Ca2+ signaling was studied using the fluorescent indicator dye Fura-2-AM. AGEs were generated by incubating bovine serum albumin with 0–250 mM glucose or glucose-6-phosphate for 0–120 days at 37°C. Under all conditions, the main AGE species generated was carboxymethyl lysine (CML) as assayed using both gas-liquid chromatograph-mass spectroscopy and high-performance liquid chromatography. In Ca2+-replete solution, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated the increase in intracellular [Ca2+] caused by ATP (100 μM). In the absence of extracellular Ca2+, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated subsequent intracellular Ca2+ release caused by ATP, thapsigargin (0.1 μM), and ionomycin (3 μM), but AGEs did not affect extracellular Ca2+ entry induced by the re-addition of Ca2+ to the bathing solution in the presence of any of these agents. The anti-oxidant α-lipoic acid (2 μM) and NAD(P)H oxidase inhibitors apocynin (500 μM) and diphenyleneiodonium (1 μM) abolished these effects of AGEs on BAECs, as did the IP3 receptor antagonist xestospongin C (1 μM). In summary, AGEs caused an acute depletion of Ca2+ from the intracellular store in BAECs, such that the Ca2+ signal stimulated by the subsequent application other agents acting upon this store is reduced. The mechanism may involve generation of reactive oxygen species from NAD(P)H oxidase and possible activation of the IP3 receptor.Post-translational modification of proteins in diabetes, including formation of advanced glycation end products (AGEs) are believed to contribute to vascular dysfunction and disease. Impaired function of the endothelium is an early indicator of vascular dysfunction in diabetes and as many endothelial cell processes are dependent upon intracellular [Ca One of the earliest signs of vascular endothelial dysfunction observed in diabetes is impairment of endothelium-dependent vasodilatation. Impaired vasodilatation has been demonstrated in a number of experimental, animal models of hyperglycemia and diabetes  is a process that is dependent on several co-factors including Ca2+, the Ca2+-binding protein calmodulin, oxygen, tetrahydrobiopterin, and NAD(P)H  represent important common elements in their function. In endothelial cells, both intermediate- and small-conductance KCa (IKCa and SKCa respectively) are involved in most established EDH mechanisms , and the activity of endothelium-derived hyperpolarizing factors (EDHFs) is dependent on an increase in endothelial cell [Ca2+] may be altered by a wide variety of neural, humoral, and physical stimuli  and subsequent inositol 1,4,5-trisphosphate (IP3)-mediated release of intracellular Ca2+ stores , activated by depletion of intracellular stores, and/or entry through other mechanisms including receptor-operated channels and membrane Ca2+-exchangers. The mechanism of CCE into vascular endothelial cells is not fully understood and is somewhat controversial  plated on glycated mixed matrix (EHS) protein, ATP-, and thapsigargin-induced increases in intracellular [Ca2+] were shown to be inhibited  were not examined. Furthermore, while endothelial cell viability was significantly reduced by the glycated EHS matrix proteins it was noticeable that ATP-induced Ca2+ signaling was not impaired in cells similarly exposed to glycated fibronectin. Interestingly, the glycated fibronectin did not markedly inhibit cell viability  followed by full characterization of the AGE species generated using a variety of techniques including assay of unmodified amine groups, gas chromatograph–mass spectroscopy (GC-MS), and reverse phase high-performance liquid chromatography (HPLC). The effects of exogenously applied AGEs on Ca2+ signaling in BAEC were then examined. As many of the deleterious effects of AGEs are mediated by the activation of RAGE and NAD(P)H oxidase H oxidase inhibitors.Only a few studies have examined the direct effects of AGEs and other glycated species on endothelial cell CaBovine serum albumin  was incubated with varying concentrations of either glucose or glucose-6-phosphate  for 5–90 days at 37°C in the dark. The glycation reaction was terminated by dialysis against fresh PBS. Prior to use in experiments, AGE preparations were passed through Detoxi-Gel AffinityPak™ columns to remove bacterial endotoxin. Endotoxin levels in samples before and after column-purification were measured using a modified Limulus Amebocyte Lysate (LAL) test  as per manufacturer’s instructions. Samples used contained <0.01 EU/ml endotoxin (lower limit of reliable detection).o-phthalaldehyde (OPA) crystals  and absorbance (340 nm) measurements together with an assessment of unmodified primary amines using fluoraldehyde 4 in 0.1 N NaOH for 16 h at 4°C. Excess of NaBH4 was discharged by addition of 10% trichloroacetic acid (w/v), samples were then centrifuged at 2000 × g and the precipitate resuspended in 0.1 N NaOH. Acid hydrolysis was performed in 6 N HCl and the samples dried in vacuo and converted to their trifluoroacetyl methyl ester (TFAME) derivatives for analysis. For preparation of TFAME, 1 ml freshly made 1 M methanolic HCl was added to the samples and incubated for 45 min at 65°C. Solvent was evaporated and the product was redissolved in 1 ml of trifluoroacetic anhydride and the mixture incubated at room temperature for 1 h to obtain the trifluoroacetyl derivatives. After removing the solvent the sample was dissolved in 150 μl of methylene chloride and 2 μl of this solution was injected for GC/MS analysis.Gas-liquid chromatograph-mass spectroscopy (GC-MS) analysis of samples was carried as described elsewhere . The injection port was maintained at 275°C and the transfer line at 290°C. The temperature program was as follows: 3 min at 130°C; ramp to 180°C at 4°/min and then to 240°C at 5°/min and to 290°C at 15°/min; hold for 5 min at 290°C. Quantification was based on isotope dilution using standard curves constructed from mixtures of a constant amount of heavy labeled internal standards  and increasing amounts of non-labeled carboxymethyl lysine (CML) and CEL. The limit of detection for GC/MS analysis of CML and CEL was 0.05 and 0.007 mmol/mol of Lys respectively, which is comparable with previous data , CML, CEL, (2S)-2-amino-5--pentanoic acid (MG-H1), (2S)-2-amino-5--pentanoic acid acetate (MG-H2), glyoxal-derived lysine dimer -3H-imidazolium acetate; GOLD), and methylglyoxal-derived lysine dimer -4-methyl-3H-imidazolium acetate; MOLD). The limit of detection for any AGE using this HPLC method is 0.1 pmol.Glycated and control BSA samples (2 mg protein) were precipitated and hydrolyzed  at 37°C in 5% CO2 (passages 3–7). Cells were allowed 72 h to adhere and become confluent on sterile, single-well MatTek™ plates . Intracellular [Ca2+] in BAEC was measured using the fluorescent, ratiometric dye Fura-2 AM. BAEC were washed three times with Krebs solution piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′- dissolved in double distilled water, pH 7.4) and incubated with Fura-2 AM (2 μM) at 37°C for 30 min. After washing, fluorescence measurements were performed in enclosed cover-slip holders perfused with Krebs solution (composition described above) at room temperature on a randomly selected area of 25 cells that were highly confluent with even dye loading. The Krebs solution used to superfuse the cells during fluorescence measurements was not gassed with CO2, our experience has shown HEPES is sufficient to buffer the Krebs and any pH changes in the environment of the cells over the brief time-course of the experiment that, including the Fura-2 incubation period, was less than an hour . Images were collected at one image per 5, 15, or 30 s based on the stage of experiment as shown on each graph. Changes in intracellular [Ca2+] were expressed as the ratio of emission (510 nm) following excitation at 340 and 380 nm. Data was normalized to the baseline, which was defined as values obtained during the period (1 min) immediately prior to intervention.Bovine aortic endothelial cells  were cultured in endothelial cell growth medium  for 1 min prior to addition of the AGE preparation or control solution. Cells were then incubated with the AGE preparation or control solution for 5 min before addition of one of ATP, thapsigargin, or ionomycin. Extracellular Ca2+ entry was induced by subsequent addition of buffer containing CaCl2 , the NAD(P)H oxidase inhibitors diphenyleneiodonium  or 4′-Hydroxy-3′-methoxyacetophenone  or the IP3 receptor antagonist xestospongin C (1 μM) were incubated with BAECs for 30 min at 37°C prior to the addition of AGE or control solutions. Where experiments varied from this protocol, such deviations are described in the text. Appropriate buffer controls were performed for these agents, the buffer controls had no effect on the parameters measured in the present study (not shown).Following Fura-2 loading, the cells were incubated with an AGE preparation or control solution  for 5 min prior to addition of a Ca® from Cambrex Bio Science Australia Pty Ltd., CML for HPLC from TRC , CEL, MG-H1, MG-H2, GOLD, and MOLD from NeoMPS . Unless otherwise stated, all other drugs and chemicals were obtained from Sigma Aldrich Pty Ltd. .Quick Start™ Bradford assay and sodium dodecyl sulfate (SDS) from Bio-Rad, Phosphate buffer saline tablets (PBS), Fura-2 AM, and trypsin-EDTA from Invitrogen Australia Pty Limited , Detoxi-Gel Affinity Pack™ Pre-Packed columns from Thermo Fisher Scientific Inc. , BSA from BOVOGEN , LAL QCL-1000 from BioWhittaker Inc., Water for Irrigation from Baxter Healthcare Pty Ltd. , Endothelial growth medium (EGM) BulletKitt-tests. Multiple comparisons were performed using two-way ANOVA with Bonferroni post-tests. All other statistical operations were performed using GraphPad Prism 4.01. n is the number of plates and represents at least three different batches of plated cells.Overall changes in normalized fluorescence ratio are expressed as AUC (area under curve) primarily because the response to AGEs (as opposed to ATP) did not feature clear “peak” or “sustained” phases, but a slowly developing and sustained increase. Calculation of AUC was performed using GraphPad Prism Version 4.01  with background fluorescence subtracted (from a region of the cover slips not containing cells). “Curves” were defined as the period between which fluorescence ratio significantly exceeded and returned to the basal level following an intervention, in other words elevations in basal fluorescence caused by AGEs were excluded. Overall, fluorescence data for an individual experiment were normalized to the baseline and presented as mean ± SEM. Comparisons of the means and SEMs of two groups were performed using unpaired Formation of AGEs in BSA solutions co-incubated with G6P or glucose was dependent on both the duration of incubation and concentration of glycating agent, as assessed by sample