Band identification and densitometry of the identified proteins were performed using Kodak MI Software (version 4.5.1 ES). Net band intensities of unsaturated blots were normalized to GAPDH expression  to correct for variations in protein loading between lanes and then expressed as fraction of the WT signal.Crude membrane preparations from lower limb muscle and primary myotubes were prepared as described previously 4 in the same buffer for 1–2 hr at 4°C, en block-stained in saturated uranyl acetate and embedded in Epon 812. Sections (about 40 nm) were cut in Leica Ultracut R microtome  using a Diatome diamond knife  and stained with lead citrate solutions. Sections were imaged in FP 505 Morgagni Series 268D  with Megaview III digital camera  and in Phillips 410  with Hamamatsu C4742-95 digital camera  electron microscopes.C57Bl/6 WT, Jct-null, Tdn-null and Tdn/Jct-null mice at 3–4 months of age were sacrificed by cervical dislocation. EDL and soleus muscles were dissected, pinned to a Sylgard dish (Dow Corning) at resting length and fixed. Sternomastoid was fixed in situ before dissecting. The muscles were fixed with 3.5% glutaraldehyde in 0.1 M Sodium Cacodylate buffer (pH 7.2) at room temperature and stored in fixative at 4°C for variable periods of time. Muscle segments were post-fixed in 2% OsOFigures were mounted and labeled using Adobe Photoshop® v7.0.The area of the jSR cross sectioned profile was measure using NIH image (ImageJ 1.45) in randomly collected images taken at a magnification of 143,000. Dimensions of feet, anchors and SR-SR bridges were measured using Photoshop from images at a magnification of 143–184.000. Statistical differences were evaluated using a Student's t test for unpaired data (Excel Software (Microsoft) and Prism 4.0 (GraphPad)). Unless otherwise indicated, EM data are presented as mean ± standard deviation (SD).2+ imaging was performed 5 days after differentiation in myotubes loaded with either 5 µM Fura-4F AM  in imaging buffer . Sensitivity to K+-depolarization and caffeine-activation were determined by 5 s perfusion with 5–6 volumes of KCl (15 mM to 60 mM) or caffeine (3 mM to 40 mM). Cell were alternately excited at 340 nm and 380 nm at a rate of 4 Hz with a DG4 multi-wavelength light source and the fluorescent emission at 510 nm captured from regions of interest within each myotube using a Stanford Photonics 12 bit digital intensified CCD. SR Ca2+ content of cultured myotubes was estimated from the peak amplitude of the Ca2+ release signal induced by 10 µM cyclopiazonic acid (CPA) from cells loaded with 5 µM Fura-2F AM. Data are displayed and analyzed using QED imaging software . Fluorescence signals are expressed as ratio of signals collected at alternating 340 nm/380 nm excitation wavelengths.Primary myoblasts were isolated from mouse skeletal muscles of each phenotype and differentiated as described previously 2+ entry during depolarization was estimated from the rate of dye quench by Mn2+ entry in myotubes loaded with 5 µM Fura-2-AM according to 2+ release from SR stores during depolarization cells were incubated overnight with 12 µM ryanodine to block RyR1 activation. Cells were depolarized with 80 mM KCl in Ca2+-free imaging buffer containing 0.5 mM MnCl  at the isosbestic wavelength for Fura-2 (360 nm) and fluorescence emission at 510 nm was then captured from regions of interest within each myotube at a rate of 15 frames per second (fps).Ca2+ concentrations of myotubes was performed with double-barreled Ca2+-selective microelectrodes assembled with ETH129 resin as described previously Determination of myoplasmic resting free Ca


Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca2+ buffering as well as its potential for modulating RyR1, the L-type Ca2+ channel  and other sarcolemmal channels through sensing luminal [Ca2+]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca2+ content and SR Ca2+ release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca2+ release with single action potentials and a collapse of the Ca2+ release with repetitive trains. Under voltage clamp, SR Ca2+ release flux and total SR Ca2+ release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca2+ release flux appears to be solely due to elimination of the slowly decaying component of SR Ca2+ release, whereas the rapidly decaying component of SR Ca2+ release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca2+] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca2+]free