Since NCX proteins contribute to numerous physiological and pathophysiological events, their pharmacological targeting has been desired for a long time. This intervention remains challenging owing to our poor understanding of the underlying structure-dynamic mechanisms. Recent structural studies have shed light on the structure-function relationships underlying the ion-transport and allosteric regulation of NCX. The crystal structure of an archaeal NCX (NCX_Mj) along with molecular dynamics simulations and ion flux analyses, have assigned the ion binding sites for 3Na+ and 1Ca2+, which are being transported in separate steps. In contrast with NCX_Mj, eukaryotic NCXs contain the regulatory Ca2+-binding domains, CBD1 and CBD2, which affect the membrane embedded ion-transport domains over a distance of ~80 Å. The Ca2+-dependent regulation is ortholog, isoform, and splice-variant dependent to meet physiological requirements, exhibiting either a positive, negative, or no response to regulatory Ca2+. The crystal structures of the two-domain (CBD12) tandem have revealed a common mechanism involving a Ca2+-driven tethering of CBDs in diverse NCX variants. However, dissociation kinetics of occluded Ca2+ (entrapped at the two-domain interface) depends on the alternative-splicing segment (at CBD2), thereby representing splicing-dependent dynamic coupling of CBDs. The HDX-MS, SAXS, NMR, FRET, equilibrium 45Ca2+ binding and stopped-flow techniques provided insights into the dynamic mechanisms of CBDs. Ca2+ binding to CBD1 results in a population shift, where more constraint conformational states become highly populated without global conformational changes in the alignment of CBDs. This mechanism is common among NCXs. Recent HDX-MS studies have demonstrated that the apo CBD1 and CBD2 are stabilized by interacting with each other, while Ca2+ binding to CBD1 rigidifies local backbone segments of CBD2, but not of CBD1. The extent and strength of Ca2+-dependent rigidification at CBD2 is splice-variant dependent, showing clear correlations with phenotypes of matching NCX variants. Therefore, diverse NCX variants share a common mechanism for the initial decoding of the regulatory signal upon Ca2+ binding at the interface of CBDs, whereas the allosteric message is shaped by CBD2, the dynamic features of which are dictated by the splicing segment.Na The evolutionary choice of Ca2+ as a universal and versatile intracellular messenger has been dictated by its coordination chemistry  is essential in every living cell, where the maintenance of resting cytosolic levels as well as the cell-specific dynamic oscillations of cytosolic Ca2+ requires tight regulation and integration of Ca2+ transport proteins. Notably, the Ca2+ transporting proteins are located in the plasma membrane and in the membranes of the organelles , thereby playing distinctive roles in the excitation-contraction coupling of cardiac  extrude Ca2+ from the cell, although their partial contributions to Ca2+ homeostasis differ among distinct cell types, depending on the functional specialization and regulatory specificity in a given cell type  and low-affinity (Km ~ 10–20 μM) system that allows rapid removal of Ca2+; it can limit Ca2+ transients over a wide dynamic range  NCX may work in the reverse mode and induce Ca2+ influx  and their splice variants are expressed in a tissue-specific manner  modulate fundamental physiological events, such as muscle excitation-contraction coupling, neuronal long-term potentiation and learning, blood pressure regulation, immune responses, neurotransmitter and insulin secretion, and mitochondrial bioenergetics  of cardiac NCX enhances the turnover rates of NCX up to 25-fold  helices and contain a large cytosolic regulatory loop (f-loop) between TM5 and TM6 , X-ray crystallography, small angle X-ray scattering (SAXS), fluorescence resonance energy transfer (FRET) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) have been utilized to study the mechanisms underlying ion transport and the allosteric regulation of NCX. Despite the tremendous progress, some important questions remain open: Why are so many isoforms and splice variants required by different cell types and why does each cell type express a specific set of isoforms and splice variants? What are the exact mechanisms underlying the function and regulation of diverse isoforms and splice variants? What is the partial contribution of distinct splice variants to specific functions in a given cell type? During the last few years, huge progress has been made in better understanding the molecular mechanisms underlying NCX regulation in tissue-specific isoforms and splice variants. This review will focus on insights into NCX ion transport and allosteric regulation mechanisms derived from structural biology techniques in recent years.2+ and three Na+ ions are translocated sequentially in separate steps rather than simultaneously across the membrane. This mechanism implies the alternating access mechanism of the NCX ion binding sites in the inward (cytosolic) and outward (extracellular) conformations   with a pseudo molecular dyad Figure . It appeext, Smid, Sint, and SCa. Interestingly, 11 ion-coordinating residues (out of twelve) are highly conserved in organisms ranging from bacteria to humans, whereas in eukaryotic NCXs, D240 is consistently replaced by glutamine  generate a tightly packed hub (which is perpendicularly inserted into the membrane), whereas two long/slanting helices (TM1 and TM6) are loosely packed in front of the rigid eight-helix core Figure  Liao et. The sli2+ and Na+, and by H+  than the increase in the steady-state to peak-current ratio (~ 10 μM)  Figure  (Hilgema) Figure .+, Ca2+, and H+ and resulted in constitutive NCX activation   and brain (AD) variants exhibit similar regulatory responses to Ca2+, they differ in their response kinetics, with the kinetics of NCX1-ACDEF being ~ 10-fold slower compared with NCX1-AD  inactivates CALX1.1  contain the large cytosolic f-loop between TM5 and TM6. At the N-terminus, adjacent to the membrane, is an amphipathic α-helical region (20 residues) termed XIP (eXchanger Inhibitory Peptide) because it inhibits NCX when applied exogenously ; the brain splice variant (AD) of CBD2 contains two Ca2+ binding sites (CaI-CaII)  are positioned at the N-terminal portion of CBD2's F-G loop, adjacent to the CBD1 binding sites , specifically to the Ca3-Ca4 sites, results in NCX activation   were measured in our laboratory by 45Ca2+ equilibrium binding assays and the rate constants of Ca2+ dissociation were measured using stopped-flow kinetics  at its Ca3-Ca4 sites and two Ca2+ ions with lower affinity (>5–10 μM) at its Ca1-Ca2 sites. Monophasic dissociation of two Ca2+ ions from the Ca3-Ca4 sites is observed in both NCX1 and CALX1, with a rate constant of ~15 s−1  at its CaI site and one with low affinity (Kd > 20 μM) at its CaII site. Ca2+ dissociates from CaI with a rate constant of ~ 150 s−1. As in CBD1, the dissociation rate constant from the low-affinity CaII site is too fast to be measured using stopped-flow kinetics   deprotonate one or more coordinating residues, thereby enabling the next Ca2+ ion to bind to the remaining sites. A similar mechanism was proposed for the C2 domain of phospholipase A2, in which two Ca2+ sites are separated by 4.1Å , thus demonstrating the physiological relevance of the NCX “proton block” under acidosis and ischemia conditions  actually contribute to [Ca2+]-dependent regulation of full-size NCX1 in the cellar system  , whereas the occupation of the CaII site by Mg2+ increases the affinity of the CaI site  fashion, with a fast component (kf ~ 5 s−1) and a slow component (ks ~ 0.5 s−1)  Figure  (Giladi ) Figure . The slo2+ binding couple with regulatory conformational transitions to decode the allosteric signal, (ii) how is the regulatory signal diversified by alternative splicing, and (iii) how does the coupling of domains contribute to the transmission of regulatory information to ion transport domains. These questions were addressed using a variety of structural approaches, which are discussed below.Crucial mechanistic questions that have emerged from the studies described above are as follows: (i) how does Ca2+ binding to conformational transitions underlying allosteric regulation, and the role played by alternative splicing, the crystal structures of CBD12-E454K (a mutant of NCX1-CBD12-AD), CALX1.1-CBD12, and CALX1.2-CBD12 were determined    were examined  additional structural elements in the regulatory f-loop and/or membrane domain are involved in decoding and specifying the regulatory effects or alternatively, (ii) the conformational dynamics differ between the NCX splice variants and CALX, despite the similar orientations observed in the crystal structures and the SAXS-EOM data obtained for diverse splice variants, because there are either positive, negative, or no responses to regulatory Ca2+.The crystallographic and SAXS data presented thus far suggested common Ca2+, CBD12 from NCX1-AD (positive response), CALX1.1 (negative response), and CALX1.2 (no response) were studied using the advanced approaches of HDX-MS   in a given splice variant. For CALX1.1-CBD12, in which a minimal response to Ca2+ occurs  in CBD1 rigidifies CBD12, whereas the domains' interactions in turn stabilize Ca2+ binding, resulting in Ca2+-dependent regulation. This represents a common mechanism for decoding the initial information upon Ca2+ binding for all NCX isoform/splice variants. The CaI site on CBD2 exhibits structural variances, while being responsible for the Ca2+-dependent alleviation of Na+-dependent inactivation (Hilge et al., 2+ binding in different orthologs and splice variants is related to the extent and strength of CBD2 rigidification upon Ca2+ binding to CBD1 (Figures Recent structural and biophysical studies have shed light on the structural basis of ion transport and the allosteric regulation of NCX proteins. The structure of NCX_Mj (Liao et al., n Figure  (Boyman n Figure ; the extn Figure . Common n Figure , 2013. T Figures . It is hAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