absorbance, fluorescence, assay of unmodified amines Figure  and GC-M2+ signaling in BAEC. Control samples consisted of 0.1 mg/ml of a 10 mg/ml BSA solution incubated without glycating agent for 60 days.The most extensively glycated BSA sample (10 mg/ml BSA incubated with 250 mM G6P for 60 days), at a final concentration of 0.1 mg protein/ml, was used routinely to examine effects on Ca2+-replete) Krebs solution, exposure of the endothelial cells to the AGE sample alone caused a significant increase in intracellular [Ca2+], whereas the control sample did not increase [Ca2+]  consisting of an early, transient increase peaking at approximately threefold the normalized basal fluorescence ratio, followed by a sustained increase in fluorescence ratio at about 1.5 times the basal level  with an apparently selective effect on the early, transient component  showed that AGEs alone could induce extracellular Ca2+ entry . In samples of BSA incubated under varying conditions , there was a strong correlation between the concentration of CML, taken as indicator of the extent of glycation, and inhibition of ATP-induced Ca2+ increase in BAEC  of CML alone, as opposed to CML adducts formed on glycated BSA, had no effect on Ca2+ signaling in BAEC were examined using the anti-oxidant α-lipoic acid (2 μM) and the NAD(P)H oxidase inhibitors apocynin (500 μM) and DPI (1 μM). Pre-incubation of the cells with each of these compounds prevented both the AGEs-induced release of intracellular Ca2+ and the inhibition of ATP-mediated Ca2+ release caused by AGEs H oxidase . In the presence of the IP3 blocker, AGEs significantly increased basal [Ca2+] in the cells, but did not inhibit ATP-induced Ca2+ release , thus ATP-induced [Ca2+] release was inhibited during this period. The sustained phase of the ATP-induced [Ca2+] increase is due to influx of extracellular Ca2+ and, as established by subsequent experiments, this component of the endothelial cell [Ca2+] increase does not appear to be altered by AGE. This is possibly because AGEs alone induced CCE or Ca2+ entry through activation of intracellular Ca2+ stores, thus complicating such observations. These effects of AGEs on Ca2+ signaling may be mediated by superoxide or its derivatives as they were prevented by inhibitors of NAD(P)H oxidase.Dysfunction of the vascular endothelium is an early and potentially damaging vascular complication of diabetes which may contribute to the vascular complications associated with diabetes. AGEs formed following prolonged exposure of plasma and matrix proteins and other macromolecules to increased blood glucose are known to cause endothelial dysfunction  and by the Ca2+ ionophore ionomycin, which acts directly upon the storage organelle membrane -releasable pool. Collectively, these observations suggest that AGEs inhibited ATP-, thapsigargin-, and ionomycin-induced intracellular Ca2+ release by depleting the pool of available Ca2+ acted upon by these agents. CCE was not affected by AGEs and, when AGEs were added to the cells following ATP, or when IP3 receptors were blocked, extracellular Ca2+ entry was enhanced, suggesting the glycated proteins may have an action to induce Ca2+ entry or enhance CCE into the BAEC. It should be noted that, unlike thapsigargin and ionomycin, ATP-induced influx of extracellular Ca2+ into vascular endothelial cells probably occurs by mechanisms additional to CCE, mostly likely receptor-operated channels such as TRPC1, 3, and 4 H oxidase as the general anti-oxidant α-lipoic acid and NAD(P)H oxidase inhibitors apocynin and DPI abolished the effects of AGE on Ca2+ signaling. Neither of these inhibitors is selective; DPI also inhibits NOS and flavin oxidase while apocynin has general anti-oxidant properties  involve the activation of NAD(P)H oxidase, and are preventable by apocynin and other inhibitors of this enzyme .Advanced glycation end products have been shown to stimulate ROS accumulation in vascular endothelial cells H oxidase. There may be a role for IP3 receptors in these actions, although this remains to be established. Clearly such rapid and significant effects on Ca2+ signaling in vascular endothelial cells may play a significant role in the effects of AGEs and diabetes on the Ca2+-dependent processes in these cells, including those which impact the regulation of vascular tone.In summary, AGEs can act acutely to release intracellular 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.


At present little is known concerning Ca2+-dependent purinergic responses in adult human astrocytes. This work has examined effects of purinergic stimulation to alter levels of intracellular Ca2+ in adult human astrocytes. Ca2+-sensitive spectrofluorometry was carried out to determine mobilization of intracellular Ca2+ following adenosine triphosphate (ATP) or 3′-O-(4-benzoyl)benzoyl-ATP (Bz-ATP) stimulation of adult human astrocytes. In some experiments pharmacological modulation of Ca2+ pathways was applied to help elucidate mechanisms of Ca2+ signaling. RT-PCR was also performed to confirm human astrocyte expression of specific purinoceptors which were indicated from imaging studies.The properties of Ca2+]i to a peak amplitude with the decay phase of response exhibiting two components. The two phases of decay consisted of an initial rapid component which was followed by a secondary slower component. In the presence of Ca2+-free solution, the secondary phase of decay was absent indicating this prolonged component was due to influx of Ca2+. This prolonged phase of decay was also attenuated with the store-operated channel (SOC) inhibitor gadolinium (at 2 μM) added to standard PSS, suggesting this component was mediated by SOC activation. These results are consistent with ATP activation of P2Y receptor (P2YR) in adult human astrocytes leading to respective rapid [Ca2+]i mobilization from intracellular stores followed by Ca2+ entry through SOC. An agonist for P2X7 receptor (P2X7R), BzATP induced a very different response compared with ATP whereby BzATP (at 300 μM) elicited a slowly rising increase in [Ca2+]i to a plateau level which was sustained in duration. The BzATP-induced increase in [Ca2+]i was not enhanced with lipopolysaccharide pre-treatment of cells as previously found for P2X7R mediated response in human microglia. RT-PCR analysis showed that adult human astrocytes in vitro constitutively express mRNA for P2Y1R, P2Y2R and P2X7R.The endogenous P2 receptor agonist ATP (at 100 μM or 1 mM) applied in physiological saline solution (PSS) evoked a rapid increase of [CaThese results suggest that activation of metabotropic P2YR (P2Y1R and/or P2Y2R) and ionotropic P2X7R could mediate purinergic responses in adult human astrocytes. An alternative pathway for entry of Ca2+ from extracellular medium is provided by activation of family members of P2XR ionotropic channels. Overall, a diversity of astrocyte functional responses such as cellular growth and proliferation, cytokine production and regulation of cerebral blood flow can depend on the characteristics of Ca2+ signaling in cellsi mobilization in stimulated cells. In addition, reverse transcription polymerase chain reaction (RT-PCR) has indicated the expression of P2Y1R, P2Y2R and P2X7R in the adult human cells. To our knowledge, this work is the first report describing changes in intracellular Ca2+ mobilization associated with activation of purinergic receptors in primary culture of adult human astrocytes.The main purpose of this study was to characterize CaATP, 3′-O-(4-benzoyl)benzoyl-ATP (BzATP), lipopolysaccharide (LPS), gadolinium and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich . ATP and BzATP were dissolved in PBS solution. Fura-2/AM (F-1221) was purchased from Invitrogen Canada  and dissolved in DMSO.Adult human astrocytes were obtained from epileptic patients undergoing temporal lobe surgery with consents of all patients. Normal brain tissues overlying the epileptic foci were obtained from a standard elective surgical procedure where, in order to remove an epileptic focus, the surgeon first removed normal brain tissue which lies superficial to the previously defined epileptic focus. The epileptic patients were a 27 year-old male, 31 year-old female, 36 year-old female and 41 year-old male. Every brain sample arrived at our laboratory within 24 h after surgery and was immediately used for astrocyte isolation. The use of human brain materials was approved by the Clinical Research Ethics Board for Human Subjects of the University of British Columbia.Astrocytes were isolated as described previously,11. They2+i in adult human astrocytes. The experiments generally employed 1 mM of ATP (application time of 200 s); this level of ATP is insufficient to activate the P2X7 subtype ionotropic receptor in human microglia[2+]i exhibiting a biphasic time course . As shown in Figure 2+ responses, induced by different concentrations of ATP in standard PSS, are mediated by a rapid release of intracellular Ca2+ followed by a secondary component of influx.Calcium-dependent spectrofluorescence was used to examine effects of ATP on i. A typical astrocytic response induced by ATP in Ca2+-free PSS is presented in Figure 2+]i in Ca2+-free solution is consistent with influx of extracellular Ca2+ mediating this component of response. In three additional experiments, the secondary slow phase of response was absent in Ca2+-free solution. Overall results  yielded a single time course of decay in Ca2+-free PSS was 28.9 ± 1.8 s. This decay time course was not significantly different (p > 0.05) from the rapid time course of the control response evoked by 1 mM ATP  added to standard PSS. Inhibition of SOC with Gd3+ has previously been demonstrated in a variety of cell types, including smooth muscle and glioma cells[2+]i change induced by 1 mM ATP in human astrocytes exposed to Gd3+. A single monophasic time course of decay for [Ca2+]i was observed, indicating that addition of Gd3+ to standard PSS inhibits the prolonged component of the ATP response. Overall , ATP induced a single time course of decay with mean value of 29.3 ± 5.2 s when Gd3+ was added to PSS. This time course of response was not significantly different (p > 0.05) from the rapid phase of decay in control induced by 1 mM ATP . The response was considerably different from that induced by ATP , the mean amplitude of [Ca2+]i was 0.21 ± 0.02 in control.Figure 2+]i induced by BzATP for cells administered LPS treatment. Overall , the amplitude of the BzATP-induced response was 0.24 ± 0.03 with LPS treatment compared with an amplitude of 0.21 ± 0.02 in the absence of LPS treatment. This difference was not significant (p > 0.05) indicating LPS was ineffective as a modulatory stimulus to enhance purinergic responses to BzATP in adult human astrocytes.Previous work has demonstrated LPS priming of BzATP responses, measured as amplitudes of fluorescent ratio, in