in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca2+ buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca2+ release.In skeletal muscle, the release of calcium (Ca During skeletal muscle activation, a single action potential initiates a release of ∼200 µM of Ca2+ (per liter of fiber volume) from the SR which causes a “twitch” contraction. As the SR [Ca2+] in fast muscle is at approximately 30–40% of maximal capacity at rest, several groups have estimated twitch release to be between 10–18% of the total releasable SR Ca2+ in fast twitch muscle from frog or mouse 2+ in the basal state and a rapid restoration of SR Ca2+ content due to increased sarco-endoplasmic reticulum ATPase activity (SERCA) and dynamic refilling of the SR through sarcolemmal Ca2+ influx  and its binding partners in the SR membrane as potential modulators of the RyR1, the DHPR and other sarcolemmal channels through sensing luminal [Ca2+] Specialized systems have evolved for the robust and rapid release of calcium  is a positive predictor of myofiber contractile force generation 2+ release from the SR, contributed to the significant ‘fade’ in force phenotype seen in muscles from CASQ1 null mice.As Ca2+ transients and Ca2+ homeostasis in single, intact WT and CASQ1 null muscle fibers stimulated by single or repetitive field stimulation. FDB fibers from 5–7 week old mice were loaded with the ratiometric Ca2+ indicator Indo1-AM and after dye conversion stimulated with field electrodes as previously described 2+ transient in CASQ1 null fibers in response to a single AP  by using Eq. 3  in the maximum peak Ca2+ release in the CASQ1 null fibers compared to WT. maxR, allowing a better visualization of effect of ablation of CASQ1 expression on the voltage dependence and steepness of the Ca2+ release. The halfV values shown here indicate a non-significant shift of 3 mV in the voltage-dependence of Ca2+ release in CASQ1 null animals  is markedly decreased in the CASQ1 null fibers, consistent with a greatly reduced total SR Ca2+ content.2+ release waveforms for larger depolarizations  is characterized by a pronounced early peak that occurs within 10 ms of the application of the pulse fibers were plotted together and normalized to the peak values at voltages that elicited Ca2+ release , further supporting the selective suppression of the slowly decaying component of release in CASQ1 null fibers.2+ release flux record  of a WT and a CASQ1 null myofiber. The maximum sustained nadir in F4-CmC was taken as the maximum depletion of SR Ca2+free as 1 mM 4-CmC releases >98% of the releasable SR Ca2+ content 4-CmC normalized to maximum SR depletion , the change in fluorescence  was normalized to Fdepetion to yield the ΔF/F4-CmC. In this presentation the ΔF/F4-CmC at the start of the experiment minus the ΔF/F4-CmC at the nadir represents the relative SR[Ca2+] content. Here we report that when compared to WT, CASQ1 null fibers exhibited a ∼24% reduction SR [Ca2+] compared to WT myofibers  was used to assay maximal depletion of SR evacuation and recovery was assayed with prolonged 50 Hz trains of AP's using field stimulation. Recently we demonstrated that this protocol could produce ∼98% depletion of the SR[Ca2+]free in WT myofibers stim/Fstim) normalized to the maximal depletion to highlight the temporal kinetics. Following the initiation of tetanic stimulation (0.5 ms pulse @ 50 Hz) in both genotypes, SR Ca2+ content reached the nadir relatively rapidly, and recovered soon after the stimulation was terminated. On an expanded time scale it was also evident that there was a delay prior to the final exponential decay of SR Ca2+ content in WT fibers that was not present in the CASQ1 null fibers. As a result, the decay time to the nadir was very much shorter in the CASQ1 null fibers  Both voluntary and involuntary exercise capacity was greatly reduced in the CASQ1 null mice. (B.) While a substantial deficit in peak force produced by the CASQ1 null EDL muscles could be almost fully accounted for by a decrease in mass these muscles exhibited a dramatic frequency dependent inability to sustain tension during a pulse train. (C.) With electric field stimulation of intact FDB fibers there is a significant decrease in the Ca2+ transients in CASQ1 null myofibers for single action potentials, and a collapse of the Ca2+ transient with repetitive trains of pulses. (D) Under voltage clamp, the peak SR Ca2+ release flux, as well as total SR Ca2+ release is significantly reduced in CASQ1 null