Ca2+ has also diverse regulatory effects on mitochondrial transporters and other enzymes. However, the consequences of these regulatory effects on mitochondrial oxidative phosphorylation (OxPhos) and ATP production, and the dependencies of these consequences on respiratory substrates, have not been investigated between the kidney and heart despite the fact that kidney energy requirements are second only to those of the heart. Our objective was, therefore, to elucidate these relationships in isolated mitochondria from the kidney outer medulla (OM) and heart. ADP-induced mitochondrial respiration was measured at different CaCl2 concentrations in the presence of various respiratory substrates, including pyruvate + malate (PM), glutamate + malate (GM), alpha-ketoglutarate + malate (AM), palmitoyl-carnitine + malate (PCM), and succinate + rotenone (SUC + ROT). The results showed that, in both heart and OM mitochondria, and for most complex I substrates, Ca2+ effects are biphasic: small increases in Ca2+ concentration stimulated, while large increases inhibited mitochondrial respiration. Furthermore, significant differences in substrate- and Ca2+-dependent O2 utilization towards ATP production between heart and OM mitochondria were observed. With PM and PCM substrates, Ca2+ showed more prominent stimulatory effects in OM than in heart mitochondria, while with GM and AM substrates, Ca2+ had similar biphasic regulatory effects in both OM and heart mitochondria. In contrast, with complex II substrate SUC + ROT, only inhibitory effects on mitochondrial respiration was observed in both the heart and the OM. We conclude that the regulatory effects of Ca2+ on mitochondrial OxPhos and ATP synthesis are biphasic, substrate-dependent, and tissue-specific.Mitochondrial dehydrogenases are differentially stimulated by Ca Even in the heart and skeletal muscle, where Ca2+ stimulation of mitochondrial respiration and ATP synthesis are well-recognized at low levels of [Ca2+]m [2+]m and the effect of such changes on mitochondrial respiration and ATP synthesis characterizing OxPhos activity [2+]m m ,60,61, cactivity . This maty [2+]m ,62,63,642+ can stimulate OxPhos in mitochondria isolated from the heart compared to that from the kidney OM of Sprague–Dawley (SD) rats. ADP-induced mitochondrial respiration was determined at different concentrations of CaCl2 in the presence of a variety of respiratory substrates. The results showed that mitochondrial respiration and ATP synthesis are dependent on both the respiratory substrates used and the concentration of CaCl2, and that this dependency is tissue-specific and biphasic with respect to the added [Ca2+]. These results strengthen our understanding of tissue-specific differences in mitochondrial bioenergetics and regulations, which, in turn, provide a basis for a deeper understanding of the roles of mitochondrial dysfunction in the pathogenesis of chronic diseases, such as hypertension. These results are also useful for building predictive mathematical models of mitochondrial bioenergetics and regulations in the heart and kidney, and to study the emergent properties of cardiac and renal metabolic systems.The present study was carried out to determine the extent to which mitochondrial respiration and ATP synthesis (OxPhos activity) is substrate- and tissue-dependent, and to determine the extent to which CaThe chemicals used in this study were purchased from Sigma–Aldrich  unless otherwise stated. Adult male Sprague–Dawley (SD) rats  were purchased from Envigo  and were housed in our animal facilities for at least a week for acclimation to the new environment before being used in this study. Rats were housed in individual cages maintained at 21 ± 2 °C with a 12:12-h of light–dark cycle, and were given water and food ad libitum. The Medical College of Wisconsin (MCW) Institutional Animal Care and Use Committee (IACUC: AUA00000851) approved the animal use and the experimental protocols described below.Mitochondria were freshly isolated from the ventricles of the heart and the OM of the kidneys of adult SD rats , as detailed in our recent study . Followi2PO4, 5 mM MOPS, 1 mM EGTA, and 0.1% BSA, with the pH adjusted to 7.15 with KOH. They were then minced, 2.5 mL of a 5 U/mL protease solution was added, and they were then homogenized for 40 s. The homogenate was first centrifuged twice at 8000× g for 10 min at 4 °C to remove the protease. The supernatant was then discarded, and the pellet was re-suspended in the IB to 25 mL and centrifuged at 700× g for 10 min. Then, the mitochondria-enriched supernatant was centrifuged at 8000× g for 10 min. The resulting mitochondrial-enriched pellet was re-suspended in IB and kept on ice. The mitochondrial protein content was measured using BSA as the standard with Biorad Quick Start Bradford Assay Kit . Depending on the protein content, the suspended pellet volume was adjusted to have a protein concentration of 12.5 mg protein/mL to allow comparison across groups or conditions without the confounding influence of differences in the concentration of mitochondria loaded into the chamber.Cardiac mitochondria were isolated via the differential centrifugation method, as described previously ,65,66,67g at 4 °C for 10 min, and the remaining supernatant was centrifuged at 12,000× g for 15 min. The resulting pellet was mixed well in IB and the volume adjusted up to 15 mL. This suspension was centrifuged again for a third time at 12,000× g for 15 min, and the final mitochondrial pellet was then transferred to a dark Eppendorf tube, and placed on ice to determine protein content, as described above, to enable adjustments of the final suspension volume to attain a protein concentration of 12.5 mg protein/mL IB.Renal OM mitochondria were isolated through the differential centrifugation method with a slight modification from that for the cardiac mitochondria . Briefly2 consumption (respiration) was measured using an Oxygraph-2k (O2k) high-resolution respirometer  with its DatLab 7 software used for data acquisition and analysis. 