H°, ΔS°, ΔG°37, and TM for each hairpin were determined. Substitution of 2-AP for an A (adenosine) at a bulge position (where either the 2-AP or A is the bulge) in the stem of a hairpin, does not affect the stability of the hairpin. For group II bulge loops such as AA/U, where there is ambiguity as to which of the A residues is paired with the U, hairpins with 2-AP substituted for either the 5′ or 3′ position in the hairpin stem have similar stability. Fluorescent melts were performed to monitor the environment of the 2-AP. When the 2-AP was located distal to the hairpin loop on either the 5′ or 3′ side of the hairpin stem, the change in fluorescent intensity upon heating was indicative of an unpaired nucleotide. A database of phylogenetically determined RNA secondary structures was examined to explore the presence of naturally occurring bulge loops embedded within a hairpin stem. The distribution of bulge loops is discussed and related to the stability of hairpin structures.Eleven RNA hairpins containing 2-aminopurine (2-AP) in either base-paired or single nucleotide bulge loop positions were optically melted in 1 M NaCl; and, the thermodynamic parameters Δ Ribonucleic acid, RNA, is central to life processes. Among these are the control of gene expression , intron The extraordinary functional capabilities of RNA can be attributed to its structural complexity. The most fundamental structural element in RNA is the duplex or stem, in which each nucleotide is base-paired with its complement. Other recurrent structural motifs include stem loops (hairpins), bulges, internal loops, pseudoknots, and multibranch loops. It should be noted that these motifs are likely to be crucial to the evolution of modern organism and studies have underscored their importance in a variety of biological events .Bulges are regions of unpaired nucleotides situated along one strand of a duplex and are often found at protein binding sites . The mosAU/3′ G A, the bold A is the bulged nucleotide. For groups II, III, and IV, the identity of the bulge and the base pairs adjacent to the bulge leads to ambiguity to the position and/or the identity of the bulge. For example, 5′CAA/3′G U, 5′ CAG/3′G U, and 5′GAA/3′U U, where the bold bases are the potential bulge, are typical group II, III, and IV bulge loop sequences, respectively.Single nucleotide bulge loops have been characterized by their structural characteristics into three classes, intercalated, extrahelical, and side-by-side (base triple) arrangements . In addiThe current work is a continuation of our studies on the stability and structure of bulge loops in the context of an RNA hairpin stem . A simpltert-butyl dimethylsilyl ether from Glen Research  and Sep-Pak C18 (Waters) chromatography. Some oligomers were ordered from Dharmacon and deprotection of the oligomers was carried out using the manufacturer's instructions. The oligomers were then purified as described above. Sample purity was determined through analytical TLC or HPLC (PRP-1 [Hamilton]), and was >95%.Most oligomers were synthesized on CPG solid supports (Applied Biosystems 392 DNA/RNA Synthesizer) utilizing phosphoramidites with the 2′ hydroxyl protected as the Research . OligomeOptical melting experiments were performed using a Beckman DU 640 Spectrophotometer and High Performance Temperature Controller at 260 or 280 nm. Absorbance changes for oligomers in 1 M NaCl melt buffer  were recorded as a function of temperature from 10°C to 90°C at a rate of 1°C/min as described previously . The expƛΔG°37(bulge), was calculated from the experimental data according to Equation ƛΔG°37(bulge hairpin) is the experimentally determined value from the melts and ƛΔG°37(parent) is the measured value for the parent strand.The free energy contribution of each bulged nucleotide, Fluorescent measurements were performed using a Shimadzu RF-501spectrofluorometer and a water bath for temperature control. Fluorescent melts were carried out at an excitation wavelength of 303 nm and an emission wavelength of 370 nm. The excitation and emission slits were 5 nm. Samples were heated from 0°C to 85°C at a rate of 1°C/min using the same buffer conditions as the optical melts. The fluorescence of each RNA hairpin was normalized to a 5 nt short strand of RNA that was analogous to the region of the RNA hairpin where the 2-AP was inserted.The RNA Strand database of RNA secondary structures  containiH°, ΔS°, ΔG°37, respectively. The average errors for the hairpins with group II bulge loops are larger  than for the fully base-paired or group I bulge loops perhaps indicative of some slight non-two-state melting (The thermodynamics for hairpin formation for 21 RNA hairpins containing either A or 2-AP in either the 5′ or 3′ stem of the hairpins are listed in  melting .Three of the fully base-paired parent hairpins (two on the 3′ side of the hairpin loop and one on the 5′ side) in Two group I bulge loop hairpins (one on the 3′ side and one on 5′ side of the hairpin) have 2-AP substituted for A as the bulge in Finally, six group II bulge loop hairpins (four on the 5′ side and two on the 3′ side of the hairpin loop) have 2-AP substituted for A as one of the possible bulged nucleotides. Substitution of 2-AP into the bulge loop for one of the A residues on the 5′ side of the hairpin stem leads to either an increase or decrease in stability by 0.8 kcal/mol. While 2-AP substitution on the 3′ side of the stem results in a slight increase in stability . Placement of the 2-AP in the proximal or distal position of the group II bulge loop, relative to the hairpin loop, has no effect on the stability of the hairpin for either the 5′ or 3′ group II bulge loops. In all six examples, the nucleotides surrounding the bulge are the same suggesting the differences must be related to non-nearest-neighbor influences such as the placement of the bulge in the hairpin stem. More studies will need to be done to elucidate the influence of 2-AP on hairpin formation.The fluorescence of 2-AP is sensitive to its local environment . The chaIn addition to local environment, the fluorescence of 2-AP is strongly influenced by temperature . TherefoThe results of fluorescent melts of the other 2-AP labeled hairpins listed in The substitution of 2-AP in either the proximal  or distal  position of the group II bulges leads to nearly the same change in thermodynamic stability of the hairpins ; therefoTo further characterize bulge loops, a database of RNA secondary structures was examined. The database contained 20,292 single nucleotide bulge loops. As previously observed , the mosThe database contained 6994 group II single nucleotide bulge loops representing 34.5% of all single nucleotide bulge loops. They are almost evenly divided between structures where the 5′ nucleotide is paired (3754) and the 3′ nucleotide is paired (3240). The AA group II bulge is the most prevalent but by a smaller percentage (25.4%) than was observed for the A bulges in total .In the case of Group III single nucleotide bulge loop, the identity of the bulge is ambiguous and the bulge may have either a Watson–Crick or wobble base pair. In the database there are 3653 group III bulge loops. Over 2/3 of the group III bulge loops have the nucleotide on the 3′ side paired. Nearly 75% (2683) of the group III bulge loops form a Watson–Crick rather than wobble base pair .Since this investigation was examining the influence of a bulge loop in the context of a hairpin stem, we next examined the natural occurrence of bulge loops in hairpin stems in the database. Group II bulge loops are very rarely embedded in hairpin stems (for hairpin loops up to 7 nt long and bulges up to 6 bp from the hairpin loop). Only 419 examples are found in the database, they are almost evenly divided as to whether the bulge is on the 5′ (195) or 3′ (224) side of the hairpin loop. There is also no discernible preference for whether the nucleotide proximal (213) or distal (206) relative to the hairpin loop is the bulged nucleotide (data not shown).2-AP can interact with uridine with Watson–Crick geometry and forms two hydrogen bonds similar to adenosine. Substitution of 2-AP for A has been shown previously to have a small influence on the stability of RNA . It was Incorporation of a 2-AP:U base pair at the terminal position of a RNA duplex increases the stability of the duplex by nearly the same amount as the addition of a terminal A/U base pair . The difΔG°37stem is the less stable stem for group I bulge loops and the second least stable stem for group II and group III bulge loops. For hairpins with embedded bulge loops, the ΔG°37stem is calculated for the distal stem  on the stability of the RNA hairpins . This analysis suggests that the terminal CG base pair does form and in fact interacts with its nearest neighbor in a similar manner whether or not the bulge is present. Our previous in-line probing results have not detected any enhanced cleavage of terminal base pairs, again indicative of the fact that the terminal base pair does not undergo any enhanced fraying as a result of the bulge loop . We are currently investigating the structural and sequence parameters that influence the positioning of the bulge loop.The group II bulge nearest-neighbor sequences in this study were chosen such that whichever nucleotide was bulged, the stability of its nearest-neighbor interactions would have similar stability. For example, for the hairpin CWe previously examined the bulge loop distribution of a much smaller database . The curExamining bulge loops in the context of a hairpin stem, the first and most striking observation is the dearth of bulge loops in hairpin stems found in the database . Only 17Escherichia coli numbering).Tetraloop hairpins have the highest number (1643) of bulge loop embedded in their stems but this is a result of the fact that tetraloops are the most abundant hairpin loop in the data base . Pentaloop hairpins have a slightly higher proportion of bulge loops embedded in their stems . Beyond hairpin loops of 9, the number of bulge loops embedded in hairpin stems drops off so that for hairpin loops of 10, only 44 occurrences are found in the database; <5% of the hairpins of this size have a bulge loop embedded in their stems. Only 80 occurrences of bulge loops are found one base pair from the hairpin loop in the database. The great majority, almost 80%, of the bulge loop occurrences are found 2–4 bp from the hairpin loop. This percentage would be even higher except for the hairpin tetraloop with a conserved bulge loop (278 occurrences) 7–8 bp from the stem at position 95 in the SSU RNA  and six having the distal nucleotide as the bulge . For the group II bulge loops with A as the bulge, five of the seven  have the distal nucleotide unpaired. Interestingly, when the group II bulge is UU, all four examples have the proximal U bulged .Of the 12  naturally occurring hairpin structures in The location and interaction of the extrahelical bulge loops were further examined in the context of the three-dimensional structures. The extrahelical bulge loops are involved in a number of tertiary interactions (data not shown). In four of the seven examples, the bulge interacts with another nucleotide, the bulged nucleotide in panels B, I, and N is stacked on an adenosine residue and the bulge nucleotide in panel G hydrogen bonds with a guanine residue. In one example, the bulge loop in panel J stacks on a histidine residue of the bound protein. In the last two examples the bulges in panels E and H are positioned into a pocket devoid of obvious interactions.In the structures considered above the geometry of the bugle loop may be influenced by tertiary and crystal packing forces in addition to the local bulge environment. To more closely examine the local influences experienced in solution by the hairpins we investigated by optical and fluorescent melting, we next examined the structure of adenosine bulge loops  whose structures were determined by solution NMR analysis. Five examples were identified . In one