fibers. The suppression of Ca2+ release flux in CASQ1 null fibers appears to be due to a selective suppression of the slowly decaying component of SR Ca2+ release (E.) There is a significant decrease in the SR [Ca2+]free and a increase in the SR [Ca2+] decay and reuptake kinetics in CASQ1 null muscles consistent with a loss of an intra-SR Ca2+ buffering capacity. Taken together we have determined that the genetic ablation of CASQ1 expression results in significant deficits in contractile activation consistent with alterations in SR Ca2+ release and/or SR [Ca2+].In the present investigation we used assays of functional performance 2+ release flux in CASQ1 null fibers appears to be solely due to elimination of the slowly decaying component of SR Ca2+ release, whereas neither the amplitude nor the time course of the rapidly decaying component of SR Ca2+ release is altered. One possible explanation for this finding could be that the rapidly decaying component of release comes from an intra-SR Ca2+ pool that is not buffered by CASQ1, whereas the slowly decaying component comes from another SR pool that is buffered by CASQ1. Alternatively, the rapidly decaying component of release could already be completed by an early time point after the start of depolarization at which the intra-SR[Ca2+] had not yet significantly declined  with no significant change in the voltage-dependence of Ca2+ release  is appreciably decreased 400 to 500 msec after the start of large depolarizing pulses in WT fibers. By contrast, our voltage clamp studies focused on events occurring during the first 10 to 20 msec following the start of similar large depolarizing steps. Independent of any possible later SR Ca2+ permeability changes in the WT fibers, our results showed that both amplitude and time course of the rapidly decaying component of SR Ca2+ release was essentially unchanged in CASQ1 null fibers, whereas the slowly decaying component of release was fully eliminated. Our use of relatively brief (20 ms) depolarizing pulses served as an empirical test of this interpretation. The late occurring events  studied by Sztretye et al. 2+ permeability changed in WT fibers but not in CASQ1 null fibers, were not examined here. In contrast, by focusing on the events early during depolarization we show that the component of release that turns off within 10–20 ms of the start of a large depolarization is independent of the presence or absence of CASQ1, whereas the more slowly decaying component of release appears to be totally dependent on the presence of CASQ1.A recent paper has also focused on differences in Ca2+ release is eliminated in CASQ1 null fibers, but the rapidly decaying component is unchanged is that the total Ca2+ release from the SR should be considerably smaller in CASQ1 null than in WT fibers. Whether the 40% suppression in measured total SR Ca2+ released in EGTA buffered CASQ1 null fibers A corollary to our conclusion that the slowly decaying component of SR Ca2+]free of between 20% 2+]free in CASQ1 null myofibers. In these experiments we used 5 mM BAPTA-AM to assist in the suppressing of movement during 4-CmC induced Ca2+ release  we identified a significant reduction of maximal twitch or tetanic force in the CASQ1 null , has been recently addressed by Zhao et al. 2+ influx (SOCE) in apparent compensation for the increased depletion of the SR during muscle fiber activation. Based on our functional assessments in vivo  it is clear that this enhancement in SOCE that decreases fatigue in vitro does not fully compensate for the EC coupling dependent processes that are needed to sustain physiologic function in vivo.It is clear that Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of the University Of Maryland School Of Medicine . C57BL/6 CASQ1 null mice All experiments involving animals were conducted according to the National Institutes of Health Voluntary exercise capacity was assessed in previously sedentary CASQ1 null (n = 6) and wild type . Mice were singly housed in a cage with a mouse running wheel  in a 12/12-hour light/dark animal facility with ad libitum access to food and water. The number of wheel revolutions was computer analyzed per 24-hour period and data was converted to mean number of meters per day and reported as descriptive data.Treadmill exercise capacity  was evaluated in previously sedentary CASQ1 null (n = 6) and WT (n = 6). The protocol was similar that of Massett and Berk Forepaw grip strength was determined on a