microglia which was attributed to inflammatory enhancement in numbers of P2X7R. This fi2+]i suggest functional expression of metabotropic and ionotropic P2R subtypes in cultured adult human astrocytes. We therefore carried out RT-PCR to examine expression for particular P2R, including P2Y1R, P2Y2R and P2X7R, which have previously been reported to mediate Ca2+ responsei change is followed by a subsequent influx of Ca2+ through SOC. RT-PCR analysis demonstrated the expression of specific subtype metabotropic P2Y1R and P2Y2R in addition to ionotropic P2X7R. Interestingly, this expression pattern of P2 purinoceptor in adult human astrocytes is consistent with observations made in fetal humani elicited by ATP in Ca2+-free and in Gd3+ with standard Ca2+ PSS (200 s treatment) were similar in magnitude (near 29 s) and somewhat longer than the rapid phase evoked by ATP in standard Ca2+ solution. This result suggests that only a partial inhibition of SOC was attained with astrocytes exposed to 2 μM Gd3+ for a duration 200 s. A possible explanation for the longer time course of decay in Ca2+-free PSS, relative to the rapid phase of control ATP response in PSS, is that residual Ca2+ could remain in nominally Ca2+-free solution. In order to minimize effects of non-physiological Ca2+-free PSS on cell viability, we employed relatively short treatment times of 60 s with this solution prior to ATP stimulation. We did not test Gd3+ at concentrations higher than 2 μM nor increase incubation time with Ca2+-free PSS to detect astrocytic responses in a robust and healthy condition. The overall results from calcium imaging experiments suggest that purinergic response to endogenous ligand in adult human astrocytes is mediated by ATP binding to metabotropic P2YR with subsequent mobilization of [Ca2+]i due to intracellular release and influx through SOC.ATP stimulation of adult human astrocytes mobilized intracellular Caular Ca2+,14. The ist, Gd3+. The sin2+ spectrofluorometry showed that application of BzATP elicited a gradual and sustained increase in [Ca2+]i in adult human astrocytes. This finding suggests influx of Ca2+ through the nonselective cationic channel coupled to activation of P2X7R[2+]i rise elicited by BzATP in fetal human astrocytesii. One postrocytes whereas trocytes.2+ signaling pathways in cultured adult human astrocytes. Metabotropic P2YR and ionotropic P2XR are putative mediators of purinergic responses in the cells. Future studies using adult human astrocytes are warranted to characterize the specific roles of the purinergic receptors in mediating cellular responses. Such work will enable clarification of downstream Ca2+-dependent and independent signaling pathways. P2X7R expression and function should be confirmed in these cells followed by examination of roles of the receptor in mediating astrocytic responses in pathological microenvironments in human brain.Our study has presented novel findings concerning expression and activation of specific purinergic CaThe authors declare that they have no competing interests.SH, YFW and JGM participated in the design of the study. SH and YFW carried out all experiments, collected the data and performed the statistical analysis. JPL and AK performed the RT-PCR analysis. HCB participated in calcium spectrofluorometry. SH, YFW and JGM interpreted the data. SH and JGM wrote the manuscript. JGM, HCB, AK and PLM revised the manuscript. All authors read and approved the final manuscript.


In this study, we developed a coupled model by integrating the spatiotemporal Ca2+ reaction-diffusion system into the cellular electrophysiological model. With the coupled model, the subcellular Ca2+ dynamics and global cellular electrophysiology could be simultaneously traced. The proposed coupled model was then applied to study the effects of rogue RyRs on Ca2+ cycling and membrane potential in failing heart. The simulation results suggested that, in the presence of rogue RyRs, Ca2+ dynamics is unstable and Ca2+ waves are prone to be initiated spontaneously. These release events would elevate the membrane potential substantially which might induce delayed afterdepolarizations or triggered action potentials. Moreover, the variation of membrane potential depolarization is indicated to be dependent on the distribution density of rogue RyR channels. This study provides a new possible arrhythmogenic mechanism for heart failure from subcellular to cellular level.Cardiac cells with heart failure are usually characterized by impairment of Ca DSR is luminal Ca2+ sensitivity parameter of Ca2+ release events, and Kmj is cytoplasmic Ca2+ sensitivity parameter of RyR clusters or rogue RyR channels. In our simulation work, Kmrogue was always set to be of the same value as Kmcluster; thus Km was used to represent the value of Kmcluster and Kmrogue.Firings of the two types of RyR channels are considered to be stochastic processes and treated by the Monte Carlo simulation in our work. To evaluate the effects of SR luminal Ca2+ handling was performed on the longitudinal section of a cardiac myocyte with the size of 100 μm × 20 μm along the cellular longitudinal direction (x-axis) and Z-line (y-axis), respectively. The number of RyR clusters was 49 × 19 along x and y axes, respectively, and the total number of rogue RyRs was Nrogue × 2000 μm2. The diffusion partial differential equation was approximated by the finite difference method (FDM) with a time-step size of 0.01 ms and a mesh size of 0.1 μm.In this study, the simulation of subcellular Ca2+ signalling were described by statistical results by carrying out repeated Monte Carlo simulations. All averaged data were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used for comparison and P < 0.05 was taken to indicate statistical significance.Because of the stochasticity of opening of RyR clusters and rogue RyRs, the properties of CaCm is the membrane capacitance, Istim is a stimulus current, and Ix denotes all kinds of ionic currents across the sarcolemma. The electrophysiological behavior of a myocardial cell is modelled based on a cardiac action potential model proposed by Ten Tusscher and Panfilov . The vol2+ dynamical system by Ten Tusscher et al., global SR Ca2+ release current Jrel at the cellular level is calculated by the summation of local Ca2+ release fluxes in the 2D subcellular model:krel is a constant multiplier equivalent to 22.25 in our coupled model.However, different from the Ca2+ cycling as well as other ionic currents have been observed in failing heart; thus we modified the parameters of our coupled model to mimic abnormal Ca2+ dynamics and electrophysiological properties in heart failure from subcellular to cellular levels.Changes of Ca2+/calmodulin-dependent-protein-kinase-I- (CaMKI-) induced hyperphosphorylation SR is ~15% lower in resting cells, and the restoring rate of SR calcium is slower than that on control condition. Due to the changes of AP morphology and calcium transient curves together with increase of the activity of Na/Ca exchanger, the curve of INaCa in heart failure differs significantly from that under normal condition. This can be seen in INaCa are increased. However, it takes a longer time to reach the peak of inward current, and the amplitude of inward INaCa in resting stage is also larger compared with that under control conditions. For the calcium handing in heart failure, it is mainly characterized by a significant impair of global Ca2+]i and [Ca2+]SR as those under normal conditions. However, the calcium release current through a RyR cluster decreases as the SR calcium store is partly unloaded, which is observed as reduction of amplitude and area of calcium sparks. Indeed, the simulation results by using our coupled model show that, although more spontaneous calcium sparks occur in failing myocytes, propagating Ca2+ waves are seldom found when there is no rogue RyRs on the 2D subcellular space without any stimulus. These spontaneous calcium sparks would slightly elevate global [Ca2+]i on the cellular level (Δ[Ca2+]i = (1.16 ± 0.06) × 10−4 mM, n = 10) and depolarize transmembrane potential with a tiny amplitude  as shown in In heart failure myocytes, the RyR channels become very unstable and are more likely to open with the same values of i and membrane potential are not affected severely by those Ca2+ release events under the condition with low density of rogue RyRs as shown in 2+]i = (1.61 ± 0.08)× 10−4 mM, ΔVm = 5.95 ± 0.29 mV, n = 10). However, how do Ca2+]i and Vm should be two groups of principal parameters to evaluate the effects of rogue RyRs on Ca2+ dynamics and electrophysiological properties. Besides Δ[Ca2+]i and ΔVm, two new parameters TpeakCa and TVmpeak are used in our simulation. TpeakCa represents the mean time to reach the peak of [Ca2+]i from the end of resting stage, and TVmpeak is the time to reach the peak of Vm. Increase rate of Ca2+ concentration and depolarization velocity could be estimated indirectly by the two parameters, which is shown in TpeakCa decreases significantly when Nrogue is upregulated from 0 to 0.25 rogue RyR/μm2 (TpeakCa = 418 ± 19.7 ms and 325 ± 15.2 ms (n = 10), resp., n = 10, P < 0.05). However, decrease of TVmpeak is slight when Nrogue is from 0 to 0.25 rogue RyR/μm2 . In Vm without any stimulus when Nrogue = 0.25 rogue RyR/μm2. The results indicate a smooth variation without a significant peak in the membrane potential morphology.Amplification and increase rate of i and membrane potential (Δ[Ca2+]i and ΔVm) as well as TpeakCa and TVmpeak. However, ΔVm elicited by intensive Ca2+ release events is often large enough to activate INa. Consequently, the voltage upstroke induced by INa would overlap the original ΔVm. Then, Δ[Ca2+]i by Ca2+ release events is also overlapped by the following inward-flowing ICaL during a triggered AP. Under those conditions, the measurement of Δ[Ca2+]i and ΔVm as well as TpeakCa and TVmpeak becomes very difficult. Thus, in our simulation, when Nrogue⩾ 1.5 rogue RyR/μm2, we set the INa to be zero after removing the external stimulus. By doing this, triggered AP will not be formed even when a DAD makes the membrane potential more positive than the threshold for INa. Therefore, we can calculate these parameters easily. Actually, our simulation results are shown in Nrogue is increased from 1.5 to 1.75 rogue RyR/μm2, the amplitude of [Ca2+]i enlarges (P < 0.05), whereas the time to reach the peak does not change obviously (P > 0.05). However, the amplitude of a DAD increases significantly (P < 0.05), while the time to observe the DAD decreases (P < 0.05).To quantitatively investigate the effect of high dense rogue RyRs on Ca2+ handling of cardiac cells in heart failure is always characterized by reduction in the amplitude as well as by slowed decay of Ca2+ transient i decay, the net current of Na/Ca exchanger might be outward current  or weak inward current, due