2 concentration in the KCl-enriched respiration buffer (RB) was equilibrated for more than 10 min with the air within the O2k chambers at 37 °C, until a steady stable signal was obtained at an O2 concentration of ~205 μM. The experiments were initiated at time t = 0 min with isolated mitochondria (0.1 mg protein/mL for heart and 0.2 mg protein/mL for kidney OM) suspended at 37 °C in 2 mL of RB containing (in mM) 130 KCl, 5 K2HPO4, 20 MOPS, 1 EGTA, and 0.1% BSA, at pH 7.2 adjusted with KOH. The mitochondrial suspension in the RB was continuously stirred (750 rpm) inside the O2k chambers, and chemicals were added to the chamber through the titanium injection port of the stoppers using Hamilton syringes.Mitochondrial Ot = 2 min. State 3 respiration was measured by adding 500 and 250 μM ADP for heart and kidney OM mitochondria, respectively, at time t = 4 min, and state 4 respiration was measured after phosphorylation of the added ADP to ATP. The functional integrity and intactness of isolated mitochondria on a given experimental day was determined using respiratory control ratio (RCR), defined as the ratio of maximum O2 consumption measured after the addition of ADP (state 3) to the state 2 O2 consumption .In the first set of experiments, state 2 respiration was initiated with the complex I substrates pyruvate + malate at saturating concentrations  at time l-carnitine + malate , and succinate + rotenone  at time t = 2 min, and ADP  at time t = 4 min, which initiates state 3 respiration proceeding to state 4 respiration after the conversion of the added ADP to ATP. To assess the effect of Ca2+ on mitochondrial O2 consumption, various concentrations of Ca2+  were added a minute after state 4 was reached. This was followed by the addition of ADP at the above concentration. O2 consumption rates  during different states of respiration were computed as the negative time derivative of O2 concentration measured in the closed O2k chambers and expressed as nmol of O2/min/mg of mitochondrial protein. Data were acquired at every 1 s, and 5 data point averages were made to compute the slope of the O2 concentration data using the DatLab 7 software version 7.4.0.4. The unique timeline protocol of 2 concentrations on mitochondrial respiration and ATP synthesis characterizing OxPhos activity.In the second set of experiments, the time-courses of states 2, 3, and 4 respiration were monitored by adding a variety of respiratory substrates  to isola2+ concentration ([Ca2+]free) in RB was controlled with 1 mM EGTA. The addition of the buffer components did not alter the total Ca2+ concentration  added to the buffer to achieve the desired [Ca2+]free. For a given [Ca2+]total, [Ca2+]free was calculated using the Ca-EGTA online calculator v1.3, along with constants from Schoenmakers’s Chelator from Fheuvenet et al. [2+]total and [Ca2+]free is given in 2+]free values are subjected to 5% uncertainty, due to a 1% uncertainty in the stability constants, or 0.01 pH unit uncertainty in the measured pH.Free Cat et al.  for 1 mM® spreadsheets of day-to-day experiments generated from the Datlab software. The programs performed the required calculations to calibrate absolute values and changes in variables from one state to another, including statistical analyses . The final data of a particular variable were expressed as mean ± standard error (SE) over at least 3 biological replicates of the same variable (n > 3), each measured with two technical replicates. Comparisons within and between groups were performed by ANOVA  with Tukey’s post hoc test for significance of means.All data were analyzed using computer programs written in MATLAB that access the raw data of specific variables from the Excel2 consumption rates  and free Ca2+ concentration ([Ca2+]free), the following empirical Equation (1) was applied to describe the state 3 JO2 − [Ca2+]free relationship:Ca is the apparent binding constant for Ca2+; V0 and Vmax are the basal and maximal state 3 respiratory rates, respectively; nH is the Hill coefficient for inhibitory term, which was set at an optimal value of 3 with several trials of the nonlinear regression fitting of the model to the data. The first term of this equation accounts for the stimulatory effect and the second term accounts for the inhibitory effect of Ca2+. V0 corresponds to state 3 JO2 when [Ca2+] is 0.To characterize the biphasic behavior of the relationship between mitochondrial O0, Vmax, KCa, peak Ca2+-stimulated state 3 JO2, and [Ca2+]free, necessary to achieve the peak Ca2+ stimulated state 3 JO2. The resulting residuals and Jacobian matrix were fed to the MATLAB function “nlparci” to calculate the 95% confidence interval for each of the parameters in Equation (1).Experimental data for each group (5 substrates and 2 tissues) were separately fitted to the above kinetic model using the MATLAB curve-fit function “lsqcurvefit” to estimate the values of V2+ stimulation of mitochondrial respiration and ATP synthesis characterizing OxPhos activity in the heart and kidney OM and the extent of their dependency on the choice of substrates. The first step was to measure the respiratory control ratio  with the PM substrates with saturated ADP concentration  to ensure the functional integrity of the isolated mitochondria, which averaged 10.40 ± 0.24 for the heart and 8.54 ± 0.25 for the kidney OM.First, we determined the substrate-dependent respiratory rates under different states using isolated heart and kidney OM mitochondria. Five different substrate combinations were used in the study, namely, PM, GM, AM, PCM, and SUC + ROT. After that, we determined the extent and the nature of Ca2 consumption rates (respiration) for the heart and kidney OM for different substrates , based on the timeline experimental protocol of 2+] on mitochondrial OxPhos (ADP-induced state 3 respiration and ATP synthesis). Importantly, the analysis of mitochondrial respiration with the second dose of ADP demonstrates that the effect of Ca2+ on mitochondrial state 3 respiration and ATP synthesis (OxPhos activity) is biphasic in the presence of all substrates studied, except for SUC + ROT, with a stimulatory effect at lower [Ca2+] and inhibitory effect at higher [Ca2+]. This is characterized by increased peak state 3 respiration and decreased state 3 duration of ADP phosphorylation at low [Ca2+] and decreased peak state 3 respiration and increased state 3 duration of ADP phosphorylation at high [Ca2+]. Interestingly, for the SUC + ROT substrate in both the heart and kidney OM mitochondria, and for the PCM substrate in the kidney OM mitochondria, only an inhibitory effect of Ca2+ was observed for all concentrations of CaCl2 added to the mitochondrial suspension.2+ addition). 