commercial rodent grip strength meter . Peak grip strength was assessed in 7 successive trials separated by 10–15 seconds by a single rater blinded to genotype. The highest two values were averaged, normalized to bodyweight and used as a measure of the peak grip strength. The decrement in force between the peak value and the value of the 7th trial was evaluated as an index of fatigue.extensor digitorum longus (EDL) was assessed in vitro using methods described previously in vitro bath between a fixed post and force transducer (Aurora 300B-LR) operated in isometric mode. The muscle was maintained in physiological saline solution  containing (in mM) 119 NaCl, 5 KCl, 1 MgSO4, 5 NaHCO3, 1.25 CaCl2, 1 KH2PO4, 10, HEPES, 10 glucose, and maintained at 30°C under aeration with 95 O2/5 CO2 (%) throughout the experiment. During a 5 min equilibration, single twitches were elicited at every 30 seconds with supra-maximal electrical pulses (200 µsec.sec) via platinum electrodes running parallel to the muscle. For brief tetanic stimuli 250 msec trains of pulses were delivered at 1, 10, 20, 40, 60, 80, 100, 150 Hz and isometric tension was evaluated. After the experimental protocol, the muscle rested for 5 min at which time muscle length was determined with a digital micrometer, and the muscle was trimmed proximal to the suture connections, blotted and weighed. The cross-sectional area for each muscle was determined by dividing the mass of the muscle (g) by the product of its length  and the density of muscle  per cross-sectional area. Statistical comparisons between groups were performed using ANOVA procedures (SigmaStat v3.1). Significance set at p<0.05.Muscle performance of the Fibers were prepared using enzymatic dissociation of flexor digitorum brevis (FDB) muscles of 6- to 7-week-old CASQ1 null and WT mice, and were cultured as previously described Indo1 AM ratiometric recording and analysis were performed as previously described 2+ current measurements were carried out using external solution containing the following (in mM): 150 TEA-CH3SO3, 2 CaCl2, 1 MgCl2, 10 Hepes, 0.001 TTX, 1 4-aminopyridine, 0.025 N-benzyl-p-toluene sulfonamide (BTS), and pH of solution adjusted to 7.4 with CsOH. The pipette solution  contained (in mM): 140 Cs-CH3SO3, 10 Hepes, 20 EGTA, 6 MgCl2, 11.5 CaCl2, 4 Na2ATP, 14 creatine phosphate, 0.3 Na2GTP, and 0.1 leupeptin and solution pH adjusted to 7.4 with CsOH to obtain a buffered solution.Membrane current measurements were performed using the whole-cell configuration of the patch-clamp technique +. Typically, the voltage depolarization steps were applied 300 ms after the start of the confocal scan sequence, thus providing control images prior to stimulation at the start of each sequence. These control images were used to determine the resting steady-state fluorescence level (F0). Average intensity of fluorescence as a function of time ) within selected areas of interest (AOIs) were measured using Image Examiner . The average F0 value in each AOI prior to test pulse is used to scale fluo4 fluorescence in the same AOI as F/F0.Fluo-4 fluorescence line-scan (100 µs/line) confocal microscopy measurements were carried out on a Zeiss LSM 5 Live system as previously described 2+] during a Ca2+ transient in these fibers the fluorescence F/F0 signal must be corrected for both dye saturation and for the kinetic delay in dye equilibrium. The kinetic lag and dye saturation can be corrected using an approach previously described for Fura-2 ratio signals 2+, the time course of cytosolic free Ca2+concentration ([Ca]cyto(t)), is given by:2+ from the indicator dye, koffd is the off rate constant for Ca2+ dissociation from the dye, F is the fiber fluo-4 fluorescence signal and Fmax and Fmin are the fluorescence at saturating and zero free Ca2+, respectively.In order to estimate the time course of free cyt (t) is derived from F(t)/F0 2+]cyto(0) is free cytosolic [Ca2+] at resting levels. This equation assumes that the dye is far from saturation by Ca2+, which appears justified for fluo-4 considering that [Ca2+]cyto was always much lower than the value of fluo-4's KD previously determined using in situ calibrations 2+]cyto(0) was set to 0.081 µm. This assumption was roughly consistent with measurements of resting [Ca2+] under similar experimental conditions using the ratiometric indicator indo-1 When the concentration is changing slowly, the following condition applies:2+ in the pipette, EGTA still the major binding site for