A neuron's record of its previous activity underlies animal memory. A new study reveals a role for the release of calcium ions from intracellular stores in mediating spatially compartmentalized memory of the activity history of a neuron. Every fact or task that we remember—the shape of the utensil we call a fork, the appropriate hand-motion needed to beat egg whites until fluffy, or the sequential steps involved in baking a cake—must be encoded by long-lasting changes in the way that our neurons function and in the strength with which they connect and communicate to each other. Current experimental evidence has led neuroscientists to propose that learning elicits a particular pattern of electrical activity in neurons, which can in turn induce changes in their morphology, their responsiveness to incoming signals, the expression of their genes, and the strength of their connection to other neurons.PLOS Biology, Friedrich Johenning, Anne-Kathrin Theis, Dietmar Schmitz, Sten Rüdiger, and colleagues provide evidence that specific electrical activity within neurons induces a long-lasting change in the amplitude of transitory increases of calcium ion concentration (Ca2+ transients) inside dendritic spines—the specialized protrusions of the dendrites of a neuron, which receive input from other neurons via synapses.These changes are the cellular counterpart of what we think of as memory. However, neuroscientists have not found many mechanisms by which a neuron can store information relative to its previous activity. In a study just published in 2+ ion is a fundamental player in the transformation of electrical to biochemical activity within neurons. The concentration of Ca2+ ions within a neuron can be increased either by influx from the extracellular milieu via ion channels and transporters or by release from intracellular stores via Ca2+-permeable channels like the ryanodine receptor (RyR). Once free in the cytosol, Ca2+ can bind to and change the activity of many proteins, including those that control gene expression, cell morphology, and the activity of other proteins via the addition or removal of phosphate groups. Because Ca2+ can have such broad impacts on the cell’s biochemistry, to ensure specificity of effect, the diffusion of the Ca2+ signal can be restricted to compartments, such as those enclosed by dendritic spines.The Ca2+ channels in the plasma membrane of the dendritic spine but also to the release of Ca2+ from intracellular stores via ryanodine receptors. More importantly, their study shows that when a dendritic spine sees a short burst of action potentials—similar to patterns of activity seen in freely moving animals—the Ca2+ transients elicited by subsequent back-propagating action potentials are enhanced for several minutes after the burst. These results indicate that the neuron can “remember” its previous history of activity and express this memory as a sustained increase in the amplitude of Ca2+ transients in a spine-specific manner  and the downstream signaling events that result in Ca2+ transient enhancement. This idea was supported by the fact that if a Ca2+ buffer was used to blunt the cytosolic Ca2+ increase during the train of back-propagating action potentials then the enhancement was eliminated.Because the spines are specialized in receiving incoming information from other neurons, it was a natural question for the authors to ask whether synaptic activity could be contributing to the enhancement of Ca2+ stores after the burst of back-propagating action potentials did not affect potentiation. This result indicates that release of Ca2+ from the stores via ryanodine receptors is important for the initial establishment, but not for the subsequent expression, of the enhancement of Ca2+ transients.The authors discovered that, surprisingly, emptying the intracellular Ca2+ ions in the tiny volume of the dendritic spines, the author used mathematical modeling to determine that the Ca2+ released via the ryanodine receptors acted on a (still unknown) target in a specific nanodomain close to the intracellular stores during the initial establishment of the Ca2+ transient enhancement induced by back-propagating action potentials.Because it is impossible to experimentally measure the temporal and spatial distribution of Ca2+ transient potentiation enhancement in only about half of the spines studied. In addition, the physiological stimuli that induce this effect—and the consequences of the sustained Ca2+ increases on the morphology and function of the dendritic spines—were not investigated. In the end, exactly how this potentiation modulates the way in which neurons communicate with each other is still unknown. This research by Johenning and colleagues is an important step forward, since it uncovers a new spine-restricted mechanism of storing the patterns of previous neuronal activity and defines the role of intracellular Ca2+ stores and ryanodine receptors in this form of cellular memory.Many questions remain open. For example, it is not clear why the back-propagating action potentials induce Ca


In most cases of chronic urticaria, non--sedating anti-histamine drugs (nsAnti-H1) control the symptoms, and the remission is obtained after 1--5 years.Here is presented a case of 54 years old woman with the history of severe chronic urticaria from over 30 yrs resistant to nsAnti-H1 and treated with plasmapheresis and intravenous immunoglobulin G.In this patient urticaria with angioedema eruptions were induced by cold, pressure, NSAID, antibiotics, but mainly appeared spontaneous, and were aggravated before menstruation. She was twice hospitalized due to the severe angioedema. nsAnti-H1 in standard and high doses were ineffective, and caused the drowsiness. Concomitant treatment with LTRA, anti-H2 and sAnti-H1 as well as diet were also ineffective. Only high doses of oral corticosteroids (oCS) partly controlled the clinical symptoms. Autologous serum skin test was positive, skin biopsy revealed oedema of derma and infiltration by eosinophils and lymphocytes. She had also the history of thyroid gland disorder. Due to autoimmunologic background plasmapheresis was applied. During first cycle adverse event (AE) -- eruption of urticaria with angioedema developed. AE was treated with intravenous CS (ivCS) and Anti-H1. Next cycles with CS premedication proceeded without AE. Remission of urticaria lasted 6 month, and then symptoms relapsed. Second series of plasmapheresis and plasmapheresis booster preceeded CS premedication passed without AE, but during intravenous immunoglobulin G infusion, applied for continuation, urtical reaction with angioedema again appeared and disappeared after treatment with ivCS. Patient was again free from symptoms through 6 months. Then symptoms turn back but they could be control by low doses of oCS with nsAnti-H1. In 2008 she had hysterectomy due to cervical cancer (in situ). Nowadays she is still symptomatic despite treatment but symptoms are mild and tolerated. She refused anti-IgE therapy due to cancerophobia.In some patients suffering from severe chronic urticaria a compromise in symptoms control and therapy intensity need to be obtain and accepted by a patient and a physician.Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


LCRs activate an inward current (likely that of the Na+/Ca2+ exchanger) that prompts the surface membrane “M-clock” to ignite an AP. Theoretical and experimental evidence has mounted to indicate that this clock “crosstalk” operates on a beat-to-beat basis and determines both the AP firing rate and rhythm. Our review is focused on the evolution of experimental definition and numerical modeling of the coupled-clock concept, on how mechanisms intrinsic to pacemaker cell determine both the heart rate and rhythm, and on future directions to develop further the coupled-clock pacemaker cell concept.At the beginning of this century, debates regarding “what are the main control mechanisms that ignite the action potential (AP) in heart pacemaker cells” dominated the electrophysiology field. The original theory which prevailed for over 50 years had advocated that the ensemble of surface membrane ion channels  is sufficient to ignite rhythmic APs. However, more recent experimental evidence in a variety of mammals has shown that the sarcoplasmic reticulum (SR) acts as a “Ca Importantly, some but not all investigators concluded that a hyperpolarization-activated “funny” current (If), is the dominant M-clock current driving early diastolic depolarization. However, since the time of If discovery its major role in cardiac pacemaking was challenged  initiate the spontaneous action potentials (AP) that are conducted to the ventricle to entrain the rate and rhythm of ventricular myocytes contractions. The identities and the relative roles of the control mechanisms within heart pacemaker cells that ignite the AP have been debated for more than 50 years. The predominant theory later named “M-clock” advocated that the ensemble of surface membrane ion channels was sufficient to ignite spontaneous AP . Nevertheless, one approach to gain further insights into the systems operation has been to artificially split the two clocks into two separate competing mechanisms  beneath the cell surface membrane; LCRs activate an inward Na+/Ca2+ exchange (NCX) current that prompts the surface membrane clock (M clock), an ensemble of sarcolemmal electrogenic molecules, to generate an AP. LCR Ca2+ signal is regulated not only by the SR Ca2+ pumping, which depends not only on SR proteins (phospholamban and RyR), and their phosphorylation status, but also by functions and phosphorylation status of M-clock proteins, e.g., L-type Ca2+ channels that regulate cell Ca2+ available for SR pumping, i.e., Ca2+-clock's substrate or “fuel.” LCR signals affect Ca2+ -dependent electrogenic processes (such as Na+/Ca2+ exchange) and voltage-dependent Ca2+ fluxes (such as via Ca2+-dependent inactivation of L-type Ca2+ channels). Therefore, the amplitude and phase of the LCR Ca2+ signal sensed by M-clock proteins reports the degree of synchronization and coupling of pacemaker mechanisms of both clocks, i.e., a stronger, more synchronized, and earlier LCR signal to M-clock proteins reports more efficient clock coupling that results in further shortening of the AP-beating interval (BI).After more than 50 years of research it is apparent that the pacemaker function is orchestrated via intrinsic signaling mechanisms originating at multiple levels of organization, including subcellular , cellular , hierarchical brain-heart signaling  and modulated by environmental mechanical, chemical and thermal factors Figure . The SR 2+ which, in turn, activates calmodulin-adenylyl cyclase (AC)-dependent protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) . Numerical model simulations predict that the diastolic LCR signal is also regulated both by the level of Ca2+ cycling, and by the phosphorylation states of coupled-clock proteins  mechanisms . Therefore, the extent to which intrinsic clock mechanisms regulates the coupled-clock determines both the steady state BI and BIV in isolated pacemaker cells.The spontaneous AP BI of single isolated pacemaker cells and SAN tissue are roughly periodic, i.e., this period varies on a beat-to-beat basis  that increases the heart rate and cholinergic receptors (CR) that decreases the heart rate Figure . In huma2+ signal that occurs early in diastole  appeared to show a minor role of Ca2+ dynamics in SANC function  were rapidly applied to single isolated rabbit SANC  is linked to that length of that cycle  was formulated by Kurata  pump blocker. It was shown that a direct perturbation of one clock inevitably affects the other due to subsequent indirect effects, resulting in mutual entrainment, i.e., clocks coupling, predicted by the theory.Both “biophysical” and “biochemical” entrainments are required to explain complex effects of clock's-specific perturbations . Numerical simulations, using a modified ML “coupled-clock” model, faithfully reproduced experimentally reported prolongation of the AP BI and associated dys-rhythmic spontaneous beating in the presence of cytosolic Ca2+ buffering  The systems has a “memory” for several beats: after flash-induced Ca2+ release the temporal rate increase are linked to changes in Ca2+ available for