to longer AP plateau and higher plateau potential in failing myocyte. Therefore, slowed decay of Ca2+ transient is mainly due to decreased SR Ca2+ pump, which removes major amount of Ca2+ at the early stage of [Ca2+]i decay. Only when the membrane potential is repolarized to a relatively negative voltage and [Ca2+]i is still high that INaCa turns to a strong inward current and accelerates decay of Ca2+ transient at the late stage of [Ca2+]i decay.Despite the increased activity of Na/Ca exchanger, at the early stage of i variation elicited by these Ca2+ release events is slight. In our simulation, when Nrogue = 0 or 0.25 rogue RyR/μm2, the amplitude of membrane potential depolarization is very small, only several mV, and the average TVmpeak is big and extensive with large SEM. The morphology of membrane potential is very smooth without a distinct peak, so that this type of depolarization could not be referred to a genuine DAD. Without rogue RyR channels or with low distribution density, occurrence of spontaneous CaVm increases gradually, while the value of TVmpeak decreases but becomes more intensive. When the value of Nrogue is elevated to 1.5 rogue RyR/μm2 or bigger, the large membrane potential depolarization tends to evoke a triggered AP, and the probability of occurrence of triggered APs increases with the larger Nrogue. The reason is that larger number of rogue RyRs would increase the amplitude and rate of DADs by initiating more Ca2+ waves which occur more synchronously. Therefore, depolarization of Vm is indicated to be dependent on the distribution density of rogue RyR channels. However, as more rogue RyRs are distributed over the 2D plane, Δ2+ release flux via a rogue RyR channel, while Ca2+ release flux via a cluster of RyRs was dependent on global luminal Ca2+ concentration ([Ca2+]SR) and local Ca2+ concentration ). Besides, different Nrogue values were used to evaluate the effect of rogue RyR on Ca2+ cycling and membrane potential in failing heart.Because the rogue RyR remains to be a hypothetical channel rather than a determinate concept, experimental parameters of rogue RyRs are lacked. Some assumptions were made regarding the density, distribution, and kinetic of rogue RyR channels. In our work, a constant was used to represent Ca2+ handling also occurs in many cardiac diseases, such as myocardial infarction, atrial fibrillation, and various arrhythmogenic paradigms. The coupled model is planned to be improved, and more relevant parameters should be added to investigate the potential mechanisms of Ca2+ dynamics in various kinds of cardiac diseases.Numerous key regulatory proteins, such as protein kinase A(PKA), Calstalin, CaMKII, and phosphatase, are bound to RyR, thus forming the junctional complex. RyR channels would be regulated via different signaling pathways . For exa2+ reaction-diffusion model into the cellular electrophysiological model, appearance of subcellular Ca2+ release events and evolution of waves together with dynamics of ionic concentration and membrane potential on the cellular level could been monitored simultaneously. By using the coupled model we investigate the effects of rogue RyRs on Ca2+ handling from subcellular to cellular level as well as electrophysiological properties in failing heart. The simulation results suggest that rogue RyR with tiny Ca2+ release flux should be an important factor in triggering arrhythmia in failing cardiac cells. Our work suggests the importance of rogue RyRs in initiation of Ca2+ release events  and consequently DADs or triggered APs. Our study indicates the arrhythmogenic effect of rogue RyRs and helps to elucidate a possible arrhythmia mechanism in failing heart.By integrating the spatiotemporal Ca


The reduction in Kd results from a reduced dissociation rate constant (from 16 s− 1 to 0.3 s− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca2+]. However, for Ca2+ transients to be effective in further promoting dissociation, the elevated Ca2+ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein–myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data.The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca 2+ binds preferentially to the EF2 domain of S100A4. ► The minimal S100A4 binding site on myosin IIA includes residues A1907-G1938. ► Aggregates of a myosin tail fragment dissociate via an S100A4–aggregate complex. ► In the presence of Ca2+, S100A4 binds the monomeric myosin tail with nanomolar affinity. ► Myosin could interact with S100A4 at resting cytoplasmic [Ca2+].► Ca This is exemplified by S100A4 , which has been implicated in a wide range of cellular events and numerous diseases and binds to several different targets in vitro.2+, S100A4 promotes the disassembly of NM-MHC IIA filaments and inhibits the assembly of monomeric NM-MHC IIA into filaments.et al. concluded that S100A4 bound tightly to the monomeric myosin tail to increase its critical concentration, thus inhibiting filament formation.Over the last two decades, there has been much interest in the role of the S100 Ca2+ binding sites, but the N-terminal domain (EF1) is a pseudo EF-hand. It contains 14 residues—instead of the usual 12 residues—in the Ca2+ binding loop, and, apart from glutamate 33, backbone carbonyl residues provide Ca2+ binding ligands instead of side chains. Literature Kd values for Ca2+ binding to S100A4 show a disparate range from the micromolar region to the millimolar region.et al.et al.2+ binding to the canonical EF2 hand, with the weaker site representing the pseudo EF1 hand. However, detailed studies on the related protein calbindin D9k indicated that both EF1 and EF2 sites bound Ca2+ with comparable affinities and that there was cooperativity between them.2+ with a very high affinity (10− 8 M) and, therefore, the weaker site cannot be assumed to be EF1 without additional evidence. Interestingly, NMR titration studies on S100A4 indicated that the pseudo EF1 hand may bind Ca2+ more tightly than EF2, as some residues in the N-terminus were perturbed at lower Ca2+ ratios compared with those in the C-terminus.S100 proteins contain two EF-hand Ca2+ binding, which have sensitivities over different concentration ranges, part of the variability in Kd values in the literature may arise from the self-association of S100A4. There is a general consensus2+ bound per mole of S100A4’ if the latter is not stated explicitly as being per monomer or per dimer). In the absence of Ca2+, monomeric S100A4 can be detected at protein concentrations below 2 μM.2+, tetrameric and higher aggregates become significant.2+, revealed a tetrameric structure in which the C-terminal extension of one S100A4 monomer bound to the target binding site of an S100A4 monomer in the apposing dimer.2+ is not required for dimerisation or further oligomerisation, Ca2+ tends to stabilise higher-order quaternary structures; therefore, the measured Ca2+ affinity will depend on the protein concentration used. Thermodynamic coupling requires the higher-order oligomerisation states to bind Ca2+ more tightly. Here, we reexamine the Ca2+ binding properties of S100A4, including mutant E33Q and D63N constructs, which are compromised in the EF1 and EF2 Ca2+ binding loops, respectively.Apart from the different methods used to study Ca2+ .et al. reported only around a 2-fold difference in binding constant between a shorter peptide comprising residues 1908–1923  and longer fragments (residues 1851–1960 and 1893–1923), and they concluded that the former represented the minimal S100A4 binding site.et al., makes a large contribution to binding energy and that the binding site extends beyond the “minimal” region of residues 1908–1923.Our kinetic studies are also used to redefine the minimal binding fragment of the NM-MHC IIA tail . Using aet al. investigated Ca2+ binding to S100A4 using isothermal titration calorimetry (ITC) and reported that, at 37 °C and 20 mM KCl, two classes of site with Kd values of 3.3 μM and 54 μM at positive and negative enthalpies, respectively, were apparent.Kd value, while the second site remained undetectable. While this might be caused by a change in affinity, it is possible that the enthalpy change for the second site is small and masked by the first. In some titrations at 10 °C, the ITC profile indicated positive cooperativity. The variability in these titrations may arise from differences in the small amounts of contaminant Ca2+ present at the start of the titration. Titration against ethylene glycol bis(b-aminoethyl ether) N,N′-tetraacetic acid (EGTA) indicated that about 20% of the S100A4 had prebound Ca2+ present. The measured stoichiometry of the higher-affinity site was 0.8–0.9 Ca2+ per monomer , as performed previously.2+ in the region of 1–10 μM, with an indication of cooperativity at 10 °C; however, the stoichiometry was reduced at 30 °C, suggesting that the protein may be less stable , suggesting some loss of stability of the Ca2+-bound form with the mutants. We also examined the E33Q and D63N mutants under similar conditions and found that the D63N mutant gave no resolved transient, suggesting that Ca2+ binding was weak and/or its release was too fast to measure (> 400 s− 1). On the other hand, the E33Q mutant gave a transient that was similar in amplitude to the wild type but with double the rate constant (− 1 cf. 22 s− 1), indicative of a small loss in the stability of the EF2 Ca2+ binding loop. The data are consistent with the ITC data in that they indicate that the D63N mutation leads to a marked weakening of Ca2+ binding and suggests that Ca2+ dissociation from EF2 is responsible for tyrosine fluorescence change. On the other hand, tyrosine residues in both the EF2 domain (Y75) and the EF1 domain (Y19) respond to the loss of Ca2+ from the EF2 site . Given the poor signal-to-noise ratio of the tyrosine signal, the difference is barely significant but may reflect the fact that the Quin-2 signal represents at least a two-step sequential reaction . The E33Q mutant showed a similar amplitude but with a slightly higher rate constant (35 s− 1), indicating that the EF2 Ca2+ binding site remained functional despite a compromised EF1 site. The D63N mutant showed a much smaller transient that was just resolved (500 s− 1) and could represent Ca2+ release from the compromised EF2 site with a 30-fold reduction in affinity, as suggested by the ITC data. Ca2+ binding to EF1 therefore appears weaker than Ca2+ binding to EF2, and its dissociation is too fast to measure by stopped-flow methods in all constructs.In order to increase the signal-to-noise ratio of the Ca2+ dissociation  with a rate constant of 0.4–4 s− 1. The amplitude of the slow phase increased on storage of S100A4 at 4 °C and probably arose from oligomerisation and/or oxidation. This factor is important to control for in Ca2+ dissociation measurements in the presence of target peptides (described below), where