2+. 2 consumed) are shown in The bar plots in 2+ effects on the heart (upper panel) and kidney OM (lower panel) mitochondrial state 2 respiration (leak respiration). Heart mitochondria exhibited no change in state 2 respiration at different concentrations of Ca2+ in the presence of PM substrates. In contrast, kidney OM mitochondria exhibited a progressive but not significant increase for added Ca2+ concentrations between 0 and 700 µM. Among the different substrate combinations studied, state 2 respiration rates with GM and AM substrates show small Ca2+-stimulated increases in both heart and kidney OM mitochondria over the range of the Ca2+ concentrations studied. This is consistent with the fact that these substrates are involved with Ca2+-sensitive metabolic pathways due to the higher activation of alpha-ketoglutarate dehydrogenation (AKGDH) compared to pyruvate dehydrogenase (PDH). The effects of Ca2+ concentration on state 2 respiration rates are relatively small in the presence of the substrate combinations PCM and SUC + ROT in both heart and kidney OM mitochondria, indicating that state 2 respiration is independent of Ca2+ in the presence of fatty acid-based or complex II-based substrates.2+ on mitochondrial state 3 respiration in the heart (upper panel) and kidney OM (lower panel). 2+ differentially regulates mitochondrial state 3 respiration and that this regulatory effect is both substrate-dependent and tissue-specific. In cardiac mitochondria with PM substrates, an ~18% increase in maximal state 3 respiration (JO2) was observed with 400 μM [Ca2+]total . For the same substrates, the effect of Ca2+ was significantly larger in kidney OM mitochondria, with an ~52% increase in maximal state 3 JO2 achieved with only 250 μM [Ca2+]total . Interestingly, with GM substrates, ~105% and ~77% increases in maximal state 3 JO2 were observed with 700 μM and 400 μM [Ca2+]total  for heart and kidney OM mitochondria, respectively. In contrast, with AM substrates, only 250 μM [Ca2+]total  was sufficient to stimulate ~80% increase in maximal state 3 JO2 in both heart and kidney OM mitochondria. The effect of Ca2+ on state 3 JO2 with PCM substrates was insignificant in cardiac mitochondria, but quite prominent (~51% increase) in kidney OM mitochondria. Finally, 2+ consistently resulted in an inhibitory effect on the state 3 respiratory rate.2+] has a stimulatory effect on state 3 respiration, whereas a large increase in [Ca2+] has an inhibitory effect. Moreover, the concentrations of Ca2+ necessary for this switching from the stimulatory to inhibitory effects are both substrate-dependent and organ-specific. For example, in the presence of PM substrates, state 3 respiration in heart mitochondria peaked at 400 µM [Ca2+]total, as compared to 250 µM [Ca2+]total in kidney OM mitochondria. When using PCM as a substrate combination, the addition of Ca2+ induced an increase in state 3 respiration in kidney OM mitochondria for concentrations between 0 and 400 μM. However, this effect was negligible in heart mitochondria. When using SUC as a substrate in the presence of ROT, the addition of Ca2+ induced a partial inhibition of state 3 respiration for most of the [Ca2+] studied in heart mitochondria, but only for the highest [Ca2+] studied in kidney OM mitochondria. Our results also show that, except for the substrate combination SUC + ROT, the effects of varying [Ca2+] on mitochondrial state 3 respiration and ATP synthesis are more prominent in kidney OM mitochondria than those in heart mitochondria.Indeed, for all complex I substrates studied, , a biphasic relationship is observed in which a small increase in [Ca2+ on the respiratory control ratio  for different substrate combinations in isolated mitochondria from the heart (upper panel) and kidney OM (lower panel). The RCR data show a biphasic pattern similar to that for state 3 respiration  in the presence of different substrate combinations for both heart (upper panel) and kidney OM (lower panel). These data suggest that small increases in [Ca2+] resulted in higher state 3 JO2 peaks and shorter state 3 durations, whereas large increases in [Ca2+] resulted in lower state 3 JO2 peaks and longer state 3 durations as compared to values with no [Ca2+] added. In general, the duration of state 3 JO2 is inversely proportional to peak state 3 JO2. This is due to the fact that if the respiration is slower, it would take more time for mitochondria to convert the added ADP to ATP. The white strips with upward arrows in 2+ greatly inhibited state 3 JO2, preventing an accurate determination of the state 3 duration.2+]free and state 3 JO2 with different substrate combinations. The data in 2 data in 2+concentrations  was converted to the free Ca2+ concentration ([Ca2+]free) present in the extra-mitochondrial buffer space, based on the Ca2+ chelator calculator [2+-stimulated state 3 JO2 and [Ca2+]free required for achieving this peak Ca2+ stimulated state 3 JO2 for ATP synthesis than those inferred from the data in 2 − [Ca2+]free curves for the heart and kidney OM mitochondria are almost the scaled versions of each other for all substrate combinations studied, except for the substrate combinations of GM and AM, where the heart mitochondria curve showed a steeper decline at higher [Ca2+]free. This indicates that heart mitochondria are more sensitive to Ca2+ inhibition when GM or AM are provided as substrate combinations.A detailed data analysis using nonlinear regression was performed to determine the relationships between free, required to achieve this peak, along with the corresponding confidence intervals (represented as error bars) are shown in 0 values (state 3 JO2 at [Ca2+]free = 0) are highest for the substrate combinations SUC+ROT and PM for both heart and kidney OM mitochondria. In general, V0, Vmax, and peak Ca2+-stimulated state 3 JO2 values for heart mitochondria are several folds higher than those for kidney OM mitochondria. However, the apparent binding constants  and [Ca2+]free required for achieving the peak Ca2+ stimulated state 3 JO2 for mitochondria from heart and kidney OM are of the same order of magnitude.The estimated parameter values, including V2+ and Mg2+ . These results further our understanding of tissue-specific differences in mitochondrial bioenergetics and regulations under physiological and