pumping into the SR that ultimately results in a temporal increase in diastolic NCX current driving the AP firing rate increase.The modified ML model provided new insights into the nature of beat-to-beat clock entrainment  observed in 3D confocal scan sections of immunofluorescence staining.Recognition of the limitations of the traditional common-pool model approach have led to novel pacemaker cell models featuring local Ca2+ dynamics within a coupled-clock system is being developed and will provide novel insights into pacemaker cell mechanisms. A “multi-scale” modeling has been put forward by James Weiss group that modeled Ca2+ dynamics in cardiac cells , describing states of all individual RyR and L-type Ca2+ channel will be helpful to approach the next level integration at the whole cell Ca2+ dynamics.A new generation of model featuring local Ca2+-clock and the tightness of this crosstalk, informed by the LCR period, determines the rate and rhythm of spontaneous AP generation. Indeed, both theoretical and experimental evidence has mounted to indicate that this clock “crosstalk” operates on a beat-to-beat basis.Mechanisms intrinsic to pacemaker cells and their modulation by the brain-heart receptor signaling determine both the heart rate and heart rate variability. Crosstalk exists between M and CaIn the level of pacemaker cells, future experiments are needed to quantify beat-to-beat regulation of cAMP/PKA signaling that drives clock coupling in order to speculate whether they take part in the mutual entrainment between the clocks in a beat-to-beat basis. Moreover, the extent to which reduction in synchronization of intrinsic clock periods within pacemaker cells is associated with cardiac diseases and aging awaits further elucidation. Finally, novel mathematical models that quantify not only the average AP firing, but also determine its rhythm await further development.2+ leak from the SR and NCX current can trigger an arrhythmia in atrial fibrillation patients (Lakatta and Guarnieri, Similar contribution of coupled-clock mechanisms to membrane firing rate and rhythm can exist in other heart tissues. In this regard, crosstalk between CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, the relevance of the Mas receptor, which along with the AT2R forms the protective arm of the renin-angiotensin system, as a target in stroke is unclear. Here we tested the efficacy of a selective MasR agonist, AVE0991, in in vitro and in vivo models of ischemic stroke. Primary cortical neurons were cultured from E15-17 mouse embryos for 7–9 d, subjected to glucose deprivation for 24 h alone or with test drugs, and percentage cell death was determined using trypan blue exclusion assay. Additionally, adult male mice were subjected to 1 h middle cerebral artery occlusion and were administered either vehicle or AVE0991 (20 mg/kg i.p.) at the commencement of 23 h reperfusion. Some animals were also treated with the MasR antagonist, A779 (80 mg/kg i.p.) 1 h prior to surgery. Twenty-four h after MCAo, neurological deficits, locomotor activity and motor coordination were assessed in vivo, and infarct and edema volumes estimated from brain sections. Following glucose deprivation, application of AVE0991 (10−8 M to 10−6 M) reduced neuronal cell death by ~60% (P<0.05), an effect prevented by the MasR antagonist. By contrast, AVE0991 administration in vivo had no effect on functional or histological outcomes at 24 h following stroke. These findings indicate that the classical MasR agonist, AVE0991, can directly protect neurons from injury following glucose-deprivation. However, this effect does not translate into an improved outcome in vivo when administered systemically following stroke.Functional modulation of the non-AT Stroke is a major neurovascular disease and a leading cause of mortality and long-term disability. While 15 million people worldwide suffer a stroke each year, the only pharmacological therapy available for stroke patients—the clot-busting agent, tissue plasminogen activator (t-PA)–must be administered strictly within a 4.5 h window from the time of stroke onset. As a result, less than 15% of stroke patients are eligible to receive t-PA and so there is a great need to identify additional therapies that may improve stroke outcome  . Ang . Under ethics committee guidance, we implemented and followed a monitoring protocol involving a clinical signs severity scoring system (approved by our ethics committee), where clinical signs  were observed and assigned a score  based on the animals’ condition per category. Animals with a total score of 0–4 were routinely monitored twice daily (at 9 am and 5 pm). Animals with a total score of 5–9 were more closely monitored—at least once every 3 hours. Animals with a total score of 10 or more were immediately euthanised.in vitro model of stroke, twelve pregnant FVB/N female mice from the Monash-bred colony were used to obtain cortical neurons from E15-17 pups of mixed sex. For the in vivo model of stroke, a total of 53 male C57BL6/J mice  from the Monash-bred colony were studied. The mice were maintained in the animal facility under specific pathogen free conditions on a 12 h light/dark cycle and had free access to water and food pellets before and after surgery. Five mice were excluded from the study because: (1) no measured increase in blood flow at reperfusion after 1 h ischemia (n = 3) and (2) death before 24 h of reperfusion had elapsed (n = 2).For the 2+/Mg2+-free Hank’s balanced salt solution  and gentamicin , then the cortices were carefully dissected out and freed from the meninges. To obtain a single cell suspension, cortices were digested in trypsin  for 10 min at room temperature, then neutralized with trypsin inhibitor . Dissociated cells were resuspended in Neurobasal medium  supplemented with B-27 , HEPES (20 mM), L-glutamine  and gentamycin , then dispensed into poly-D-lysine  coated 60-mm2 Petri dishes . Cells were then incubated overnight in a humidified incubator . Medium was replaced with fresh NBM (+ supplements), and the cells were maintained for a further 7–9 d with renewal of half of the medium every 3–4 d.Cortical neuronal cultures were prepared, as previously described . Brieflyin vitro) were incubated in glucose-free Locke’s buffer  supplemented with gentamicin (25 μg/ml) for 24 h in a humidified incubator. Neurons received one of the following treatments: (i) control (no treatment); (ii) vehicle (Locke’s buffer only); (iii) AVE0991  or 1x10-7 M at 0 h, 1 h, 4 h, or 8 h after glucose deprivation; MedChem Express; Monmouth Junction, NJ, USA); (iv) A779 ; (v) AVE0991  in combination with A779 (1x10-6 M). Cell viability was determined using trypan blue exclusion assay. Following 24 h treatment, cells were incubated in 0.2% trypan blue  for 15 min in a humidified incubator. Cells were rinsed twice with pre-warmed phosphate-buffered saline  then fixed with ice-cold 4% paraformaldehyde for 10 min at room temperature. Twenty images of random fields at 200x magnification were photographed in each dish using an inverted microscope and camera  and associated computer software . Approximately one thousand cells were counted manually from each dish with the assistance of image analysis software . In order to determine the amount of cell death naturally occurring during the experimental treatment period (i.e. independent of treatment), one neuronal culture dish was randomly chosen and a cell viability assay performed 1 d prior to the treatment. Percent cell death was the primary endpoint of this experiment. Investigators were blinded as to the treatment groups when obtaining images and counting cells.For glucose deprivation, cultured primary neurons  and also by hanging grip test, as described previously , 22. BriLocomotor activity and motor coordination were assessed on a parallel rod floor apparatus using ANY-maze software coupled to an automated video-tracking system used previously in mouse models of Alzheimer’s disease , duplicaCerebral infarct and edema volume were estimated as described previously . Infarctin vitro) or Bonferroni’s multiple comparison tests (in vivo) as appropriate, using GraphPad Prism version 6.Data are presented as mean ± standard error of the mean (S.E.M), except for neurological deficit scores (median). Statistical significance (P<0.05) was determined by one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test (-7 M reached near-complete protection of neurons in comparison to control cells (-7 M) was applied 1 h after the onset of GD the protection was maintained, whereas the protective effect was lost if application occurred at later time points   . Moreovefter GD) . Note, Ain vivo, mice were systemically administered vehicle or AVE0991 at the time of reperfusion. The regional cerebral blood flow profile did not differ between vehicle- and AVE-treated mice during 60 min of MCAo and for at least the first 15 min of reperfusion .The results demonstrate that AVE0991 strongly protects neurons from ischemic injury in vitro observed here, we previously found that direct stimulation of AT2R using CGP42112 can also protect cultured neurons exposed to glucose-deprivation. Moreover, systemic administration of CGP42112 improved functional outcome and reduced infarct volume in the same model of stroke as was used here [Our finding that AVE0991 increased neuronal survival following glucose-deprivation in a MasR-dependent manner, is novel. Analogous to the neuroprotective effect of AVE0991 in vitro effects on primary neurons, our in vivo data suggest that the systemic administration of a selective MasR agonist does not provide protection following stroke. Thus, it seems likely that the systemic route of administration is greatly inferior to local injection of AVE0991 in terms of limiting cerebral post-ischemic injury.Previous studies have reported that Ang (1–7) reduced infarct size, prevented the loss of neurons, and improved neurological outcome in rats subjected to endothelin-1-induced MCAo . In addiin vivo was due to an inadequate (or possibly excessive) dose of AVE0991 . Nonetheless, similar doses of AVE0991 (9 mg/kg and 15 mg/kg) have been reported to be protective in other models of disease, such as arthritis and renal ischemia-reperfusion [in vivo (data not shown). Another potential limitation of the in vivo part of our study may have been the timing of AVE0991 administration, i.e. after initiating post-ischemic reperfusion, as opposed to an earlier administration (i.e. pre-treatment) that might be more effective. However, our interest here was to evaluate potential clinical utility of the drug by simulating post-stroke treatment. Furthermore, the potential for non-specific activation of MasR on other cell types, including astrocytes and endothelial cells, should not be overlooked as a possible confounding factor. Nevertheless, this is the first study to have tested post-treatment of AVE0991 in stroke and our data do not support the hypothesis that the MasR is a viable target for therapy in acute stroke.Previous studies have reported that AVE0991 crosses the blood-brain barrier , 33. It erfusion , 35. In in vitro. However, such protective effects do not appear to translate to an in vivo stroke model in which the compound is administered systemically early after ischemia.In conclusion, this study has found that the classical MasR agonist, AVE0991, can directly and powerfully protect neurons from ischemia-like injury S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.