the observed rate constants are comparable to this slow phase.Some S100A4 preparations showed a second slow phase of Ca2+ bound. In a separate experiment, the Quin-2 fluorescence signal obtained in the stopped-flow apparatus was calibrated by the addition of known Ca2+ concentrations, and the baseline fluorescence in the absence of contaminating Ca2+ (+ 1 mM EGTA) was determined. These data indicated that the resolved signal corresponded to one Ca2+ bound per S100A4 monomer  with S100A4 depends on  + k− 2) as a function of [F-M16N] yielded association (k2) and dissociation (k− 2) rate constants of 4.9 μM− 1 s− 1 and 6.2 s− 1, respectively , with subsequent rapid dissociation of F-M16N via k− 0, because the S100A4 concentration (> K2 = 1.3 μM) would ensure that the flux through the other route (via k− 2 and k− 1 in kobs through this route: kobs = 16 × 1.3/(1.3 + 10) = 1.8 s− 1). From the overall thermodynamic balance of k3 value of > 5 × 108 M− 1 s− 1, which is close to the diffusion-controlled limit, for Ca2+ binding to the preformed F-M16N–S100A4 complex. The kinetics of M32 binding, as far as determined, were comparable to those of F-M16N  when premixed with S100A4  increasing at the expense of the fast phase (k− 1 = 17 s− 1) with increasing [M32]. A comparison of fits to a single exponential and fits to a biphasic exponential with fixed rate constants and variable amplitudes  to the data obtained at 0.25 μM M32 showed small deviations in the residuals with similar magnitudes but opposite signs. This suggests that the kinetics of Ca2+ and M32 dissociation from the M32–S100A4–Ca2+ complex are comparable (k− 2 ≈ k− 3), as might be expected from the kinetics of competition with F-M16N binding (Kd for M32 binding (K2 = 0.4 μM), which is smaller than that deduced by the anisotropy competition assay, and also suggested a stoichiometry closer to 0.5 mol of M32 per S100A4 monomer than 1:1.Ca k− 1 in . The obsh S100A4 b, wherea0A4 , while the amplitudes interchanged. This is indicative of a slow equilibrium between free M111 and bound M111 relative to the rate constant for Ca2+ release. In the case of M200, the measurement was complicated by the fact that, in 100 mM NaCl, this fragment forms filamentous aggregates (see the text below). However, on addition of twice the stoichiometric amount of S100A4 in the presence of Ca2+, the aggregates were solubilised and the solution had low turbidity. On mixing with excess Quin-2, free Ca2+ was rapidly chelated, and the M200–S100A4–Ca2+ complex dissociated. The liberated M200 then formed filaments. Simultaneous measurement of Quin-2 fluorescence and light scatter showed that the latter reaction had a substantial lag and that the Ca2+ dissociation phase was essentially complete before the aggregation phase began  of 0.3 s− 1 attributed to Ca2+ dissociation from the M200–S100A4–Ca2+ complex , but the increase in light scatter is not observed immediately because filament formation has a slow nucleation event (see the text below). The slower dissociation of Ca2+ from the M111 and M200 complexes (0.3 s− 1), compared with the M32 complex (3.2 s− 1) and the F-M16N complex (9.4 s− 1), could be a consequence of the stabilisation of a multimeric complex due to the coiled-coil nature of the fragments and/or because myosin residues beyond the minimal binding region identified above (residues 1907–1938 ≡ M32) are involved , while binding of target peptides to S100A4 in the presence of Ca2+ (K2) is around a micromolar or less for longer fragments. Knowing that Ca2+ binding to S100A4 has K1 = 2–5 μM (from the ITC data above and the literature2+ binding to the M–S100A4 complex be very tight (K3 < 0.15 μM and even lower for M200). The latter cannot be easily determined directly as it requires protein concentrations greater than K0  so that the proteins remain bound together throughout the titration with Ca2+. However, K3 is readily determined by calculation because values or limits for K0, K1, and K2 are available  spectrum exhibiting highly dispersed cross-peaks of uniform intensity that correspond to a stable globular fold  comparable to the exchange rate constant kex = kon + koff. The shift difference of 0.12 ppm at 600 MHz gives the exchange rate constant estimate of kex ∼ 450 s− 1 at 35 °C, in fair agreement with the k2[S100A4] + k− 2 value determined by stopped-flow analysis at 20 °C in The contributions of different myosin regions to the interaction with S100A4 were analysed by NMR. To observe the direct perturbations of protein resonance on binding, we used truncated Δ10-S100A4, which lacks 10 C-terminal residues. From our datalar fold . No inten of M32 a. In then of M32 a, inset.N or M15C demonstrated fast exchange between the free state and the bound state , consistent with about 170 residues forming a stable coiled coil and 30 residues comprising the nonhelical C terminus, which were not visualised by electron microscopy (Rotary shadowed images of M200 and M111 deposited from high-ionic-strength solutions (0.5 M ammonium acetate) confirmed that these myosin fragments formed coiled-coil structures . The meacroscopy . Using acroscopy .2+, no trace of filamentous bundles was observed , as shown in The kinetics of the polymerisation and depolymerisation of M200 are likely to be complex, as M200 monomers could add to the ends or the sides of filaments, and the filaments themselves may bundle, fragment, or anneal. For the purposes of discussion, we will consider the dissociation of one M200 monomer (M) from a filament bundle (denoted Mk− 4 and k5), or whether S100A4 also binds to the filament bundle and actively depolymerises an M200 unit (via k6 and k− 7). It is known that S100A4 can bind to myosin rod fragments,k− 7. In an attempt to address this question, the dissociation rate of the M200 filament was followed on jumping the NaCl concentration from 100 mM to 120 mM in the absence and in the presence of S100A4 . Therefore, at the end point of this reaction, the filament concentration decreased by about 2-fold %). In the presence of 20 μM S100A4 (the concentration after the NaCl jump), the reaction had almost double the amplitude (indicating near-full dissociation of the M200 filament) and was closer to a single exponential . Significantly, the initial rate  was five times faster than in the absence of S100A4 . For a significant decrease in the concentration of myosin filaments  measurements on A431 cells expressing green fluorescent protein (GFP)–myosin IIA . From our measurements, a 32-mer peptide (corresponding to A1907-G1938 of the myosin sequence) represents a minimal binding region. Longer fragments (M111 and M200) bind with even higher affinity, indicating that additional amino acids of the myosin heavy chain contribute to the binding interaction and/or the capacity of the longer fragments to form coiled-coil structures enables higher-order structures to form with increased avidity. We are currently exploring longer peptides to address this question and consider that both factors are involved.We confirm that a 16-amino-acid myosin peptide derivative, F-M16 binding  suggestset al.  fragment with the S100A6 dimer, as determined by NMR.Based on the S100A4 concentration determined from the absorbance at 280 nm, one mol of M32 peptide binds to two S100A4 monomers. In support of this conclusion, the ITC titration curve of an R1893-R1923 myosin fragment of Malashkevich et al.et al.Kd values would be altered by just a statistical factor but would more likely extend the potential for cooperativity. However, until accurate stoichiometries are determined by direct structural methods, 2+ requires the target-bound form of S100A4 to bind Ca2+ with a very high affinity . The physiological consequence of this is that the myosin tail and S100A4 could bind at resting cytoplasmic Ca2+ levels and cause filament disassembly, provided at least one of the protein components is present at a concentration exceeding several micromolars  b. Murakaaffinity . In the lars cf. . Unfortu2+ , mixing S100A4 with excess Quin-2 produced a small-amplitude signal (corresponding to 0.1 mol of Ca2+ per mole of S100A4 monomer) with a rate constant of around 1 s− 1. This is similar to the rate constants obtained for target-bound S100A4 and suggests that this process might correspond to the dissociation of Ca2+ from a small fraction of the S100A4 tetramers. The crystal structure of the Ca2+-bound form of the S100A4 tetramer shows that the C termini of two of the monomers are positioned in the target binding sites of two opposing subunits.2+ than for the dimer. Indeed, ultracentrifuge data2+Kd value of around 0.1 μM for the tetramer would suggest that overexpression of S100A4 could lead to oligomerisation of S100A4 at resting cytoplasmic [Ca2+]. In addition, phenothiazine drugs have been reported to induce S100A4 oligomers and to inhibit interaction with NM-MHC IIA.In the absence of added Ca2+; in terms of kinetics, this construct serves as a model system for studying the mechanism of depolymerisation of intact NM-MHC IIA filaments. S100A4 binds to M200 filament aggregates and actively depolymerises them, rather than just binding to the monomer and perturbing the myosin filament equilibrium. However, the (M200)n.S100A4.Ca2+ filament complex is only a transient species that leads to a solubilised M200.S100A4.Ca2+ complex. This has implications for in vivo studies, since the degree of colocalisation of S100A4 with myosin filaments may be rather limited under steady-state conditions, and any solubilisation would lead to a more general cytoplasmic distribution of both proteins. Zhang et al. found that an eCFP-S100A4 construct did interact with a NM-MHC IIA–eYFP rod construct in the cytoplasm of HeLa cells, using fluorescence lifetime imaging.2+ concentration was likely to be close to resting levels and thus corroborates the potential for these proteins to interact at Ca2+ concentrations significantly lower than the Kd for S100A4 in the absence of target. However, the in vivo case is complicated by further regulatory mechanisms such as myosin phosphorylationS100A4 solubilises M200 filament aggregates in the presence of CaN, and M15C peptides  and was identical with FITC-MIIA1908–1923 as used by Malashkevich et al.M32, M16peptides  with an The mutant forms of S100A4 were made using the QuikChange Site-Directed Mutagenesis kit (Stratagene) and the following oligonucleotides:5′ CCACCTTCCACAAGTTCTCGGGCAAAGAGG 3′and 5′ CCTCTTTGCCCGAGAACTTGTGGAAGGTGG 3′ for Y19F S100A4;5′ GGACTTCCAAGAGTTCTGTGTCTTCCTGTC 3′and 5′ GACAGGAAGACACAGAACTCTTGGAAGTCC 3′ for Y75F S100A4;5′ GCTCAACAAGTCACAGCTAAAGGAGCTGC 3′and 5′ GCAGCTCCTTTAGCTGTGACTTGTTGAGC 3′ for E33Q S100A4; and5′ GATGAGCAACTTGAACAGCAACAGGGAC 3′and 5′ GTCCCTGTTGCTGTTCAAGTTGCTCATC 3′ for D63N S100A4. To check that no spurious changes had arisen as a result of the mutagenesis reactions, we resequenced the