pathophysiological conditions. These results are also useful for building predictive mathematical models of mitochondrial bioenergetics and regulations to investigate the emergent properties of cardiac and renal metabolic systems.The heart is challenged by rapid changes in energy demands from a resting to an exercise state, and the mitochondria of myocytes face great fluctuations of Ooduction ,36,76. Toduction ,79. Yet,ic state ,79. To e2 consumption rates  for isolated heart and kidney OM mitochondria following the timeline protocol of 2 addition. The OCR (states 2 and 3), state 3 durations (sec), RCR, and PO ratios were derived from the dynamic data shown in 2 addition. The state 2 respiration was significantly higher in the presence of the FADH2-linked substrate SUC in the presence of ROT (SUC + ROT), in comparison to the NADH-linked substrates provided to heart and kidney OM mitochondria , while Ca2+ efflux occurs through the Na+-dependent electrogenic 3Na+/Ca2+ exchanger (NCE) and/or Na+-independent electroneutral Ca2+/2H+ exchanger (CHE) at the inner mitochondrial membrane (IMM) m is controlled by complex mitochondrial Ca2+ buffering mechanisms [2+]m, a dynamic balance is required between the MCU-based Ca2+ influx, the NCE- and/or CHE-based Ca2 + efflux, and Ca2+ buffering within the mitochondrial matrix [2+]c to [Ca2+]m and the influence of [Ca2+]m on the TCA cycle was evaluated in the presence of different ionic concentrations, such as Na+ and Mg2+, in the buffers by m is crucial for regulating mitochondrial energy metabolism in response to changes in workload, the Ca2+ buffering level in the mitochondrial matrix is critical to reaching the desired steady-state [Ca2+]m level. Steady-state [Ca2+]m controls mitochondrial ATP production via Ca2+ regulation of the mitochondrial DH enzymes that activate mitochondrial metabolic machinery and, in the process, stimulate ATP production to meet the cellular energy demands [2+]m controls the mitochondrial energy metabolism without considering the role of cytoplasmic Ca2+ ([Ca2+]c) on the mitochondrial substrate transport that ultimately impacts the mitochondrial energy metabolism.The three consequential functional phases of mitochondrial Cae (mPTP) ,29,94,95 in vivo ,97. Sinc demands ,98. Ther2+]m, are responsible for the regulation of OxPhos [2+ to regulate metabolic activities [2+]c stimulates OxPhos to respond to increased cellular workload by adjusting the rate of pyruvate transport from the cytosol to the mitochondria [2+]c controls ~85% of OxPhos rates when pyruvate is provided as a substrate, and the remaining ~15% is regulated by [Ca2+]m. The mitochondrial glutamate–aspartate carrier , an essential component of MAS with a regulatory Ca2+-binding site facing the inter membrane space (IMS), promotes Ca2+ sensitivity of the MAS [2+]c and not the [Ca2+]m  on mitochondrial state 3 respiration in heart and kidney OM in the present of the different respiratory substrate combinations as mitochondrial fuel. Our results suggest that Ca2+ differentially regulates mitochondrial state 3 respiration, and that this differential regulation is both substrate-dependent and organ-specific  may vary in the extent of MAS activation and MCU-mediated Ca2+ uptake.Several studies using MCU knockout mice showed that deleting MCU did not alter OxPhos, suggesting that other factors, in addition to m . In contransport , Aralar ransport ,111. Thespecific . Kidney 2+ and metabolic substrates in a manner similar to that of the heart, or whether they respond differently. The free Ca2+ level in cardiac mitochondria is 50–100 nM at rest, but can increase to >1µM during exercise m. The differential regulation of these DH enzymes by Ca2+ lies in their different binding activation constants (KCa) for Ca2+ and, therefore, these are sensitive to different ranges of [Ca2+] [2+ in the IMS (KCa = 0.1 µM) [2+]m are the pyruvate dehydrogenase (PDH) phosphatase , isocitrate dehydrogenase (ICDH), and oxoglutarate dehydrogenase (OGDH). PDH and OGDH are the primary DH enzymes responsible for pyruvate and α-ketoglutarate oxidation, respectively. High mitochondrial [Ca2+] stimulates the activity of PDH phosphatase, which further dephosphorylates PDH and increases its activity [Ca) ranges from 5 to 50 μM depending on the ATP/ADP ratio [2+ (KCa = 1 μM). ICDH and OGDH enzymes become more sensitive to Ca2+ with a decrease in the NADH/NAD+ ratio [Ca) makes ICDH less relevant since inhibitory effects occur before [Ca2+] reaches the KCa for ICDH. This may explain the relatively moderate activation effects of Ca2+ with PM and PCM as substrate combinations in heart mitochondria. The differences in the Ca2+ binding activation constants (KCa) also explain the reason behind the substrate- and organ-dependent [Ca2+] required to reach maximum respiration. Interestingly, Ca2+ also increases the MAS activity [2+], as summarized in 2+ on DH enzymes in the TCA cycle and the activation effects of extra-mitochondrial Ca2+ on MAS activity  . FAD-gly 0.1 µM) . The othactivity . The affDP ratio . OGDH, aD+ ratio ,119. Theactivity ,120 thatactivity .2+ upon mitochondrial state 3 respiration are determined by the factors through which the components of the TCA cycle reactions are activated  .It is evident that the effects of Cactivated ,23. For  of ICDH . When NA via MCU ,105, DH MCU [2+] . Therefo2+ and metabolism is essential for maintaining cellular energy homeostasis. It starts with maintaining ΔΨm, which is essential for balancing cellular Ca2+ homeostasis [m is responsible for maintaining the fine balance between the relative rates of Ca2+ uptake and efflux responsible for maintaining the equilibrium between [Ca2+]c and [Ca2+]m. This, in turn, prevents [Ca2+]m from reaching levels that can induce an irreversible disruption of mitochondrial structure and function. As clearly seen in the present studies, the effects of Ca2+ on mitochondrial state 3 respiration in both heart and kidney OM are biphasic. A reduction in mitochondrial state 3 respiration in both heart and OM , resulting in decreased mitochondrial state 3 respiration [2+ overload can result in a reduction of the ETC activity and compromised ATP production capacity through the respiratory uncoupling and disruption of the IMM [m and the inhibition of mitochondrial dehydrogenases [2+]free concentrations >5 µM [2+]free in the buffer