released Ca2+. In that case d [Ca-EGTA](t)/dt provides a good approximation to the rate of SR Ca release in the fiber, and will be used in the Ca2+ release calculations presented here. For Ca2+ binding to EGTA,one and koffe are on and off rate constants for Ca2+ binding to EGTA.2+]cyto(t) from Eq. 3, and using the Euler method to calculate the time course of d[Ca-EGTA](t)/dt, which was used as the time course of the rate of Ca2+ release from the SR during the voltage clamp pulses. [EGTA] and [Ca-EGTA] inside the fiber at the start of each pulse were assumed to be 70% of that estimated for the pipette solution (obtained with the programs Maxchelator offd = 90 s-1 offe = 1.2 s-1 (EGTA KD = 0.08 µM).Under the conditions of 20 mM EGTA with added CaN-benzyl-p-toluene sulfonamide , to prevent contraction during imaging. During 37°C loading of fluo-5N, fibers were plated on cover slips coated with Matrigel . BAPTA-AM also reduced the fluorescence arising from fluo-5N molecules remaining in the cytosol and minimized the loss of fibers caused by the maximal release of Ca2+ from the SR. Fibers were studied within 2 hr after loading was terminated.2, 0.3 NaH2PO4, 5 HEPES, 5.5 glucose, 1.8 CaCl2, and 5 KCl, pH of7.4 with NaOH. Confocal images were acquired on an inverted microscope  equipped with an oil immersion objective  and a 488 nm excitation laser in line scan (xt) mode.Fibers loaded with fluo-5N were imaged in normal Tyrode's solution (NT), containing (in mM), 140 NaCl, 0.5 MgClChanges in the fluo-5N signal in response to application of 1 mM 4-chloro-m-cresol (4-CmC) in the bath were recorded with the confocal microscope in line scan mode. Fluo-5N was excited with a 488 nm laser and emitted light was filtered through a long pass, 505 nm filter.2, 1–50 Hz for 10 s) was used to trigger incremental SR Ca2+ release. Stimulation was produced with a Myopacer  through platinum electrodes placed on either side of the fiber of interest. Following a brief period of inactivity, we rapidly perfused 4-CmC onto the fiber (see above) to measure its maximal release of Ca2+ from the SR. Stimulation elicited SR release was then normalized to the 4-CmC result.Electrical stimulation (0.5 ms pulse duration, 40 V/cmStatistical analyses were performed using OriginPro 8.0 or SigmaStat 3.5. All data are presented as mean ± S.EM. unless otherwise noted. Statistical significance was assessed using either parametric two sample t-test, ANOVA or with the non-parametric Mann-Whitney rank-sum test as indicated. Significance was set at P<0.05.


Previous studies have shown that maternal malnutrition affects the reproductive tracts of adult male offspring. However, little is known about the effects of CM, a widespread life-long condition that persists from conception throughout growth to adult life.The aim of this work was to investigate the mechanisms by which chronic malnutrition (CM) affects vas deferens, hypertrophy of the muscle layer in the epididymal portion of the vas deferens and intense atrophy of the muscular coat in its prostatic portion. At a molecular level, the vas deferens tissue of CM rats exhibited a huge rise in lipid peroxidation and protein carbonylation, evidence of an accentuated increase in local reactive oxygen species levels. The kinetics of plasma membrane Ca2+-ATPase activity and its kinase-mediated phosphorylation by PKA and PKC in the vas deferens revealed malnutrition-induced modifications in velocity, Ca2+ affinity and regulation of Ca2+ handling proteins. The severely crippled content of the 12-kDa FK506 binding protein, which controls passive Ca2+ release from the sarco(endo) plasmic reticulum, revealed another target of malnutrition related to intracellular Ca2+ handling, with a potential effect on forward propulsion of sperm cells. As a possible compensatory response, malnutrition led to enhanced sarco(endo) plasmic reticulum Ca2+-ATPase activity, possibly caused by stimulatory PKA-mediated phosphorylation.Young adult male rats, which were chronically malnourished from weaning, presented decreased total and haploid cells in the vas deferens were an accentuated reduction in fertility and fecundity.The functional correlates of these cellular and molecular hallmarks of chronic malnutrition on the  It is generally accepted that reproductive performance in the adult is determined by a wide variety of influences, including nutritional status. It is clinically known that among the several forms of undernutrition, the life-long