entire S100A4 gene in each mutant construct.Escherichia coli BL21(DE3) cells. Transformed cells were grown overnight at 37 °C in 2× YT media supplemented with 100 μg mL− 1 ampicillin. The overnight culture (15 mL) was used to inoculate 1 L of 2× YT media containing 100 μg mL− 1 ampicillin. The cultures were grown at 37 °C until the OD600 had reached a value of ∼ 0.8, and then isopropyl 1-thio-β-d-galactopyranoside  was added. The temperature was reduced to 30 °C, and incubation continued overnight. Cells were harvested by centrifugation , and cell pellets were resuspended in lysis buffer  supplemented with two Complete Protease Inhibitor Tablets (Roche). Cells were lysed by sonication . After sonication, DNase I and MgCl2  were added, and the suspension was stirred for 30 min at 4 °C. The lysate was centrifuged at 18,000 rpm for 50 min, and the cell-free extract was loaded onto a 20-mL column of Ni-NTA Superflow resin (Qiagen) equilibrated in lysis buffer. The resin was washed with 400 mL of wash buffer , and the protein was eluted using a linear gradient ranging from 20 mM to 250 mM imidazole in 50 mM NaH2PO4 and 300 mM NaCl (pH 8.0). S100A4-containing fractions were pooled and concentrated using a centrifugal 3000 molecular weight cutoff filter unit (Millipore). The protein was further purified using a Superdex 75 gel-filtration column equilibrated in 10 mM Hepes, 20 mM NaCl, and 2 mM DTT (pH 7.5) buffer. Pure S100A4-containing fractions eluted from the column were frozen in aliquots and stored at − 80 °C. Expression and purification of M111 and M200 were similar to those for S100A4, except that the buffer used for the Ni-NTA column contained 500 mM NaCl for M200 and the final gel-filtration column (and subsequent protein storage) contained 100 mM NaCl (for M111) or 500 mM NaCl (for M200), respectively, and lacked DTT. Uniformly 15N-labelled S100A4 was expressed in 2× M9 minimal medium using 4 g L− 1 glucose and 1 g L− 1 15NH4Cl. Uniformly 15C,15N-labelled M111 was expressed in 2× M9 minimal medium using 2 g L− 1 [13C]glucose and 1 g L− 1 15NH4Cl. Labelled proteins were purified following the same protocols as for unlabelled proteins.Recombinant wild-type and mutant forms of S100A4 were expressed and purified from ITC data were collected using a VP-ITC microcalorimeter  and analysed by fitting to a single-site binding equation or a two-site binding equation using MicroCal Origin software.− 1 cm− 1 (M200 and S100A4) and 1490 M− 1 cm− 1 (M111), based on their tyrosine content . Myosin fragments were measured in 0.5 M NaCl buffer, and care was taken to correct for the small contribution from turbidity. Protein concentrations are stated in terms of their monomeric polypeptide chain concentration. Under the conditions used for kinetic analysis, S100A4 is predominantly a dimer,N concentrations were based on fluorescein absorbance . Other peptide concentrations (M32 and M16: > 95% purity) were based on their dry weight concentration. Quin-2 concentrations were calculated from ɛ354 = 5000 M− 1 cm− 1 in the absence of Ca2+.2+ concentration, Fura-2 fluorescence excitation spectra were measured in the same cuvette using the 2-mm path for excitation to minimise inner filter effects.Absorption spectra were recorded using a Cary 50 spectrophotometer . Protein concentrations were calculated from their absorbance at 280 nm, using molar coefficients of 2980 MCD spectra were measured on a JASCO J-715 spectropolarimeter equipped with a JASCO PTC-348WI temperature-controlled unit at 0.05 mM protein solutions using a 1-mm pathlength microcuvette.Ivv + 2Ivh) and anisotropy (Ivv − Ivh/(Ivv + 2Ivh)), corrected for the g factor (hh/hv), were performed using an Excel spreadsheet. Competitive equilibrium binding data were analysed by an iterative fit to a single-site model using the Excel Solver routine (Microsoft) in which the Kd for M32 was a free parameter, while that for F-M16N was fixed at 2 μM. The latter was determined by a fit to a hyperbola to an independent titration in the absence of M32 (N] = 0.1 μM ≪ Kd). Note that the small correction for the emission intensity weighting to the observed anisotropy was not applied to these competitive titrations as the error will tend to cancel.Fluorescence spectra and anisotropy measurements were recorded using an SLM 8000 spectrofluorimeter . Fluorescein anisotropy measurements were carried out in L-format using a 515-nm emission filter. The four combinations of vertical and horizontal polarisers  were each recorded for 30 s, and then the initial setting (vv) was re-read to check for signal stability. Calculations of the total fluorescence  with a dead time of 1.5 ms.A clone of human epidermoid carcinoma A431/SIP1 cells,FRAP was carried out using a custom-built total internal reflection fluorescence (TIRF) apparatus based on a Zeiss 135 TV Axiovert microscope.2, and 5% D2O buffer (pH 6.1). DTT (2 mM) was added to the samples containing S100A4. Titration experiments were carried out at 0.2 mM S100A4 using 10 mM peptide solutions. Spectra for M111 backbone resonance assignments were measured using 0.4 mM protein solution. NMR spectra were obtained at 298 K using Bruker AVANCE DRX 600 or AVANCE DRX 800 spectrometers, which are both equipped with cryoprobes. Proton chemical shifts were referenced to external 2,2-dimethyl-2-silapentane-5-sulfonate. The 15N and 13C chemical shifts were referenced indirectly using recommended gyromagnetic ratios.α, and Cβ resonances.All NMR samples were prepared in 20 mM 4-morpholineethanesulfonic acid, 20 mM NaCl, 5 mM CaCl2, 0.1 mM CaCl2, 10 mM imidazole, and 5 mM NaH2PO4 (pH 7.0), with or without 14 μM S100A4, to give a final concentration of 7 μM M200. Following dilution, 5 μl of the mixture was applied to a grid with a thin carbon film supported by a holey carbon film, and the grid was negatively stained using 1% uranyl acetate. For metal shadowing, the stock solutions of the M200 and M111 tail fragments were diluted with 0.5 M ammonium acetate solution to give a final concentration of 5 μM protein. This was mixed with an equal volume of glycerol, and the resulting mixture was sprayed onto freshly cleaved mica then rotary shadowed with platinum at an angle of 6°.− 1). The lengths of nonoverlapping well-shadowed molecules were measured as described by Burgess et al.For negative staining, the stock solution of the M200 tail fragment was diluted with a solution consisting of 0.1 M Na-acetate, 3 mM MgCl


Here, we review the mechanisms through which Ca2+ regulates mitochondrial ATP synthesis. We focus on cardiac myocytes and pancreatic β-cells, where tight control of this process is likely to play an important role in the response to rapid changes in workload and to nutrient stimulation, respectively. We also describe a novel approach for imaging the Ca2+-dependent regulation of ATP levels dynamically in single cells.Stimulation of mitochondrial oxidative metabolism by Ca We discuss this form of regulation in detail here, taking as examples two cell types with contrasting energetic needs, the cardiac myocyte and the pancreatic β cell.Mitochondria are the major site of oxidative metabolism, and hence ATP synthesis, in eukaryotic cells. Thus, whereas the glycolytic metabolism of glucose generates 2 ATP molecules, 36–38 are generated by the reactions of the citrate cycle and the oxidation of the resulting NADH and FADH22+ has emerged in recent years as a further important means of controlling this vital aspect of cell function. Mammalian mitochondria have a huge capacity for Ca2+ uptake and for many years it was thought that these organelles served as mobilisable intracellular reservoirs of Ca2+ ions 2+ content was low and that increases in cytosolic free Ca2+ ([Ca2+]c) in response to extrinsic agents , were likely to provoke increases in intramitochondrial free Ca2+ ([Ca2+]m) concentrations. Thus, based on the properties of a group of key intramitochondrial dehydrogenases (later characterised in some detail – see the following sections) 2+ ions from the cytosol increased [Ca2+]m from ∼0.1 to 10 μM or more. The consequent activation of oxidative metabolism would then provide an increased supply of reducing equivalents to drive respiratory chain activity and ATP synthesis. Export of mitochondrial ATP in exchange for ADP was anticipated then to meet an increased ATP demand to fuel energy-requiring processes in the cytosol such as ion pumping, contraction, exocytosis etc. The biochemical analyses  and that the free concentrations of the ion increased within mitochondria in response to Ca2+-mobilising agonists. Subsequent refinements of these approaches, including the use of GFP-based probes, highlighted the importance of close contacts between the endoplasmic reticulum and mitochondria in the uptake of Ca2+ by the latter 2+ increases stably elevate mitochondrial [ATP] Mitochondrial oxidative metabolism has long been recognised as subject to complex regulation by several factors, notably the concentrations of ADP and substrate(s) ses e.g.  upon whi32+ transport proteins were not purified or cloned, and that use of the inhibitors of these pathways is problematic in living cells. However, recent work has very likely identified the proteins responsible for both the Ca2+ influx and Ca2+ efflux pathways in mitochondria (see below).An impediment to progress in this field has been that the mitochondrial Ca2+]c are relayed to the mitochondria by the mitochondrial Ca2+ uniporter (MCU), resulting in activation of mitochondrial dehydrogenases and stimulation of ATP synthesis. Average [Ca2+]m is controlled by the activities of the MCU and the efflux pathway, the mitochondrial Na+/Ca2+ exchanger (mNCX). Ca2+ uptake is respiration-dependent, thus high rates of uptake can effectively lower the mitochondrial membrane potential (ΔΨm) . The other paper 2+ uptake, and purified MCU reconstituted in bilayers exhibited Ca2+ channel activity that was sensitive to RuR.Research into the mitochondrial Ca2+ efflux was enhanced upon over-expression of NCLX in HEK 293 cells and inhibited by CGP 371457, and that Ca2+ efflux was greatly reduced using an anti-NCLX siRNA.In 2010, Palty et al. 44.12+]c regulate FAD-GPDH  in permeabilized INS1 β cells Physiological levels of c, albeit in the supraphysiological range Pyrophosphatase activity in liver and heart mitochondria is inhibited by Ca2+, due to competition of CaPPi with MgPPi (K0.5 = 12 μM) i is expected to result in an increase in mitochondrial volume and activation of the respiratory chain.The ATP-Mg2+/Pitransporter (SCaMC1). This transporter is stimulated by extramitochondrial Ca2+ and may regulated the total content of adenine nucleotides inside mitochondria, and hence alter Ca2+ buffering. SCaMC1 is overexpressed in some cancer cell lines, and this has been proposed to contribute to the ability of the cells to withstand Ca2+ loads that would otherwise