was around 1 µM, which may not be enough to attain a matrix [Ca2+]free of >5 µM to initiate the mPTP opening. In addition, mPTP opening should significantly affect mitochondrial respiration under a leak state  [2+]free upon mitochondrial state 3 respiration. Second, it has been proposed that Ca2+ and its derivatives could inhibit mitochondrial enzymes. For example, Lai et al. free curve to a kinetic model (Equation (1)) to provide a clearer representation of the effects of different [Ca2+]free on mitochondrial state 3 respiration. As seen in 2+]free can be well described by an equation that accounts for both stimulatory and inhibitory effects of [Ca2+]free on state 3 OCR. It should be noted that only one apparent KCa value and mitochondrial matrix binding site was considered for data analysis since differentiating the activation binding constant  from the inhibition constant did not improve the fits.The mitochondrial state 3 respiration rates obtained in the present studies were analyzed by fitting each state 3 OCR − free used in experiments, which suggests a biphasic relationship between state 3 OCR and [Ca2+]free.The estimated Vntervals , indicatv et al. , who exabination . Importa2+  than that of the kidney OM mitochondria. The effects of varying [Ca


Increased Ca2+ is exported by the Na+–Ca2+ exchanger (NCX) and plasma membrane Ca2+–ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1–4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4–8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.Intracellular Ca The data show that intracellular Ca2+ signaling participates in sensory signal transduction sequences that produce dentinal pain or promote dentinogenesis. Therefore, in odontoblasts, the regulation of [Ca2+]i is important for regulating cellular function.Odontoblasts play critical roles in the generation of dentinal sensitivity (known as the odontoblast hydrodynamic receptor model ) and denchannels . This intoblasts ,8. ATP rsitivity . In additoblasts  to maint2+ concentrations are tightly controlled by multiple plasma membrane/intracellular Ca2+ transport proteins. Transport mechanisms are mediated by transmembrane Ca2+ influx and intracellular Ca2+ mobilization, as well as Ca2+ extrusion mechanisms. The [Ca2+]i increase induced by Ca2+ influx from the extracellular space or Ca2+ release from Ca2+ stores subsides and returns to resting [Ca2+]i via the sarco/endoplasmic reticulum Ca2+–ATPase (SERCA)-mediated uptake of Ca2+ into Ca2+ stores or Ca2+ extrusion to the extracellular space [2+]i is catalyzed by NCX or plasma membrane Ca2+–ATPase (PMCA) i measurement, enzymatically treated dental pulp slices were plated onto a culture dish, soaked in alpha-minimum essential medium containing 5% horse serum and 10% fetal bovine serum (Life Technologies Japan), and maintained at 37 °C in a 5% CO2 incubator. [Ca2+]i in odontoblasts located on the periphery of the primary cultured dental pulp slices was measured within 24 h of isolation. In a previous study, cells isolated using the same protocol were validated as odontoblasts with positive immunofluorescence for odontoblast marker proteins, dentin matrix protein-1 (DMP-1), dentin sialoprotein, and nestin within 24 h of isolation i was determined using the fluorescence ratio (RF340/F380) of F340 to F380, which is represented as F/F0 units; the RF340/F380 value (F) was normalized to the resting value (F0). The F/F0 baseline was arbitrarily set at 1.0. All experiments were carried out at room temperature (25 ± 1.0 °C). Hypotonic, isotonic, high-pH, and Krebs solutions and those containing PMCA inhibitors were applied by superfusion using a rapid gravity-fed perfusion system . It is worth noting that these solutions included 2.5 mM extracellular Ca2+. At the start of the experiment using caloxin 1b1, the odontoblasts in the recording chamber were perfused with a standard extracellular solution or isotonic solution. By changing the standard extracellular solution or isotonic solution to high-pH or hypotonic solution, respectively, with caloxin 1b1 , followed by changing the solution into a standard or isotonic solution, we analyzed hypotonic- or high-pH-mediated increases and extrusion of [Ca2+]i. For the experiments using CE, the odontoblasts were perfused by standard extracellular or isotonic solution with CE, and the solution was changed to high-pH or hypotonic solution with CE, followed by changing the solution to a standard extracellular or isotonic solution with CE . For the control, the cells were perfused by standard extracellular or isotonic solution, and the solution was changed to high-pH or hypotonic solution, followed by changing the solution to a standard extracellular or isotonic solution.Odontoblasts in dental pulp slices and HOB cells were loaded with 10 μM fura-2-acetoxymethyl ester   and 0.1%2. To determine the effects of PMCA activity on mineralization, HOB cells were cultured in mineralization medium without (as control) or with PMCA inhibitors CE (10 µM) or caloxin 1b1 (100 µM) for 28 days. During the 28-day culture period, mineralization media with or without PMCA inhibitors was changed twice a week i increases, we measured [Ca2+]i and analyzed the extrusion efficiencies following the application of hypotonic or high-pH (pH 8) solutions with or without non-selective PMCA inhibitors  i2+, hypotonic stimulation transiently increased [Ca2+]i in both the absence  to acutely isolated rat odontoblasts  in the presence of 10 μM CE  of both stains were normalized to the mean intensities of adjacent areas without cells (I0). We compared the mineralization levels in HOB cells cultured in mineralization medium for 28 days in the presence or absence of PMCA inhibitors. The application of 10 µM CE  and von Kossa (right) staining A,C were 10 µM CE A,B or 1010 µM CE C,D to miWe analyzed the changes in the mRNA levels of non-collagenous extracellular matrix proteins, such as odontoblast markers, dentin matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP), following 3 days of HOB cell culture under physiological or high-pH conditions as well as isotonic or hypotonic conditions with or without 10 µM CE. High-pH stimulation significantly increased the mRNA levels of DMP-1 A and DSPWe further observed the presence of the ubiquitously expressed PMCA1 ,14 protePMCA4 gene causes male infertility i in the resting state [2+]i