The aim of this study was to determine whether the overexpression of mIGF-1 can modify functional features of satellite cells during the differentiation process, particularly in relation to modifications of intracellular Cain vitro, and morphological analyses, intracellular Ca2+ measurements, and ionic current recordings were performed.Satellite cells were isolated from wild-type and MLC/mIGF-1 transgenic mice. The cells were differentiated 2+ handling. In particular, the differentiated MLC/mIGF-1 transgenic myotubes have reduced velocity and amplitude of intracellular Ca2+ increases after stimulation with caffeine, KCl and acetylcholine. This appears to be due, at least in part, to changes in the physico-chemical state of the sarcolemma  and to increased expression of dihydropyridine voltage-operated Ca2+ channels. Interestingly, extracellular ATP and GTP evoke intracellular Ca2+ mobilization to greater extents in the MLC/mIGF-1 transgenic satellite cells, compared to the wild-type cells.mIGF-1 overexpression accelerates satellite cell differentiation and promotes myotube hypertrophy. In addition, mIGF-1 overexpression-induced potentiation of myogenesis triggers both quantitative and qualitative changes to the control of intracellular CaThese data suggest that these MLC/mIGF-1 transgenic satellite cells are more sensitive to trophic stimuli, which can potentiate the effects of mIGF-1 on the myogenic programme. It has been hypothesized that supplemental mIGF-1 produces a qualitatively different environment for the sustaining of more efficient muscle regeneration and repair Insulin-like growth factors (IGFs) represent a system of circulating and/or locally secreted factors that have key roles in cell growth, differentiation and regeneration. Skeletal muscle is a target of the circulating IGFs, and a source of muscle-derived IGF-1 (mIGF-1). IGF-1 also has a critical role in activation, proliferation and differentiation of muscle stem cells (satellite cells), and in tissue repair mIGF-1 gene is regulated by the MLC promoter. This restricts the expression of the transgene selectively to skeletal muscle, and predominantly in muscle with a high ratio of fast-twitch fibres mIGF-1 transgene expression also sustains hypertrophy and regeneration in senescent skeletal muscle Several processes that are modulated by mIGF-1 have been studied and described in myosin light chain (MLC)/mIGF-1 transgenic mice, an animal model in which the 2+ sensitivity of the contractile apparatus Functional studies have revealed that the hypertrophic phenotype of skeletal muscle in these MLC/mIGF-1 transgenic mice is associated with an increase in muscle strength (tetanic force) and maintained specific force 2+ concentration ([Ca2+]i), that results from motor activation, has a key role in both contractile-activity-dependent and fibre-type-specific gene expression. Mounting evidence has shown that the Ca2+/calmodulin-dependent kinases have key roles in the regulation of expression of some of the oxidative enzymes, genesis and activity of the mitochondria, and expression of fibre-type-specific myofibrillar proteins 2+-activated serine/threonine phosphatase calcineurin is activated by IGF-1 2+-regulated systems have received little attention as regulators of muscle trophism. Compared to other signalling pathways, the relatively few studies that have examined the role of Ca2+ in the regulation of muscle size, have produced discordant results In skeletal muscle, the increase in intracellular Cain vivo growth response related to in situ mIGF overexpression. For this reason, we used satellite cells from these MLC/mIGF-1 transgenic mice as an experimental model, to determine whether there are any differences in the functional behaviour of these transgenic satellite cells during myogenesis, as compared to those of wild-type mice. The aim of this study was thus to investigate the relationships between increased levels of extracellular mIGF-1 and [Ca2+]i handling, as two potential positive factors in the homeostasis of muscle tissue.Therefore, the satellite cells are conditioned by the extracellular muscle microenvironment and are involved in the The care and use of FVB wild type (WT) and MLC/mIGF-1 transgenic mice (from Musarò Laboratories) strictly followed “The Guiding Principles for the Care and Use of Animals”, in accordance with the principles of the Declaration of Helsinki and with the European Community Council (86/609/CEE) and the Italian Government law on the protection of animals for experimental procedures in research laboratory (92/116). The experimental protocol “Characterization of factors involved in muscle diseases”, which was aimed at the production of the transgenic mice that were used in the present study, was reviewed and approved on 14 February, 2011, by the Institutional Animal Care and Use Committee of the Unit of Histology and Medical Embryology . The animals (3 months old) were sacrificed under sodium pentobarbital anaesthesia and all efforts were made to minimize suffering.Unless otherwise indicated, cell culture media, sera and antibiotics were from Life Technologies Italia , cell culture plastic-ware was from Becton Dickinson Falcon , and the reagents and standards were from Sigma-Aldrich .−1 collagenase/dispase  in phosphate-buffered saline (PBS). After centrifugation (150× g for 5 min), the pellet was digested for 15 min at 37°C with 0.1 mg ml−1 collagenase type II (Sigma-Aldrich) in PBS. The enzymatic reaction was blocked by adding cell growth medium , then the suspension was filtered , and then centrifuged at 200× g for 15 min. The pellet of cells was re-suspended in growth medium, and the cells were pre-plated twice for 1 h, to remove the fibroblasts. The satellite cells were then plated on collagen-coated dishes and grown in growth medium −1 penicillin, and 100 µg ml−1 streptomycin) for 10 days The animals (3 months old) were sacrificed under sodium pentobarbital anaesthesia. The bulk of hind-limb muscles were harvested under sterile conditions, and then manually shredded and digested for 30 min at 37°C with 1 mg mlThe level of successful differentiation of the satellite cells into myotubes was determined using immuno-staining protocols, with an antibody against myosin heavy chain , and an antibody against MyoD (Santa Cruz Biotechnology Inc), accompanied by Hoechst staining of the nuclei. To assess the level of myogenic differentiation, the number of Hoechst-stained nuclei located within the sarcomeric myosin-positive cells, and of MyoD-positive cells was determined and expressed as:the myogenic differentiation (differentiation %), calculated as the number of Hoechst-stained nuclei located within sarcomeric myosin-positive cells, and expressed as a percentage of total analysed myogenic cells, positive to MyoD;the fusion index, quantified as the percentage of Hoechst-stained nuclei located within multinucleated cells , positive to sarcomeric myosin, and calculated on the total analysed MyoD-positive nuclei;myonuclear accretion, expressed as average number of nuclei per myotube, and relative percentage of mature myotubes (containing at least 5 nuclei) The morphometric analyses were performed calculating the size and length of MyHC-positive myotubes, using Image J  To better define the percentage of different populations present in culture we performed cytofluorimetric analysis, following protocols described by Carosio et al Total RNA extraction was performed using TriRiagent (Sigma-Aldrich) and was reverse-transcribed using the QuantiTec Reverse Transcription kit . Relative quantitative PCR was performed on ABI PRISM 7500 SDS , using premade 6-carboxyfluorescein (FAM)-labelled TaqMan assays for HPRT1 (Mm00446968_m1), MyoD1 (Mm00440387_m1), Myogenin (Mm00446194_m1), Pax-7 (Mm00834079_m1), Myh-2 (Mm00454991_m1), IGF-1 (Mm00710307_m1) and MRF4 (Mm00435126_m1) (Applied Biosystem). Relative quantitative real time PCR (RT-PCR) sample value was normalized for the expression of HPRT1 mRNA. The relative level for each gene was calculated using the 2-ddCt method and reported as mean fold change in gene expression.2+]i was measured using the dye Fura-2-acetoxymethyl ester , with an inverted microscope  connected to a high-speed wavelength switcher  equipped with a 75-W stabilized xenon lamp  and a cooled charge-coupled device (CCD) camera . The wild-type and MLC/mIGF-1 transgenic satellite cells and myotubes plated on collagen-coated special-optics 96-well plates  were incubated with 5 µM Fura-2/AM for 30 min at 37°C in normal external solution -NaOH, pH 7.3) supplemented with 1% (w/v) bovine serum albumin. Fura-2/AM loaded cells were sequentially and repetitively excited at 340 nm and 380 nm, the fluorescence images were acquired with a CCD camera and stored on an interfaced computer. The acquisition time was one image ratio  per second. The image-ratio calculations were carried out pixel-by-pixel on a pair of corresponding 340 nm and 380 nm image files. The temporal plots  were calculated from the image ratios. The [Ca2+]i in a single cell field was recorded according to the [Ca2+]i calibration plot of the 340/380 ratio, as calculated using the Calcium Calibration kits for video imaging (Molecular Probes) The -PN200-110. The membrane proteins (40 µg) were incubated in a final volume of 250 µl binding buffer  in the presence of 1 nM [3H]-PN200-110 for 1 h at room temperature. The samples were then filtered through Whatman GF/C filters and rapidly washed with six volumes of ice-cold washing buffer . The radioactivity associated with the filters (membrane-bound [3H]-PN200-110) was then determined by liquid scintillation counting , and is expressed as pmol mg−1 protein. The non-specific [3H]-PN200-110 binding to the membranes was determined in the presence of 10 µM unlabelled nifedipine, and this value was subtracted from each experimental point This binding assay was performed with membranes purified from the wild-type and MLC/mIGF-1 mature myotubes sonicated in sodium phosphate buffer (20 mM NaPO−1), as indicated for the DHPR binding assay above. The samples were incubated in 250 µl binding buffer in the presence of 5 nM [3H]-ryanodine for 120 min at 37°C. The samples were then filtered through Whatman GF/C filters and rapidly washed with six volumes of ice-cold washing buffer. The radioactivity associated with the filters (membrane-bound [3H]-ryanodine) was then determined by liquid scintillation counting , and is expressed as pmol mg−1 protein. The non-specific [3H]-ryanodine binding was determined in the presence of 100 µM unlabelled ryanodine, and this value was subtracted from each experimental point This ryanodine receptor (RyR) binding assay was performed