trigger opening of the mitochondrial permeability transition pore 2+ reportedly potentiates fatty acid oxidation to β-hydroxybutyrate Ca55.12+ uptake in the control of ATP synthesis in the heart.Oxidative phosphorylation in mitochondria provides over 90% of ATP production in the heart + and ADP/ATP. It was originally proposed that changes in the ADP/ATP ratio were the main regulator of ATP synthesis ATP production can potentially be regulated by several different mechanisms, such as increases in NADH/NAD2+, following the discovery of the activation by Ca2+ of mitochondrial enzymes, as discussed above, namely the matrix dehydrogenases PDH 2 resulting from Ca2+ activation of the dehydrogenases would increase ATP production; thus suggesting a parallel activation model of stimulation of contraction and ATP synthesis by Ca2+1F0ATP synthase (ATPase) may also be stimulated by Ca2+ in this setting This role was later found to be fulfilled by intramitochondrial Ca2+ transport into mitochondria occurs in the heart, and whether or not [Ca2+]m changes on a beat-to-beat basis in this organ, has remained controversial for some time. Based on work in isolated mitochondria, the MCU and mNCX pathways could certainly not respond quickly enough to the very rapid (ms) changes in [Ca2+]c that occur during excitation-contraction (EC) coupling. However, more recent work using isolated myocytes has suggested that mitochondrial Ca2+ transients do occur during EC coupling in neonatal and adult cardiac myocytes 2+ accumulation occurred over tens of seconds in cardiac myocytes 2+ spiking occurs in some species but not others remains unknown. However, there is agreement that the main role of MCU and mNCX under physiological conditions is to translate or decode cytosolic Ca2+ signals in the mitochondria, so that Ca2+ activation of mitochondrial metabolism occurs.Whether Ca2+ directly to mitochondria (measured with rhod-2 fluorescence) upon stimulation with caffeine , and of increasing the NADH autofluorescence signal, suggesting activation of the dehydrogenases. This was the first direct evidence that Ca2+ transfer directly from the SR to mitochondria is capable of activating oxidative metabolism In addition to the known proximity of mitochondria to the ER in a variety of cell types, there is now evidence for a direct physical coupling that persists during isolation of mitochondria .Activation of ATP synthesis in the heart by mitochondrial Ca2+ regulates substrate supply into mitochondria via the aspartate–glutamate carrier  and the malate–aspartate shuttle (see above). This newer mechanism has been proposed to act as a “gas pedal, supplying…substrate on demand” There is also evidence to suggest that extramitochondrial Ca2+]m and [ATP] was that although ATP has been measured in whole hearts, and now in animals and humans using non-invasive 31P-NMR  followed by recovery to higher than initial levels m before a time-dependent activation of mitochondrial oxidative metabolism by Ca2+ stimulated ATP synthesis c were much smaller than those in the mitochondrial matrix, again suggesting that even when [ATP]m does change, the cytosolic energy supply is very rapidly matched to the increased demand. The lag phase where [ATP]m falls before recovering, exactly matches the time course for [Ca2+]m to increase However, when we allowed cells to rest, then suddenly stimulated to contract in the presence of isoproterenol (“standing-start” experiments), [ATP]2+]mSimilarly it has been generally observed that under conditions of adequate oxygen consumption, NAD(P)H levels remain constant in beating hearts and myocytes, again highlighting that ATP supply and demand are extremely well-coupled. The only time NAD(P)H was observed to decrease was in similar experiments to the “standing start” experiments described above, where there was an initial drop in NAD(P)H levels before recovery, again being slightly preceded by an increase in [Ca2+ have joint role in regulating ATP production in myocytes? Balaban 2+]m is absolutely required to maintain ATP levels during continuous or large increases in energy demand is not yet known and the idea of [Ca2+]m being the only factor that is important physiologically in regulating ATP supply and demand is not universally accepted. Our own studies show that in rat cardiac myocytes when stimulation rate is increased from 0.2 Hz to 4 Hz, which presumably causes a large increase in ATP demand, there is very little change in [Ca2+]m unless an adrenergic agonist is also present 2+]c sufficiently that MCU is activated to increase [Ca2+]m.Do ADP and CaKM of ADP for stimulating respiration, and removing diffusion limits of ADP that occur in resting cells , intracellular [Na+] was 16 mM compared with 5 mM in control cells 2+]m and dehydrogenase activity since adding Na+ to isolated mitochondria shifts the activation curves for PDH and OGDH by Ca2+ to the right The mNCX has also been considered a target for cardioprotection in heart failure. Recent work from O’Rourke's group has shown that dysregulation of Na5.2KM glucose transporters (Glut2 in rodents) and glucokinase Insulin secretion from pancreatic β-cells is prompted by increases in blood glucose over the physiological range (4–8 mM), thanks to the expression of high + (KATP) channels on the plasma membrane 2+ influx into the cytosol via voltage-gated Ca2+ channels 2+]c then triggers exocytosis of insulin granules ATP-independent” effects of glucose, possibly involving the inhibition of AMP-activated protein kinase The metabolic configuration of β-cells is adapted to favour the complete oxidation of glucose by mitochondria 2+ influx with EGTA or through the inhibition of voltage-gated Ca2+ channels substantially inhibited glucose-induced ATP/ADP increases, implicating a role for Ca2+ influx in metabolic control In contrast to most tissues including the heart (see above), cytosolic ATP changes over a relatively wide range in β-cells 2+]m can be imaged in real time in single primary living β-cells using a mitochondrially targeted, adenovirally-delivered and GFP-based reporter  simultaneously with [Ca2+]c measured with the trappable small molecule, Fura Red  whilst amplifying the response as glucose levels rise – thus contributing to the well-known highly sigmoidal response of insulin secretion to blood glucose levels 2+]c and [ATP/ADP]c, using the genetically encoded fluorescent sensor Perceval c in response to high glucose, with Ca2+ entry into cytosol being the key factor for the second phase In light of the above limitations, we have recently established a technique whereby [CaFura Red . Importa2+]c leads firstly to the activation of a number of processes  and exploration of the potential therapeutic use of new information flowing from the identification of the molecular players in Ca2+ transport.There is no doubt that [CaNone of the authors declares any conflict of interest.


In this study we have further explored the SC-791 action on ion channels and heartbeat in Drosophila, which lacks cyclooxygenases and thus can serve as a convenient model to study COX-2-independent mechanisms of coxibs. Using intracellular recordings in combination with a pharmacological approach and utilizing available Drosophila mutants, we found that SC-791 inhibited voltage-activated K+ and L-type Ca2+ channels in larval body-wall muscles and reduced heart rate in a concentration-dependent manner. Unlike celecoxib and several other K+ channel blockers, SC-791 did not induce arrhythmia. Instead, application of SC-791 resulted in a dramatic slowing of contractions and, at higher concentrations, in progressively weaker contractions with gradual cessation of heartbeat. Isradipine, a selective blocker of L-type Ca2+ channels, showed a similar pattern of heart arrest, though no prolongation of contractions was observed. Ryanodine was the only channel modulating compound of those tested additionally that was capable of slowing contractions. Like SC-791, ryanodine reduced heart rate without arrhythmia. However, it could not stop heartbeat completely even at 500 µM, the highest concentration used. The magnitude of heart rate reduction, when SC-791 and ryanodine were applied together, was smaller than expected for independent mechanisms, raising the possibility that SC-791 might be interfering with excitation-contraction coupling in Drosophila heart.Recent findings suggest that modulation of ion channels might be implicated in some of the clinical effects of coxibs, selective inhibitors of cyclooxygenase-2 (COX-2). Celecoxib and its inactive analog 2,5-dimethyl-celecoxib, but not rofecoxib, can suppress or augment ionic currents and alter functioning of neurons and myocytes. To better understand these unexpected effects, we have recently investigated the mechanism of inhibition of human K Selective inhibitors of cycloxygenase-2 (COX-2) are important non-steroidal anti-inflammatory drugs (NSAIDs), widely prescribed for treatment of arthritis and acute pain. Four coxibs, celecoxib (Celebrex), etoricoxib (Arcoxia), rofecoxib (Vioxx), and valdecoxib (Bextra) were developed and marketed as NSAIDs with reduced gastrointestinal side effects. However, increased risk of cardiovascular side effects, including myocardial infarction, cardiac arrhythmias and stroke, led to eventual withdrawal of rofecoxib and valdecoxib in 2004 and 2005, respectively.