has been shown to be between pCa 6.4 and 6.6 in rat incisor odontoblasts [2+]i below 100 nM in odontoblasts under resting conditions. In the present study, we also found that intracellular Cathan NCX ,13,37. Tng state ,37. The toblasts ,39. Thus2+]i after hypotonic or high-pH stimulation-induced [Ca2+]i increases in both rat and human odontoblasts, showing that PMCA extrudes intracellular Ca2+ increased by membrane stretch or high-pH stimuli. Thus, PMCA participates in the maintenance of low [Ca2+]i not only during rest but also after intracellular signaling events by stimuli to the dentin surface. Hypotonic and direct mechanical stimuli  activate Ca2+ influx via mechanosensitive ion channels, including TRP vanilloid (TRPV), TRP ankylin 1 (TRPA1), and Piezo1 channels, increasing [Ca2+]i in rat and mouse odontoblasts [2+ release-activated Ca2+ channels that induce store-operated Ca2+ entry in odontoblasts [2+ influx via high-pH- and mechanosensitive-TRP channels and NCX plays an important role in regulating [Ca2+]i homeostasis and driving cellular functions in rat, mouse, and human odontoblasts, including the induction of reactionary dentin formation i was driven by PMCA activity; however, we did not observe any significant differences in levels of DMP-1 and DSPP mRNA under hypotonic conditions as compared to under isotonic conditions. Interestingly, PMCA inhibitors did not affect their mRNA levels in the normal-/high-pH and isotonic/hypotonic extracellular conditions, whereas PMCA activity mediated mineralization under physiological conditions by odontoblasts. Although further studies are needed, these results indicate that Ca2+ extrusion processes via PMCA and the secretion of extracellular matrix proteins are regulated by independent signaling pathways. In the mineralization assay with Alizarin red and von Kossa staining, the PMCA inhibitors CE and caloxin 1b1 significantly decreased the mineralization levels. Mineralization in odontoblasts cultured in mineralization medium without PMCA inhibitor (as a control experiment) mimicked the physiological or developmental conditions of dentinogenesis. Thus, these results also demonstrate that CaOB cells , it is c2+]i homeostasis in odontoblasts. PMCA was found to play critical roles not only in physiological dentin formation but also in the pathological tertiary (reactionary) dentin formation induced by multiple external stimuli applied to the dentin surface, as well as by the application of high-pH dental materials on the dentin surface. It has been reported that the activation of neural activity in the brain causes a rapid rise in extracellular pH by removing H+ from the external space. This has been proposed to arise from neuronal PMCA activity, which exchanges internal Ca2+ for external H+ [2+-H+ exchanging ability of PMCA in odontoblasts, compounds that alter PMCA activity may be good candidates for inclusion in dental materials to promote dentinogenesis.In conclusion, this study demonstrates that PMCA contributes to the maintenance of [Caernal H+ . Therefo


Treatment failures of acute myeloid leukemia (AML) have been attributed to the persistence of leukemia stem cells (LSCs), which are refractory to conventional treatments. Venetoclax (VEN), currently FDA-approved in combination with low-dose cytarabine or hypomethylating agents, is highly effective in inducing disease remission in patients with de novo AML; however, most of these patients eventually relapse; thus, novel VEN combinations are urgently needed. In this regard, 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets AML cells, including LSCs. We demonstrate that VEN and 8-Cl-Ado cooperate in targeting ribosomal RNA synthesis and mitochondrial metabolism in LSCs, thereby decreasing LSC survival. Given the emerging concept that LSC behavior is strongly associated with protein synthesis regulation and mitochondrial metabolism, our results suggest that the VEN/8-Cl-Ado combination is a promising regimen for the treatment of patients with relapsed AML.It is known that 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets leukemic stem cells (LSCs). In a phase I clinical trial with 8-Cl-Ado in patients with refractory or relapsed (R/R) AML, we observed encouraging but short-lived clinical responses, likely due to intrinsic mechanisms of LSC resistance. LSC homeostasis depends on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. We recently reported that 8-Cl-Ado and the BCL-2-selective inhibitor venetoclax (VEN) synergistically inhibit FAO and OXPHOS in LSCs, thereby suppressing acute myeloid leukemia (AML) growth in vitro and in vivo. Herein, we report that 8-Cl-Ado inhibits ribosomal RNA (rRNA) synthesis through the downregulation of transcription initiation factor TIF-IA that is associated with increasing levels of p53. Paradoxically, 8-Cl-Ado-induced p53 increased FAO and OXPHOS, thereby self-limiting the activity of 8-Cl-Ado on LSCs. Since VEN inhibits amino acid-driven OXPHOS, the addition of VEN significantly enhanced the activity of 8-Cl-Ado by counteracting the self-limiting effect of p53 on FAO and OXPHOS. Overall, our results indicate that VEN and 8-Cl-Ado can cooperate in targeting rRNA synthesis and OXPHOS and in decreasing the survival of the LSC-enriched cell population, suggesting the VEN/8-Cl-Ado regimen as a promising therapeutic approach for patients with R/R AML. Chemotherapy induction therapy followed by consolidation therapy with allogeneic stem cell transplantation  is a treatment strategy with the highest chance for long-term survival for the majority of patients with acute myeloid leukemia (AML). However, older and/or unfit patients and those with other medical comorbidities are frequently not candidates for this treatment program. In addition, disease relapse still occurs in a relatively large percentage of patients undergoing alloSCT. Recently, several molecularly targeted therapies have been approved by the US Food and Drug Administration (FDA) for the treatment of AML ,2,3,4. ATreatment refractoriness or disease relapse of AML patients is broadly attributed to the persistence of the treatment-resistant quiescent leukemia stem cells (LSCs) . RecentlNucleoside analogs have long represented a backbone in the treatment of AML. Emerging novel nucleoside analogs possess novel mechanism(s) of action and metabolic properties. Furthermore, 