condition characterized by mild-to-moderate protein-energy malnutrition is the most common and is referred to as chronic malnutrition. This condition is defined by a deficiency of energy and nutrients over a period of several years that frequently impairs the growth and development of the child into adulthood ,2. Evidevas deferens, located in the accessory male genitalia, is a paired organ that provides a link between the epididymis and prostate gland. It comprises circular and longitudinal smooth muscles with columnar epithelium lining the lumen  and were also approved by the Committee for Experimental and Animal Ethics at the Federal University of Rio de Janeiro (protocol CEUA DFBCICB 007).All procedures were performed in accordance with “The guide for care and use of laboratory animals’’ -inorganic phosphate (32Pi) was obtained from the São Paulo Institute of Energetic and Nuclear Research. [γ-32P]-adenosine triphosphate ([γ-32P] ATP) was prepared as described by Maia and coworkers  ATP , 4 mM MgCl2 and 60 mM KCl, in the presence or absence of 3 µM thapsigargin. Enough CaCl2 was added to give free Ca2+ concentrations in the range 1 nM–10 μM (or a fixed 10 µM concentration), calculated according to Sorenson et al.  ATP  to a medium (0.1 ml) containing 20 mM HEPES-Tris (pH 7.0), 4 mM MgCl2, 1.5 mg/mL histone and 0.7 mg/ml protein. After 5 min, the reaction was stopped by adding 0.1 ml of 40% (w/v) TCA and the samples were immediately placed on ice. After vigorous stirring, an aliquot of 0.1 ml was removed, filtered through a Millipore filter (0.45 µm pore size) and successively washed with ice-cold 20% (w/v) TCA and 0.1 M phosphate buffer (pH 7.0). The radioactivity was quantified by liquid scintillation. The results were expressed as nmol of P ~ histone incorporated per mg of vas deferens tissue protein at 5 min (for both the PKA and the PKC activities).The protein kinase activities were analyzed in homogenized escribed ,28. The 2+-ATPase in homogenized vas deferens tissue was determined as previously described  ATP (specific activity ~3.0 × 1011 Bq/mmol) and 120 mM KCl . Ten min later the reaction was stopped by adding 50 µl of sample buffer [32P]-phosphorylated 140 kDa (PMCA) and 110 kDa (SERCA) bands recognized by their specific primary antibodies. Specific phosphorylation of Ca2+-ATPase by PKA or PKC was quantified by the difference between band intensities in the absence and presence of the respective inhibitors, and normalized by the intensities of the same bands immunodetected using the corresponding antibodies. The results were expressed as the ratio between the 32P signals from the autoradiograms (at 140 or 110 kDa), corrected by the PMCA and SERCA2 contents in the corresponding bands of the same nitrocellulose membrane. For both PKA- or PKC-mediated regulatory phosphorylations of PMCA and SERCA, the levels obtained with control rats were taken as 100%.Kinase-mediated phosphorylation of Caescribed ,29. The e buffer , and thet-tests. P<0.05 was considered the criterion for statistical significance.The data are presented as means ± S.E.M. Differences between the parameters studied in the CTRL and CM groups were analyzed using unpaired Student’s vas deferens were counted (-1 per 100 mg of wet whole tissue) that chronic malnutrition did not alter the total number of cells in the testis. However, the total cell counts were reduced by ~65% and ~80%, respectively, in epididymis and vas deferens (-1 per 100 mg of wet tissue) and vas deferens   were considerably higher in the undernourished group  was seven-fold higher than in CTRL  and the thapsigargin-sensitive Ca2+-ATPase .The presence of the molecular entities responsible for Cacade ago . Here we2+-ATPase activity demonstrated increased overall active Ca2+ handling across the membranes of the CM rats, which was quantified in the following experiments. 