on whole homogenates obtained from sonicated wild-type and MLC/mIGF-1 transgenic mature myotubes (25 µg protein assay2+/Mg2+ ATPase activity was carried out on whole homogenates obtained from sonicated myotubes (as for the radiolabelled binding assays). Each sample contained 25 µg protein incubated in 1 ml containing 2.5 mM ATP, 100 µM CaCl2, 60 µM K-EGTA, 10 mM KCl, 5 mM MgCl2, 300 mM sucrose, 10 mM HEPES, pH 7.4, for 30 min at room temperature. The reactions were terminated with the addition of 1 ml 12.5% trichloroacetic acid, and the precipitate was removed by centrifugation at 5000× g for 10 min. The released phosphates were estimated from 1 ml of the cleared supernatant, according to the method described previously 2+/Mg2+ ATPase activity was calculated as µg released inorganic phosphate (Pi) min−1 ml−1 mg−1 protein.The assay for the determination of the Ca−1 phenylmethylsulfonyl fluoride, 10 µg ml−1 leupeptin, 5 µg ml−1 pepstatin A, and 10 µg ml−1 benzamidine. The sonicated cells were centrifuged at 1000× g for 10 min at 4°C, the supernatants were collected, and the protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories). Samples containing 50 µg protein were resuspended in Laemmli buffer , boiled for 5 min, and separated by SDS–PAGE on 6% (w/v) gels for DHPR and RyR1 protein detection, and 8% (w/v) gels for that of Ca2+-ATPase. The proteins were electroblotted onto a hydrophobic polyvinylidene difluoride membrane . These membranes were blocked in TBS-T  containing 5% (w/v) fat-free milk, and then incubated individually with mouse monoclonal antibodies against DHPR , against RyR1 , and against Ca2+-ATPase . The membranes were then incubated with horseradish-peroxidase-conjugated anti-mouse IgG , which was detected by chemiluminescence . An anti-actin antibody  was used as a loading control. All of the antibodies were diluted in TBS-T.The wild-type and MLC/mIGF-1 transgenic myotubes were washed with pre-cooled phosphate-buffered saline and sonicated in 20 mM sodium phosphate buffer (pH 7.0) containing 100 µg mlTo define the oxidative status of the cell membrane lipids, malondialdehyde levels were determined. Malondialdehyde forms an adduct with thiobarbituric acid that can be measured spectrophotometrically. The measurement of such thiobarbituric acid reactive substances is a well-established method for screening and monitoring of lipid peroxidation. These lipid peroxidation measurements on the sarcolemma and sarcoplasmic reticulum membranes isolated from the wild-type and MLC/mIGF-1 transgenic skeletal muscle, were performed using the OXItek ‘TBARS’ Assay kits , as described by the manufacturer. The sarcolemma and sarcoplasmic reticulum membrane fractions were prepared as previously described Membrane fluidity was assayed using the MarkerGene Membrane Fluidity Kit , that contains a lipophilic pyrene probe that undergo excimer formation upon spatial interaction, and consequently shifting its emission spectrum. This property makes it a useful tool to monitor on live cells, changes in membrane fluidity or viscosity. The protocol was optimized using different cell number , 1–50 µM fluorescent lipid reagent concentrations, dye-incubation times (15–30 min) and temperatures (20–45°C). The results, shown here, come from experiments performed on wild-type and MLC/mIGF-1 transgenic satellite cells plated (3000 cells/well) on collagen-coated special-optics 96-well plates (Corning-Costar). After 0, 3 and 7 days in differentiation medium, cells were incubated with 10 µM fluorescent lipid reagent (Membrane Fluidity Kit) for 20 min at 25°C also in accordance with the manufacturer instructions. At the end, the probe was removed, cells washed twice with PBS and the residual fluorescence read at 360 nm excitation wavelength, and 470 or 400 nm emission wavelength, using a microplate reader . Membrane fluidity was expressed as the ratio of fluorescent intensities at 470 and 400 nm emission wavelength (excimer/monomer).2, 2 mM MgCl2, 10 mM glucose, and 10 mM HEPES-HCl, pH 7.4). The patch pipette was filled with 130 mM KCl, 2 mM MgCl2, 10 mM glucose, 5 mM EGTA and 10 mM HEPES-NaOH (pH 7.4). The electrophysiological recordings were carried out at room temperature using the whole-cell configuration of the patch-clamp technique −1 was used, and the currents were filtered at 5 kHz.In voltage-clamp experiments, the total currents were recorded using an extracellular solution containing 130 mM NaCl, 3 mM KCl, 1 mM CaClAll of the quantitative data are expressed as means ± standard error of the mean (SEM). Differences between groups were analysed using Students' t-tests, with the Prism 4 software .Satellite cells were isolated from the hind-limb muscles of wild-type and MLC/mIGF-1 transgenic mice. As expected, and previously published We then analysed the effects of mIGF-1 on myogenic program, performing a time course experiment. We observed that mIGF-1 expression accelerates the differentiation of satellite cells and promotes a precocious mature phenotype even at early stage of the myogenic program (4 days in growth medium) . In partThe accelerated process of myogenic differentiation, induced by mIGF-1 expression, was also supported by the significant up-regulation of relevant markers involved in myogenic differentiation, such as MyoD1, Myogenin, MRF4 and myosin, in MLC/mIGF-1 transgenic mouse-derived cells compared to wild-type cell cultures .Morphometric analyses, performed at 1 day in differentiation medium, revealed that the MLC/mIGF-1 transgenic mouse-derived myotubes appeared longer and with a bigger area compared to the wild-type mouse–derived myotubes .The effect of mIGF-1 overexpression on the myogenic process was also confirmed at 5 days in differentiation medium: at this time MLC/mIGF1 transgenic mouse-derived cultures showed an increased myogenic differentiation and larger myotubes compared to the wild-type mouse–derived cultures .These data correlate with the muscle phenotype of the transgenic mice 2+]i was monitored in these isolated satellite cells, using fluorescence video-imaging techniques. 2+]i increased by about 50% in the wild-type differentiated cells and 30% in the MLC/mIGF-1 transgenic differentiated cells. This increase was significant , although there was no significant difference in the [Ca2+]i rise between the wild-type and MLC/mIGF-1 transgenic models . Similarly, in the presence of extracellular Ca2+, KCl induced a significantly slower [Ca2+]i increase in the MLC/mIGF-1 transgenic myotubes compared to the wild-type myotubes ; this was also seen with KCl in Ca2+-free medium . In the absence of extracellular Ca2+, only the myotubes with complete maturation of their excitation-contraction coupling showed intracellular Ca2+ level increases, like those illustrated for myotubes in The quantitative analysis of the ion kinetics  shows th2+ level increases (as the immediate response) was significantly lower in the myotubes derived from the MLC/mIGF-1 transgenic mice compared to that measured in the wild-type cultures  compared to that observed in the wild-type myotubes (2.50±0.17). Moreover, also the amplitude at 1 min stimulation was very similar to that induced by KCl, showing no significant differences between MLC/mIGF-1 transgenic myotubes (1.28±0.06) and wild-type myotubes (1.14±0.05).The initial peak of the caffeine-induced and KCl-induced intracellular Cacultures . However similar . In ordeThe responsiveness to acetylcholine mimicked that for caffeine and KCl, with a decreased response velocity in MLC/mIGF-1 transgenic myotubes compared to wild-type myotubes  in the wild-type and MLC/mIGF-1 transgenic cells. In particular, both 100 µM ATP and 300 µM GTP induced significantly more rapid intracellular Ca2+ level increases in MLC/mIGF-1 transgenic myoblasts compared to those evoked in wild-type myoblasts  compared to the wild-type myoblasts (99%±1%); conversely, the MLC/mIGF-1 transgenic myoblasts were significantly more responsive to GTP (87%±11%) compared to the wild-type myoblasts (56%±9%) . In addiery 30 s , inset. 2+ level increase parameters  in the presence of these purines. In particular, the intracellular Ca2+ level increases stimulated by both ATP and GTP showed significantly lower cell responsiveness in the MLC/mIGF-1 transgenic myotubes  in comparison to the wild-type myotubes  , while the GTP-responsive cells in the MLC/mIGF-1 transgenic myotubes showed a significantly higher amplitude of the intracellular Ca2+ level increase with respect to the wild-type myotubes   . The amp p<0.01) . Under tstration . Concernmulation .To support the functional background of the satellite cells, the membrane electrical properties and the inward and outward currents were also measured in myoblasts and myotubes from the wild-type and MLC/mIGF-1 transgenic samples. These analyses were performed using the patch-clamp technique in the whole-cell configuration After the satellite cell differentiation, the resting potential (Vm) was significantly lower in the myotubes with respect to the myoblasts, for both the wild-type and MLC/mIGF-1 transgenic samples . AlthougThe functional activity of myotubes depends not only on the differentiative status, but also on the environmental conditions. The oxidative balance in skeletal muscle is one of the main features that is representative of its metabolic activity as a whole, and this also conditions the satellite cell activity. For these reasons, the oxidative status of cell membranes was assayed in mature skeletal muscle biopsies from the wild-type and the MLC/mIGF-1 transgenic mice.The oxidative status of lipids can affect the membrane biophysical properties, and for this reason, membranes' fluidity was assayed on live MLC/mIGF-1 and wild-type differentiating satellite cells at 0, 3 and 7 days in differentiation medium, using a fluorescent probe. The results showed some differences in membranes' fluidity between MLC/mIGF-1 transgenic and wild-type satellite cells. The differences were significant at 3 days of the differentiation period .mIGF-1 gene expression induces local skeletal muscle hypertrophy, which improves muscle mass and strength by activating calcineurin-mediated Ca2+ signalling and GATA-2/NF-ATc1 It has been previously demonstrated that Interestingly, Zheng and co-workers proposed a model in which CaM kinase and calcineurin are involved in IGF-1-induced DHPRalpha expression in C2C12 cells. IGF-1 receptor activation triggers CaM kinase and calcineurin activity to regulate the balance of phosphorylated/dephosphorylated CREB, that in turn regulates DHPRalpha transcription 2+ sensor and activates Ca2+ entry by the Orai channels. STIM1 has a fundamental role in postnatal skeletal muscle