8-chloro-adenosine (8-Cl-Ado) is one such analog that is RNA-directed and targets LSCs without significantly inhibiting HSCs ,16, inhiWe previously reported that 8-Cl-Ado synergizes with VEN in the inhibition of energy metabolism and survival of LSC-enriched blast cells ,19. HereHuman samples. Human specimens were collected from patients registered at City of Hope (COH) National Medical Center who had consented to the City of Hope Institutional Review Board approved protocol (IRB#18067); specimens from healthy donors were collected under COH IRB#06229. The study was conducted in accordance with the Declaration of Helsinki. Patient characteristics of primary AML samples are listed in –ΔCt method . Primer sequences for q-PCR are listed in RT-PCR and q-PCR analysis. To measure pre-rRNA and GAPDH expression, total RNA was extracted using the RNeasy Mini Kit . For pre-rRNA and GAPDH expression, first-strand cDNA was synthesized using the SuperScript III First-Strand Kit. q-PCR was performed using TaqMan Gene Expression Assays . GAPDH was used as an internal control and the results are presented as log2-transformed ratio according to the 2ChIP assay. Chromatin immunoprecipitation (ChIP) was performed as described by the manufacturer . Precleared chromatin was incubated overnight by rotation with 4 μg of Pol I antibody or IgG antibody as a negative control. Immunoprecipitates were resuspended in 50 μL TE buffer. Inputs and immunoprecipitated DNA samples were quantified by q-PCR on a 7900T Fast real-time PCR system . Antibodies are listed in 32P] orthophosphate . Total RNA was extracted with TRIzol  according to the manufacturer’s protocol. Equal amounts of RNA (10 μg) were separated on a 1.2% MOPS formaldehyde gel. The gel was dried and visualized by autoradiography.RNA labeling and analysis. The cells were washed and incubated in phosphate-free DMEM (Gibco) supplemented with 10% FBS for 2 h, followed by one-hour labeling with 0.5 mCi -uridine  per 1 mL for 1 h. For DNA synthesis, cell cultures were incubated with 5 μCi [3H]-thymidine  per 2 mL for 3 h. The cell cultures were then transferred onto Whatman GF/C glass microfiber filters  pre-treated with 1% aqueous sodium pyrophosphate on a multiscreen vacuum assay system . The filters were then washed with cold PBS, followed by two washes with 0.4 N perchloric acid, then dried with 70% ethanol. The glass filters were then added to a scintillation vial with 7 mL scintillation fluid; the level of radioactivity was measured using a liquid scintillation analyzer  and expressed as percent of control.RNA and DNA incorporation assay. AML cell lines were incubated with 0–10 μM 8-Cl-Ado for 24 h. Mononuclear cells derived from peripheral blood of AML patients were incubated with 10 μM 8-Cl-Ado for 24 h. For RNA synthesis, cell cultures were incubated with 1 μCi -labeled uridine, prior to evaluation via scintillation counting. As compared to vehicle-treated controls, RNA synthesis decreased in a dose-dependent manner in both KG-1a and MV4-11 cell lines  pre-rRNA E and decg of RNA F. Given g of RNA G. Taken We next dissected the molecular mechanism through which 8-Cl-Ado deregulated rRNA synthesis in AML cells. Transcription of rDNA by Pol I requires a pre-initiation complex consisting of RNA Pol I, upstream binding factor (UBF), selectivity factor 1 (SL-1) and TIF-IA transcription initiation factor. While TIF-IA is required to recruit Pol I to the rDNA promoter, UBF and SL-1 are primarily responsible for DNA binding ,30. We oOur previous work indicated that TIF-IA negatively regulates p53 expression . In LSC-We previously reported that 8-Cl-Ado treatment of LSCs resulted in decreased FAO and a diminished oxygen consumption rate (OCR), a marker for OXPHOS, but did not significantly alter the extracellular acidification rate (ECAR), a marker for glycolysis . Of noteWe recently reported that VEN and 8-Cl-Ado cooperate in the inhibition of OXPHOS and induction of apoptosis in LSC-enriched blasts . The 8-CPrevious studies showed that LSCs from de novo AML patients are susceptible to the VEN/azacitidine combination, whereas LSCs derived from patients with R/R AML are less sensitive to this treatment ,10,35. OThe schematic diagram  represenVEN combination treatments with either an HMA drug or low-dose cytarabine demonstrate superior clinical outcomes compared to single-agent treatment in AML patients. Relapse of AML is attributed to the presence of the LSC population. Recently, progress has been made in understanding the mechanisms of action of current VEN combination treatments. We and others demonstrated that the VEN/HMA combination augments oxidant stress in AML cells and we provided a molecular mechanism of a VEN-HMA-regulated NF-E2-related factor 2 (Nrf2) antioxidant pathway that could explain the impressive results observed in early clinical studies in AML , but mecPrevious studies showed that the VEN/azacitidine combination targets amino acid metabolism/OXPHOS and inhibits LSCs from de novo AML patients; however, LSCs from R/R AML patients can also utilize fatty acid metabolism to feed into OXPHOS and are thus less sensitive to VEN/azacitidine. Importantly, it has been demonstrated that dual inhibition of both amino acid metabolism and fatty acid metabolism using VEN/azacitidine together with the fatty acid transport inhibitor sulfo-N-succinimidyl oleate re-sensitizes resistant AML blasts to VEN/azacitidine treatment . TherefoWe have previously reported that the nucleoside analog 8-Cl-Ado possesses in vitro and in vivo activity against AML as a single agent and that it synergizes with the FLT3-ITD inhibitor quizartinib . IntracerRNA synthesis is a fundamental process utilized by all cells and targeting of this process for therapeutic intervention in cancer was long deemed not feasible. However, the dependence on increased rDNA transcription appears to selectively render cancer cells vulnerable to therapeutic intervention in the rRNA synthesis process ,39. In cWe demonstrate that the combination of VEN plus 8-Cl-Ado targets LSCs through the inhibition of rRNA synthesis and FAO/OXPHOS metabolic processes, thus potentially also targeting LSCs otherwise resistant to the currently used VEN combinatory regimens. To this end, a phase I/II clinical trial with VEN plus 8-Cl-Ado in patients with R/R AML will be initiated at our institution shortly.