2+-ATPase activity – i.e. one way to evaluate ATP-dependent Ca2+ transport – can be described as a first-order process in both experimental groups, thus allowing the rate constant k to be measured  to evaluate the contributions of PMCA and SERCA, and their respective kinetic parameters were investigated in assays carried out at different free Cavas deferens of CM rats  in the SERCA2 amino acid sequence is phosphorylated by Ca2+/calmodulin-dependent protein kinase (CaM kinase). This phosphorylation induces an increase in the Vmax of Ca2+ transport [vas deferens tissue via a pathway in which CaM kinase participates. However, further studies regarding Ca2+ concentrations in muscle cells of the vas deferens and physiological motility of the whole organ in chronically malnourished rats are needed to elucidate the detailed mechanisms.The alterations in thickness of the muscle coat of the h muscle . The incnd SERCA Table 3 icus L.  and in ricus L. ,29, whericus L. , and theransport , suggestvas deferens function provoked by chronic malnutrition. Therefore, they convey an advance in the understanding of how this systemic disorder can negatively affect male reproductive capacity in populations worldwide.In conclusion, the findings described here provide experimental evidence regarding the molecular mechanisms involved in the compromised


Electrophysiological and confocal imaging experiments showed that the voltage-dependence of L-type Ca2+ current (ICa) was shifted to higher potentials in ventricular myocytes from ground squirrels vs. rats. The elevated threshold of ICa did not compromise the Ca2+-induced Ca2+ release, because a higher depolarization rate and a longer duration of action potential compensated the voltage shift of ICa. Both the caffeine-sensitive and caffeine-resistant components of cytosolic Ca2+ removal were more rapid in ground squirrels. Ca2+ sparks in ground squirrels exhibited larger amplitude/size and much lower frequency than in rats. Due to the high ICa threshold, low SR Ca2+ leak and rapid cytosolic Ca2+ clearance, heart cells from ground squirrels exhibited better capability in maintaining intracellular Ca2+ homeostasis than those from rats and other nonhibernating mammals. These findings not only reveal adaptive mechanisms of hibernation, but also provide novel strategies against Ca2+ overload-related heart diseases.Heart tissues from hibernating mammals, such as ground squirrels, are able to endure hypothermia, hypoxia and other extreme insulting factors that are fatal for human and nonhibernating mammals. This study was designed to understand adaptive mechanisms involved in intracellular Ca Impaired regulation of intracellular Ca2+ under pathologic, ischemic and hypothermic conditions leads to cellular dysfunction and global disorders. For example, the inability to elevate [Ca2+]i to a required level following an excitation is a major pathogenic mechanism involved in heart failure 2+, a status known as Ca2+ overload, has been proven to be deleterious to almost all cell types, and can be associated with either necrotic or apoptotic cell death 2+ may induce severe arrhythmias and ventricular fibrillation Intracellular Ca2, combined procaine and adrenaline treatment or ligation of coronary artery branches cannot trigger the ventricular fibrillation that would commonly occur in non-hibernator hearts Heart tissues from hibernating mammals demonstrate enhanced resistance against many insulting factors that cause heart dysfunction in humans and non-hibernators 2+ homeostasis is maintained against so many pathogenic/insulting factors. In heart cells, most of the Ca2+ cycling components are involved in E-C coupling, where the Ca2+ influx thorough the voltage gated L-type Ca2+ channels (ICa) activates the Ca2+ release from the ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) 2+ entering the cytosol is either taken back to the SR by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), or removed via Na+/Ca2+ exchange and other Ca2+ transporting systems 2+ current and up-regulate the SR Ca2+ uptake and release capacity when they hibernate 2+ handling is a part of the adaptive preparation for maintaining forceful contraction under hibernating conditions. However, previous studies have not fully explained why hibernating mammals, even in their awake state, are still resistant against arrhythmogenic and hypoxic perturbations.Given these unique adaptations, it is intriguing how the intracellular Ca2+ homeostasis than other mammals, we systemically characterized the Ca2+ cycling and E-C coupling processes in heart cells from ground squirrels in comparison with commonly used animal models.In order to test the hypothesis that hibernators are adapted for better maintaining intracellular Ca2+ dynamics during field stimulation in ventricular myocytes from rats and ground squirrels at varying temperature are illustrated in 2+ transients exhibited an increased background and decreased amplitude in rat cells but not in ground squirrel cells when the temperature was lowered from 30°C to 10°C. Below 15°C, most Ca2+ transients