growth in relation to the calcineurin/NF-AT signalling pathways, through the regulation of the levels of Ca2+ in the sarcoplasm and sarcoplasmic reticulum cisternae. Based on this mechanism, it was proposed a model whereby STIM1-mediated store-operated Ca2+ entry regulates the Ca2+ signalling required for cellular processes that are necessary for muscle growth and differentiation, even though the excitation-contraction coupling remains unperturbed More recently, Li and co-workers 2+-dependent signalling and muscle hypertrophy, and that support the working hypotheses developed in the present study. The data presented here relating to some of the systems involved in intracellular Ca2+ homeostasis in myotubes , show that the differences in the behaviour observed in MLC/mIGF-1 transgenic myotubes in comparison to the wild-type myotubes arise from the different kinetics of the Ca2+-controlling system, rather than from any further specific component.Many evidences demonstrate the deep relationship between growth factor-induced intracellular CaVice versa, in the MLC/mIGF-1 transgenic myotubes, only a small, and not significantly different, decrease in the functional capacity of the sarcoplasmic reticulum was observed, in terms of the RyR channels and Ca-pumps.In the myotubes derived from the MLC/mIGF-1 transgenic mice, we observed increased capacity of the T-tubule–associated DHPR channels, together with increased outward potassium currents, with respect to the myotubes derived from the wild-type mice. 2+ mobilization was slower under all of the conditions tested here , even if after longer times (≥1 min), the differences between the MLC/mIGF-1 transgenic and wild-type cultures were significantly reduced.Overall, these data suggest that in the myotubes derived from the MLC/mIGF-1 transgenic cultures, Ca+ currents are a possible consequence of external membrane modifications that do not involves the sarcoplasmic reticulum structures. The data derived from the oxidative status of the sarcolemma appear to confirm this hypothesis. These show that in the membranes derived from mature myotubes, the lipid oxidation is greater in the MLC/mIGF-1 transgenic samples compared to wild-type ones, which suggests modification of the membrane fluidity These data indicated that in the MLC/mIGF-1 transgenic myotubes, there might be different sarcolemmal organization, as both the increased DHPR functionality and the increased output K+ channels, leads to increased Ca2+ influx, which promotes myoblasts fusion and consequently multinucleated myotube formation + currents) in comparison to the wild-type myotubes, which supports the hypothesis that these cells are more prone to differentiate.The electrical events are also very important during myoblast differentiation. It has been reported that during differentiation of human muscle satellite cells, the resting potential is hyperpolarized, due to the sequential expression and activation of ionic channels. Such hyperpolarization, which is mainly due to the activation of ether-a-go-go and inward rectifier K2+-activating system that can influence myogenesis: the extracellular pool of micromolar concentrations of purines, such as ATP and GTP 2+ level increases that are due to the release of Ca2+ from intracellular inositol-1,4,5-trisphosphate-sensitive stores 2+ mobilization, the consequent activation of the intermediate Ca2+-activated K+ channels (IKCa), and hyperpolarization of the plasma membrane 2+ released from internal stores induces a proliferative boost, and increases the number of cells that express the myosin heavy chain proteins and at least three genes involved in myogenesis: Pp3ca, Gsk3b and Pax7In skeletal muscle, there is another Ca2+ mobilization in muscle-derived cells derived from the mIGF-1 transgenic mice, as compared to the wild-type cells. During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, which activates purinergic receptors, and thus increases Ca2+ influx, and enhances the contractile force, a response that is known as potentiation 2+ rise rather than a higher Ca2+ level, which might be due to a more controlled in vitro extracellular environment with respect to what happens in vivo. This evidence might explain the potentiation induced in the MLC/mIGF-1 transgenic mice by ATP and GTP, which induced higher and more rapid Ca2+ responses. In addition, considering that GTP can also potentiate muscle activity Our data show that at physiological external concentrations, ATP and GTP induce a more rapid CaThe data in the present study show that about 50% of the wild-type myoblasts are responsive to 300 µM GTP, which increases to about 90% in the MLC/mIGF-1 transgenic myoblasts. This response appears to provide evidence of the better myogenic properties of the MLC/mIGF-1 transgenic cells, considering that GTP acts via two specific binding sites with different affinities, and in particular, that only the activation of the low-affinity GTP-binding site favours the fusion and myogenesis processes 2+ oscillations, which was not seen in the MLC/mIGF-1 transgenic myoblasts. It has been hypothesized that the molecular mechanisms responsible for intracellular Ca2+ oscillations depends on the biphasic (release/uptake) regulation of the inositol 1,4,5-trisphosphate receptor by the cytosolic Ca2+. Generally, high frequency cytosolic Ca2+ oscillations regulate fast responses, such as synaptic transmission and secretion, whereas lower frequency oscillations regulate slower processes, such as gene transcription 2+ influxes, and not global intracellular Ca2+ oscillations 2+ level increase. Under the experimental conditions of the present study, the GTP-induced intracellular Ca2+ oscillations, observed in wild-type myoblasts, are replaced by the more rapid intracellular Ca2+ level increase in the MLC/mIGF-1 transgenic cells.Of note, in the wild-type cells, GTP stimulated Ca2+ handling, these are both quantitative  and qualitative (absence of the GTP-induced intracellular Ca2+ oscillations). This is due, at least in part, to changes in the physico-chemical state of the sarcolemma  and to increased expression of DHPR voltage-operated Ca2+ channels.In conclusion, the data provided by the present study indicate that the functional properties of these MLC/mIGF-1 transgenic satellite cells are mostly different from those of the wild-type cells. In particular, this mIGF-1 overexpression leads to changes in the control of intracellular Ca2+ mobilization to a greater extent in the MLC/mIGF-1 transgenic cells, with respect to wild-type cells, and this might explain both the potentiation and activation of myogenesis. Although further investigations are necessary to define whether these changes are directly involved in the mechanism(s) underlying the hypertrophic status of the transgenic muscle, it can be hypothesized that the sensitivity of these MLC/mIGF-1 transgenic cells towards non-canonical myogenic factors, such as the purines, appears to be a specific mechanism involved in the effects induced in vivo by the overexpression of mIGF-1.Of particular interest, ATP and GTP evoked intracellular Ca


Aplysia. We unveiled ApLRRTK as the first member of the FGFRs family deprived of Ig-like domains that instead contains extracellular LRR domains. We describe that ApLRRTK exhibits properties typical of canonical vertebrate FGFRs, including promotion of FGF activity, enhancement of neuritic outgrowth and signaling via MAPK and the transcription factor CREB. ApLRRTK also enhanced the synaptic efficiency of neurons known to mediate in vivo memory-related defensive behaviors. These data reveal a novel molecular regulator of neuronal function in invertebrates, provide the first evolutionary linkage between LRR proteins and FGFRs and unveil an unprecedented mechanism of FGFR gene diversification in primeval central nervous systems.Fibroblast Growth Factor (FGF) Receptors (FGFRs) regulate essential biological processes, including embryogenesis, angiogenesis, cellular growth and memory-related long-term synaptic plasticity. Whereas canonical FGFRs depend exclusively on extracellular Immunoglobulin (Ig)-like domains for ligand binding, other receptor types, including members of the tropomyosin-receptor-kinase (Trk) family, use either Ig-like or Leucine-Rich Repeat (LRR) motifs, or both. Little is known, however, about the evolutionary events leading to the differential incorporation of LRR domains into Ig-containing tyrosine kinase receptors. Moreover, although FGFRs have been identified in many vertebrate species, few reports describe their existence in invertebrates. Information about the biological relevance of invertebrate FGFRs and evolutionary divergences between them and their vertebrate counterparts is therefore limited. Here, we characterized ApLRRTK, a neuronal cell-surface protein recently identified in The online version of this article (doi:10.1007/s00726-014-1803-2) contains supplementary material, which is available to authorized users. Aplysia receptor recently identified by Kassabov et al.  are cell-surface tyrosine kinase proteins that regulate a variety of essential biological functions, including embryological development, cellular growth, nervous system formation, adult neurogenesis and learning-related long-term synaptic plasticity  as an index of synaptic strength Kandel  before aAplysia  in sensory-motor neuron co-cultures Kandel . 5HT is a Kandel . We founa Kandel d.n = 4; GFP = 1.5 ± 0.6 n = 4; ApLRRTK-ΔK-GFP = 1.4 ± 0.3 n = 4; ApLRRTK-GFP = 3.8 ± 0.7 n = 4). We additionally explored the possibility that the actions exerted by ApLRRTK were related to the MAPK/CREB pathway, which is known to be involved in FGFRs signaling . These observations are in agreement with previous reports relating LRR proteins with the actions of FGFs  following methods that we previously described , respectively.We retrieved the tyrosine kinase-containing protein sequences  from the NCBI database. Sequences were identified by BLASTP searches using ApLRRTK (NP_001232922) and ApNTRK (NP_001232923) sequences as query. We downloaded the hidden markov model (HMM) for the TK domain from the Pfam database  treatment. Ganglia were plated in L-15 culture media overnight to allow recovery from protease treatment. The next day, culture media was washed out and ganglia were treated with five pulses of 5-HT, as previously described .Supplementary material 1 (DOC 156 kb)Supplementary material 2 (TIFF 4687 kb)Supplementary material 3 (TIFF 946 kb)Supplementary material 4 (TIFF 1726 kb)Supplementary material 5 (TIFF 876 kb)Supplementary material 6 (TIFF 3434 kb)Supplementary material 7 (DOC 36 kb)Below is the link to the electronic supplementary material.


Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca2+-flux and sarcoplasmic reticulum Ca2+-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca2+-concentration, trans-sarcolemmal Ca2+-flux via L-type Ca2+-channels and Na+-Ca2+-exchanger, and Ca2+-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca2+-channels by verapamil (1 μM) decreased force of contraction to 22±7% compared to baseline . Ni2+ was the only substance to prolong relaxation . Surprisingly though, inhibition of the sarcoplasmic Ca2+-release decreased force development to 54±3% in ventricular  and to 52±8% in atrial myocardium  suggesting a substantial role of SR Ca2+-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s  and 10 s  and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca2+-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.Zebrafish ( Danio rerio) as a model organism in cardiovascular research is a result of features such as a high degree of genetic homology with mammals and especially humans, ease of handling and reproduction, and straightforward techniques for genetic manipulation like gene-specific antisense knockdown using morpholino oligonucleotides ec) were performed in Tyrode’s solution allowing the exposure to low [Ca2+]ec beneath 1.97 mM. Composition of Tyrode’s solution was equal to the one used for slicing except for the addition of 2 mM sodium pyruvate. All pharmacological experiments were performed during constant field stimulation at 1 or 2 Hz. After recording of baseline conditions for 10 minutes, drugs were administered and recorded for 10 minutes for each concentration. For data analysis, signals were filtered  and baseline correction was achieved by an automated algorithm (moving arithmetic average of 1000 samples after above described high- and low-pass filtering). To investigate changes of twitch morphology, single contractions were averaged during steady state conditions  using an automated algorithm.To determine the force-frequency relationship (FFR), ventricular slices were paced according to a stimulation protocol of alternating higher and lower frequencies . The last 30 seconds of each stimulation period were analysed to evaluate the effect of frequency on the twitches. Post-pause potentiation behaviour of the slices was studied by interrupting field stimulation for 5 or 10 seconds after 2 minutes of constant electrical stimulation at 2 Hz. The first beat after quiescent periods was analysed and normalised to the steady-state condition represented by the mean of the developed force of the last 10 beats before the rest interval. Measurements to assess the response to ascending extracellular calcium concentration . Pictures were acquired with a Zeiss Axiovert 200 microscope . For immunohistochemical investigations, enzymatic antigen retrieval was carried out by using trypsin. Sarcomeric-α-actinin antibody  was applied to examine sarcomere morphology. Anti-mouse IgG1 Alexa Fluor 555  was used for secondary detection followed by nuclear staining with Hoechst 33342. Stained sections were imaged using a Zeiss Axiovert 200 microscope and the image processing software AxioVision release version 4.6 (both Carl Zeiss).2 stock solution was dissolved in Tyrode’s solution at a concentration of 100 mM. NiCl2 was dissolved in IMDM at a concentration of 200 mM. (±)-Verapamil (VER) stock solution was prepared in demineralised water at a concentration of 1 mM. Ryanodine (Ry) stock solution was prepared in ethanol at a concentration of 2 mM. (-)-Isoproterenol (ISO) was dissolved in Aqua dest. at a concentration of 1 mM with 10 mM ascorbic acid.All chemicals for buffer and stock solution preparation were purchased from Sigma-Aldrich Chemie GmbH, Steinheim, Germany, unless otherwise stated. CaClResults from pooled data are presented as mean ± SEM. Statistical analysis was performed using an ANOVA test followed by a Student-Newman-Keuls test, a Student t-test or ANOVA repeated measures, as appropriate, using IBM SPSS Statistics Version 22 . p < 0.05 was considered statistically significant.2, 5% CO2, 37°C), cross-striation was almost completely lost after 2 days  using a nominally Ca2+-free Tyrode’s solution under constant field stimulation at 1–2 Hz. Withdrawal of extracellular Ca2+ abolished generation of force completely in all preparations (n = 5). The force of contraction increased with ascending [Ca2+]ec .Myocardial contractility strongly depends on [Ca+]ec Fig  and 3B. Application of the LTCC blocker VER decreased force of contraction in a dose-dependent manner  . Twitch 2+ loading during rest. We studied post pause potentiation after 5 s and 10 s . Ry decreased force of contraction to 54 ± 3%  compared to baseline  before and after application of Ry  23.4 ± 4.3 s-1 vs ventricular (n = 13) 11.8 ± 0.3 s-1, p < 0.05; maximum relaxation velocity normalised to twitch amplitude: atrial (n = 4) 15.6 ± 1.5 s-1 vs ventricular (n = 13) 10.9 ± 0.3 s-1, p < 0.05), application of ryanodine decreased force of contraction to 52 ± 8%  compared to baseline     . In cont.05) Fig  and 5D.  < 0.05) . Relaxat all Fig  and 5D.-9 M, to 101 ± 4% at ISO 10-8 M, to 112 ± 6% at ISO 10-7 M, 144 ± 14% at ISO 10-6 M and 154 ± 18% at ISO 10-5 M . Force did not further increase at ISO 10-4 M . In addition to the positive inotropic effect, ISO accelerated relaxation mildly . Shortening velocity was significantly increased only at ISO 10-4 M , but not at lower concentrations.The β-adrenergic agonist ISO increased force of contraction Fig  and 6B. n mildly . Maximum-1 at 23°C, 14.5 ± 1.0 s-1 at 32°C and 21.3 ± 2.0 s-1 at 37°C vs. 11.7 ± 0.8 s-1 at 29°C, n = 5, p < 0.05; relaxation velocity relative to amplitude: 8.5 ± 0.2 s-1 at 23°C, 12.8 ± 0.3 s-1 at 32°C and 14.9 ± 0.6 s-1 at 37°C vs. 11.6 ± 0.2 s-1 at 29°C, n = 5, p < 0.05; Normalised to 29°C, force of contraction decreased to 88 ± 7% at 23°C and increased to 108 ± 6% at 32°C and to 129 ± 11% at 37°C  application of NiCl2 but not VER prolonged relaxation, and most importantly, (5) inhibition of SR Ca2+-release by application of Ry significantly reduced force of contraction.In the present study, we used isometric force measurements of zebrafish ventricular heart slices in order to characterise intrinsic and extrinsic regulators of force of contraction and to determine whether zebrafish ventricular myocardium has similar contractile properties to mammalian myocardium. In summary, we observed that (1) viable slices of zebrafish ventricular myocardium could be prepared, (2) zebrafish ventricular myocardium showed an overall-negative FFR, (3) contractility was regulated by extracellular CaDue to the simplicity of the preparation and experimental approach, heart slices have become a popular technique especially in the recent years and have been prepared from many mammalian species . While aWe observed an overall negative FFR . This is2+ cycling and mechanical reconstitution are generally considered a requirement for a positive FFR ec and the LTCC blocker VER to modify trans-sarcolemmal Ca2+-flux, Ry to inhibit SR Ca2+-release, ISO to stimulate β-adrenergic effects on inotropy and lusitropy, and NiCl2 to inhibit NCX. Following this order, we discuss the effects of pharmacological interventions and putative mechanisms in the following paragraphs.In order to further characterise the role of several components of EC coupling, we used specific pharmacological interventions including changes of [Ca2+]ec and by the LTCC inhibitor verapamil, we were surprised by a ~50% reduction of force both in atrial and ventricular myocardium after application of Ry. This points to an important role of SR Ca2+-release in zebrafish myocardium. The role of SR Ca2+-release in EC coupling of teleost fish is controversial as data are highly dependent on species and experimental conditions [et al. reported that SR Ca2+-release contributes less than 20% of the total Ca2+-transient and that RyR expression is at least 72% lower in isolated zebrafish than in rabbit ventricular cardiomyocytes [2+ was released from the SR after application of caffeine. A high SR Ca2+-load has also been found in other teleost species including rainbow trout [2+-release significantly contributes to force development at 22°C to a higher extent than at 12°C [2+-release under our experimental conditions. This is further supported by the finding that zebrafish ventricular myocardium showed a strong post pause potentiation already after 5–10 seconds. Interestingly, inhibition of SR Ca2+-release reduced force of contraction stronger at higher frequencies. Thus, FFR became more negative with inhibition of SR Ca2+-release. Although this finding is difficult to interpret given potential frequency-dependent changes in myofilament sensitivity to cytosolic Ca2+-concentrations, and a potential use-dependence of RyR inhibition [2+-release is increased in order to compensate frequency-dependent inhibition of LTCC. This would further underline the important role of SR function in zebrafish ventricular myocardium.While it was not suprising that force of contraction was strongly influenced by varying [Canditions . Recentlmyocytes . Interesow trout  and seemow trout . In isol at 12°C . In the hibition , one mig2+-cycling and especially SR function strongly depend on temperature in the fish heart [2+-release contributes to EC coupling in vivo at least under laboratory housing conditions.In our study, and other published work in the zebrafish , contracsh heart , we chos2+-cycling, we observed a mildly positive lusitropic effect of the β-adrenergic agonist ISO in addition to a positive inotropic response , but not at lower concentrations. While the dose-dependent reduction of force of contraction might also underlie additional inhibition of LTCC and T-type calcium currents, prolongation of relaxation can only be explained by the inhibitory action of Ni2+ on NCX which occurs at high concentrations (5–10 mM) [2+-induced deceleration of relaxation is mainly caused by NCX inhibition, and that NCX significantly contributes to Ca2+-extrusion under our experimental conditions.The importance of NCX for myocardial relaxation, however, has been firmly established for many species and preparations , althougchannels . Ni2+ re5–10 mM) . In supp5–10 mM) , does noIn the present study, force data can only be compared between measurements of the same preparation, but not between different preparations as force was not normalised to the cross-sectional area of the preparation. However, as fibre orientation in the zebrafish ventricle is not aligned to a specific axis but non-parallel, a large proportion of fibres will be oriented with an unknown angle to the axis of the measurement setup. Therefore, we avoided normalisation to the cross-sectional area as this might give false impression of comparability between measurements in different preparations. As our main aim was to study the role of different force regulatory mechanisms but not to give absolute values of generated force, this limitation does not interfere with the aims of the study.2+-release substantially contributed to force generation in the zebrafish ventricle, although force-frequency relationship was negative. We are not able to explain why the force-frequency relationship is negative in the majority of fish species. It can, however, be assumed that the influences of external variables on the mechanical environment in the fish heart is much more complex than in mammalian hearts. For example, ventricular compliance is influenced by diving depth and temperature as well as the elasticity of the bulbus arteriosus and thus, afterload and resonant frequency of the vessel downstream to the heart. To fully understand these complex mechanical dynamics, a completely different experimental or numerical approach is required.The main finding of our study is that SR Ca2+, trans-sarcolemmal Ca2+ influx, NCX-driven Ca2+ extrusion, and SR Ca2+-release. Thus, although some regulatory mechanisms of force development seem to be similar between zebrafish and mammals, including SR Ca2+-release, one of the most important regulatory principles of contractile force—the relationship between force and frequency—is contrary between the mammalian and the zebrafish heart. Beyond a per se interest in fish cardiac physiology, these aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.In conclusion, we observed that despite the overall-negative FFR, regulation of contractile force in zebrafish ventricular myocardium strongly depends on extracellular CaS1 Fig2 for 2 days) (D). Cyrosections stained against sarcomeric-α-actinin (green) at day 4 (B-C) or cultured under hypoxic conditions (E-F). Nuclei are counterstained with Hoechst (blue).HE staining of cryosections (8 μm) obtained from a myocardial tissue slice at day 4 (A) and cultured under hypoxic conditions (1% O(TIF)Click here for additional data file.S2 FigBox plots of the ratio of change in resting tension and change in amplitude after frequency switches. If frequency was reduced from 2 Hz to 1 Hz, a decrease in resting tension contributed to 16± 5% of amplitude increase, if increased from 1 Hz to 3 Hz, an increase in resting tension contributed to amplitude change by 6 ± 1%, and if decreased from 3 Hz to 2 Hz, resting tension contributed to 17 ± 4%.(TIF)Click here for additional data file.S3 Figtmax) and relaxation (dF/dtmin) velocities normalized to amplitude increased with temperature. (D) Relaxation of the myocardial slices was also accelerated at elevated temperatures. All data are expressed as mean ± SEM; asterisks indicate statistical significant differences .(A) Force of contraction increased with elevated temperature. Force of contraction was normalized to values at 29°C. Results are expressed as mean ± SEM (B) Original averaged twitches of a representative experiment showing the influence of investigated temperatures on twitch amplitude as well as contraction and relaxation kinetics. (C) Maximal contraction (dF/d(TIF)Click here for additional data file.