+, K+ and Ca2+ channels Several studies have demonstrated that celecoxib can interact with various molecular targets, including cellular and enzymatic mechanisms other than cyclooxygenases. For instance, the drug inhibits carbonic anhydrases with nanomolar affinity + channels in fruit flies and mammals, reducing beating rate and inducing arrhythmia in Drosophila heart and cultured rat ventricular cardiomyocytes + and K+ channels in isolated rat retinal neurons with a strong suppression of spontaneous spiking activity v2.1 and Shab channels via modification of gating at lower concentrations and channel block at higher concentrations v7.5 (KCNQ5) cardiac channels in the isolated rat mesenteric artery myocytes and A7r5 rat aortic smooth muscle cells, while simultaneously inhibiting other currents v1.5, Kv4.3, Kv7.1, and hERG channels and to alter action potential duration in mouse and guinea pig cardiomyocytes We have previously shown that celecoxib can inhibit Kv2.1channels expressed in HEK-293 cells 50 of 4 nM and hCOX-1 with an IC50 of 114 μM v2.1 via gating modification, but, unlike celecoxib, it does not induce channel block. In this study we have examined effects of SC-791 on ion channels and heartbeat in Drosophila melanogaster. The fruit fly, a widely used model organism, apparently lacks cyclooxygenases + and L-type Ca2+ channels and reduced heart rate in concentration-dependent manner. Unlike celecoxib and several other K+ channel blockers, SC-791 did not induce arrhythmia. Instead, application of SC-791 resulted in a dramatic slowing of contractions and, at higher concentrations, in progressively weaker contractions with a gradual cessation of heartbeat.A surprising variety of coxibs' molecular targets requires further research to better understand possible risks associated with these important drugs. We have recently examined effects of SC-791 on human KDrosophila larval body-wall muscles: T-type and L-type Ca2+ currents + currents, and three voltage-activated K+ currents A; Shab channels, mediating relatively slow delayed rectifier IKS; and rapidly activating IKF with unknown molecular basis + currents, IA, IKS, and IKF  from a holding potential (HP) of −40 mV (to inactivate T-type Ca2+ channels) with Ba2+ ions as charge carrier  KF can be recorded from the Shab strain in the presence of 4-AP to block the IA; the IKF can be completely abolished by 200 μM Cd2+ in the external solution KS from the wild-type larvae. The IA can be recorded from wild-type animals with Cd2+ (to inhibit IKF) and 100 μM quinidine (to selectively inhibit the IKS) in the bath or from the Shab in the presence of Cd2+ only.Several ionic currents have been previously identified in  and IKF A, 2A. 2+ channels in a concentration-dependent manner with an IC50 of 54 μM for the peak currents  of 30 μM . Effects of exposure of heart preparation to 1 or 3 μM isradipine strongly resembled the effects of SC-791: a gradual decrease in heart rate with decreasing amplitude of contractions , quinidine (blocks Kv2 and other channels), 4-AP (Kv1), heteropodatoxin (Kv4), linopirdine (Kv7), E-4031 (erg or Kv11), paxilline (KCa1), and apamin (KCa2). Some of these compounds , nor by slowing of L-type current activation -mediated Ca2+ release in RyR loss-of-function mutant flies dramatically slows the muscle contraction velocity and reduces heart rate to 25% of control 2+-ATPase (SERCA), which is responsible for replenishment of Ca2+ stores in the sarcoplasmic reticulum, can dramatically reduce heart rate and prolong contractions (unpublished observations); this observation is consistent with the previously published report on the crucial role of SERCA in Drosophila heartbeat 2+ were also able to alter heartbeat drastically, transforming individual contractions into prolonged spastic events with contracted heart muscle unable to relax for many seconds (unpublished observations). We therefore hypothesized that slowing of contractions in the presence of SC-791 could result from modulation of Ca2+ release from sarcoplasmic reticulum. As previous studies have described interaction of celecoxib with numerous ion channels and other targets, and this study showed that SC-791 is similar to celecoxib in this regard, and can modulate several dissimilar ion channels, it appeared possible that SC-791 could interact with Ca2+ release channels, specifically with the RyR Ca2+ release channel, a major component of excitation-contraction (EC) coupling in myocytes, which mediates Ca2+ release in response to activation of dihydropyridine (DHP) receptors L-type Ca2+ channels. We tested this hypothesis by examining effects of ryanodine on heartbeat separately and in combination with SC-791.Prolongation of contractions in the presence of SC-791 could not be explained by a reduced Caeed 3 ms C, i.e. 2+ release channels. At nanomolar concentrations ryanodine promotes opening of the RyR, at low micromolar concentrations it locks the RyR in the half-open state, and at concentrations around 100 μM it completely and irreversibly inhibits the RyR 2+ release is an important but not the essential aspect of EC coupling in the larval heart. This conclusion is consistent with the previously published observations Ryanodine is a highly selective modulator of the RyR CaBIAB. The BIC is derived from probability theory as a measure of total effect of two pharmacological agents acting via independent mechanisms. The equation for the zero-interaction effect is EBIAB = EA+EB−EA*EB, where EA and EB are the fractional single-agent effects of compounds A and B at given concentration versus 25±5% when SC-791 was applied after exposure to 100 µM ryanodine  Eonly via the RyR, then no additional effects of the SC-791 on heartbeat would be observed in the presence of 100 μM ryanodine, a saturating concentration almost completely inhibiting the receptor. Yet two such effects were observed: a further reduction in heart rate and slowing of contractions  2+ channels  and also the IKF (about 50% inhibition at 100 μM) 50 of 11 μM; its effects on heartbeat are very similar to those observed in Shab flies, with a decrease in heart rate, strong arrhythmia and an abrupt ceasing of beat observed at higher concentrations. In contrast, SC-791 inhibited Shab current with IC50s of 21 μM (the end of 500 ms pulses) and 74 μM (at 50 ms), and L-type current with an IC50 of 54 μM. The heart rate was reduced with an EC50 of 30 μM, but in a different manner: with a much slower development of the effect, without substantial arrhythmia, virtually irreversibly, and with prolongation of contractions.This study demonstrates that even highly structurally similar coxibs, celecoxib and SC-791, can dramatically differ in their effects on KS, whereas SC-791 – the L-type current and the IKS. This difference can explain the absence of arrhythmia in the presence of SC-791: celecoxib and SC-791 perturb the balance of ionic currents during action potential in dissimilar ways. By inhibiting K+ channels with a higher potency than Ca2+ channels, celecoxib might induce arrhythmia by compromising repolarization in the pacemaker cells without measurably affecting depolarization. SC-791, on the other hand, inhibited both Shab and L-type Ca2+ channels with a similar potency, which could result in less misbalanced depolarizing and repolarizing currents and, therefore, trigger no arrhythmia . Such scenario could also explain the differences in the character of heart pauses and stops: blocking repolarization, celecoxib often stops heartbeat abruptly (with a similarly sudden resumption of beating), whereas SC-791 stopped heartbeat after a gradual decrease in the beating rate and contraction amplitude, and without pauses.Thus, at lower concentrations (<50 μM) celecoxib mainly affects the I2+ channels are not important for the function of the pacemaker, while OPQ-type Ca2+ channels appear to be critical Drosophila, but celecoxib blocked similar channels in rat PC12 cells with lower affinity than the L-type Ca2+ channels 2+ channels, application of relatively low concentrations of isradipine ; similar observations of dysrhythmic heartbeat were made in the KCNQ knock-out flies Another explanation for the lack of arrhythmia in the presence of SC-791 could be that as the body-wall muscle and heart channellomes are not the same, the drugs could differentially inhibit other ion channels expressed in heart but not in body-wall muscles. Two such channels associated with arrhythmic heartbeat are KCNQ and erg. Experiments in mammalian experimental systems have demonstrated that celecoxib can inhibit these and many other channels 2+ channel blocker and an anti- hypertensive medicine, strongly suggest that inhibition of L-type Ca2+ channels is a major determinant of SC-791 action on heartbeat. However, what could be the mechanism behind the gradual weakening of contractions?Our experiments with high concentrations of isradipine, a selective L-type Ca2+ entering the cell during the action potential is essential not only for maintaining depolarization, but also for excitation-contraction coupling 2+ stores in the sarcoplasmic reticulum (SR). In the presence of a L-type Ca2+ channel blocker, reduced influx of Ca2+ through these channels will lower the overall Ca2+ load in the lumen during the action potential, which can itself compromise the contraction directly or via reduced Ca2+ induced Ca2+ release. Intracellular Ca2+ is subsequently removed back into the SR by SERCA, and ejected from the cell by the sodium-calcium exchanger or the plasma membrane calcium ATPase. As the share of luminal Ca2+ coming from the outside would be lower than in control, the Ca2+ removal mechanisms might disproportionally wash out Ca2+ coming from the SR, thus gradually depleting the sarcoplasmic Ca2+ store and impairing contractions. Interestingly, the notion that coxibs can interfere with muscle contractions by blocking L-type Ca2+ channels is supported by experiments with celecoxib in a different experimental system, rat aortic smooth muscle A7r5 cells, where celecoxib inhibits L-type Ca2+ channels while augmenting the repolarizing KV7.5 current. There, inhibition of L-type Ca2+ channels eliminates the arginine vasopressin (AVP)-induced Ca2+ spiking and causes concentration-dependent dilation of mesenteric arteries Reduction in the L-type current is more than just a decrease in a depolarizing current, because Ca2+ channels by SC-791 could explain most of the observed changes in heartbeat, it could not explain the slowing of contraction velocity. Our experiments with ryanodine suggest that prolongation of contractions in the presence of SC-791 might result from the altered EC coupling, that is, mainly, but not exclusively, from SC-791's interference with the RyR Ca2+ release pathway. Application of a saturating concentration of ryanodine (100 μM) could eliminate the rapid component of Ca2+ release from intracellular stores, dramatically slowing the contraction velocity and reducing the heart rate. When SC-791 was supplemented to the bath already containing ryanodine, the resultant changes in heartbeat were smaller than expected for independent mechanisms. Although reduction in the Ca2+ influx due to inhibition of L-type Ca2+ channels by SC-791 could in principle interfere with the RyR-mediated Ca2+ induced Ca2+ release and slow down the contractions, experiments with isradipine ruled out this possibility. Therefore, it is possible that SC-791 interacted with RyR directly or through one of its regulatory pathways 2+ release mechanism(s). As a side note, because the effects of ryanodine were also irreversible, such interaction might help explain the irreversibility of SC-791 action on heartbeat  using two-microelectrode voltage-clamp technique as described previously + currents contained (in mM): NaCl (77.5), KCl (5), MgCl2 (4), NaHCO3 (2.5), trehalose (5), sucrose (115), and HEPES (5), pH 7.1 2, 20 mM TEA, 1 mM 4-AP, and 100 µM quinidine. SC-791 was purchased from Calbiochem  and all other chemicals from Sigma-Aldrich Co. . Celecoxib was purified as described previously Voltage-activated KP<0.05, (**): P<0.01.All experiments were performed at 21°C on a temperature-controlled headstage using a circulating water bath from Forma Scientific. In figures, error bars indicate ± s.e.m. Statistical analysis was performed using a single factor ANOVA test. All values are means ± SEM; (*):