were accompanied by “after-transients”  from the holding potential of −50 mV  was roughly sequenced, and deeper sequencing is ongoing. With the application of modern research tools on hibernation research, the mechanisms underlying the adaptation of hibernating mammals, including those involved in Ca2+ homeostasis, will be understood in more detail. These mechanisms will be expected to provide new ideas for developing therapeutic strategies against diseases/disorders involving intracellular Ca2+ mishandling.In 2007, the entire genome of thirteen-lined ground squirrels . All surgery was performed under 20% ethylurethanm (i.p. injection 5 ml/kg) anaesthesia, and all efforts were made to minimize suffering.Citellus dauricus, is a species dominant in Northeast Asia, which belongs to the same genus as those live in North America, e.g., Spermophilus tridecemlineatus. The animals were trapped in the field of Zhanbei County in Hebei Province, China. Ground squirrels were kept in active state in an environment of natural photoperiod at room temperature. The Sprague-Dawley rats were housed by and purchased from the Laboratory Animal Center of Peking University. The hearts were rapidly excised from adult ground squirrels (200–250 g) or rats (200–250 g) under anaesthesia and mounted on a Langendorff apparatus. Ventricular cardiac myocytes from animals were enzymatically isolated as previously reported 2, 1.2 MgCl2, 1.2 NaH2PO4, 10 glucose and 10 HEPES, pH 7.35 adjusted with NaOH, and were used the same day they were isolated. The functional features of the prior non-hibernating state were assumed to be preserved after cell isolation, because isolated cardiomyocytes from hibernating and non-hibernating individuals exhibited distinct functional performances The ground squirrel used in the present study, 2+ concentration, cells were incubated in Tyrode's solution containing 2.5 µmol/L indo-1 AM for about 10 min in the dark at 37°C. The fluorescence was exited by a UV laser and measured by ASCS meridian 575UV confocal microscope using 485 nm and 405 nm band-pass filters as reported previously 2+ concentration was measured at different temperatures, and calculated with formula [Ca2+]i = kA(R−Rmin)/(Rmax−R), where kA was an apparent constant determined by calibration max and Rmin were the R in Ca2+ free and Ca2+-saturated buffers, respectively.When indo-1 fluorescence was used to determine intracellular Ca2+ current were recorded at room temperature with 4 mmol/L 4-aminopyridine and 0.02 mmol/L tetrodotoxin in the bath solution, as reported reviously 2 5, Na2ATP 5, TEA-Cl 15, EGTA 11, CaCl2 1, and HEPES 10 (pH was adjusted to 7.2 with CsOH). When Ca2+ transients were recorded simultaneously, the pipette electrode was filled with (in mmol/L) CsCl 127, NaCl 10, MgCl2 1, MgATP 5, TEA-Cl 15, HEPES 10 and fluo-4 pentapotassium 0.2 (pH 7.2 adjusted with CsOH). Leak current subtraction was performed Offline.Whole-cell Ca2 6, Na2ATP 5, EGTA 0.2 and HEPES 10 (pH was adjusted to 7.2 with KOH). Action potentials were evoked by 2-ms current pulses.Membrane potential was recorded at room temperature under whole-cell current clamp mode. The pipettes were filled with intracellular solution which contained (in mmol/L) KCl 125, MgCl2+ indicators were loaded into myocytes either via the pipette electrode in patch-clamp experiments 2+ level was either reported as the fluorescence normalized by its resting level (F/F0), or calculated according to the formula [Ca2+]i = kd·R/(kd/C0+1−R) 0, kd was set to 1.1 mol/L for fluo-4 and 2.3 mol/L for fluo-5N. The threshold for Ca2+ spark detection was set at 1.25 0F/F. Cell contraction was measured by edge detection and presented as the fractional shortening of cell length.Cat test unless otherwise specified. A P value<0.05 was considered to be statistically significant.The data are presented as means ± s.e.m. Statistical significance was determined using Student-Figure S1Contraction of papillary heart muscles in rats (left) and ground squirrels (right) in response to 0.2 Hz field stimulation at 8°C. Note the after-contractions and elevated resting tension in the rat.(TIF)Click here for additional data file.Figure S22+ load of ventricular myocytes at room temperature (RT) and 10°C were measured in rats and ground squirrels by perfusing the cells with 20 mmol/L caffeine after 15 min indo-1 AM loading. SR load was reported as the ratio of fluorescence at 405 nm vs. 485 nm.SR Ca(TIF)Click here for additional data file.