Our results reveal an interrelationship between the metabolically driven low-frequency component and the electrically driven high-frequency component, with the latter exhibiting the highest bursting rates around the peaks of the slow component and the lowest around the nadirs. Moreover, the activity, as well as the average synchronicity of the fast component, considerably increased with increasing stimulatory glucose concentration, whereas the stimulation level did not affect any of these parameters in the slow component domain. Remarkably, in both dynamical components, the average correlation decreased similarly with intercellular distance, which implies that intercellular communication affects the synchronicity of both types of oscillations. To explore the intra-islet synchronization patterns in more detail, we constructed functional connectivity maps. The subsequent comparison of network characteristics of different oscillatory components showed more locally clustered and segregated networks of fast oscillatory activity, while the slow oscillations were more global, resulting in several long-range connections and a more cohesive structure. Besides the structural differences, we found a relatively weak relationship between the fast and slow network layer, which suggests that different synchronization mechanisms shape the collective cellular activity in islets, a finding which has to be kept in mind in future studies employing different oscillations for constructing networks.Beta cells within the pancreatic islets of Langerhans respond to stimulation with coherent oscillations of membrane potential and intracellular calcium concentration that presumably drive the pulsatile exocytosis of insulin. Their rhythmic activity is multimodal, resulting from networked feedback interactions of various oscillatory subsystems, such as the glycolytic, mitochondrial, and electrical/calcium components. How these oscillatory modules interact and affect the collective cellular activity, which is a prerequisite for proper hormone release, is incompletely understood. In the present work, we combined advanced confocal Ca We focus particularly on the relationship between both oscillatory components and to what extent their collective rhythmicity is coordinated on the multicellular level. For this purpose, we combine time series analysis with network-theoretical approaches to examine glucose-stimulated oscillatory Ca2+ dynamics measured in beta cells from acute mouse pancreas tissue slices.In the present study, we, therefore, aim to further explore the multimodal nature of oscillatory activity in pancreatic beta cells that is governed by interactions of various physiological regulatory systems. We distinguish between the metabolically driven low-frequency component of CaThe study was carried out in strict accordance with all national and European recommendations related to work with experimental animals, and all efforts were made to minimize the suffering of animals. The protocol was approved by the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection (permit number: 34401-35-2018/2).2, the abdomen was exposed via laparotomy and low-melting-point 1.9% agarose  in extracellular solution  125 NaCl, 26 NaHCO3, 6 glucose, 6 lactic acid, 3 myo-inositol, 2.5 KCl, 2 Na-pyruvate, 2 CaCl2, 1.25 NaH2PO4, 1 MgCl2, 0.5 ascorbic acid continuously bubbled with a gas mixture containing 95 % O2 and 5 % CO2 at barometric pressure to ensure oxygenation and a pH of 7.4) at 40°C was retrogradely injected into the pancreatic ductal tree via the proximal common bile duct clamped at the papilla of Vater. Subsequently, following immediate cooling with ice-cold ECS and extraction, small blocks of tissue (0.1–0.2 cm3 in size) were cut and embedded in agarose at 40°C. The tissue was cut at 0.05 mm s–1 and 70 Hz into 140 μm-thick slices , and the obtained slices collected in HEPES-buffered saline at room temperature  150 NaCl, 10 HEPES, 6 glucose, 5 KCl, 2 CaCl2, 1 MgCl2; titrated to pH = 7.4 using 1 M NaOH) until incubation in the dye-loading solution. All chemicals were obtained from Sigma-Aldrich  unless indicated.Pancreas tissue slices were prepared from adult NMRI male mice kept in individually ventilated cages  on a 12 light/12 dark cycle, as described previously . In brie2+ imaging. For Ca2+ imaging, individual tissue slices were transferred to the perfusion system delivering carbogenated ECS with varying glucose concentrations, according to the stimulation protocol, and kept at 37°C. The protocol consisted of initial exposure to the non-stimulatory 6 mM glucose, followed by either 8 or 12 mM glucose for 45 min, and washout with 6 mM glucose. The Ca2+ imaging was performed on a Leica TCS SP5 AOBS Tandem II upright confocal system  and a Leica TCS SP5 DMI6000 CS inverted confocal system . The acquisition was set to 10 Hz at 512 × 512 pixels to make the precise quantification of Ca2+ oscillations feasible. The dye was excited by argon 488 nm laser line and emitted fluorescence was detected by Leica HyD hybrid detector in the range of 500–700 nm , as described previously (F–F0)/F0 ratios, where F0 is the initial fluorescence intensity, and F is the fluorescence signal recorded at an individual time point during the experiment.Slices were incubated in the dye-loading solution  at RT for 50 min. Following the staining protocol, the slices were transferred into HBS containing 6 mM glucose and stored for up to 8 h until Caeviously . Additio2+ signals were first corrected for photobleaching of the dye employing a combination of linear and single exponential fit as described previously  and fj(t) represent the slow or fast traces of the i-th and j-th cell. By computing Ri,j among all node pairs, we create the correlation matrix, R, with the ij-th element being the correlation coefficient Ri,j. To enable a direct comparison between different networks, we used variable thresholds to extract the binary adjacency matrix, so that the average node degree in each network was k=8. Conventional tools from the complex network theory were then used to quantify functional beta cell networks . All significances are expressed as exact values and the number of islets included in analyses indicated accordingly. All significances are expressed as exact p values (p) and the number of islets included in analyses is indicated accordingly. We estimated effect sizes by calculating the values of Cohen’s d (d) by dividing the difference in sample means by the pooled standard deviation, according to the original definition . We compared groups by using the finition . Our judfinition .in vivo, i.e., 8 mM, and a supraphysiological concentration, i.e., 12 mM. First, we focused on the temporal aspect of the glucose-evoked oscillatory Ca2+ activity measuring the classical physiological measures, whereas in the second part of our analyses, we examined the synchronicity and collective activity of beta cell populations utilizing correlation analysis and network-based approaches. Particular emphasis was devoted to the interrelationship between the slow and the fast oscillatory component in both approaches.We studied the effect of stimulation with two glucose concentrations: a physiological concentration that is commonly observed 2+ response to glucose stimulation was recorded by means of multicellular confocal imaging in acute tissue slices as described in Materials and methods. The cells responded to stimulation with a delay in the onset of Ca2+ activity (TS), and they reached a state of sustained activity after a slightly longer time interval .Ca2+ signaling parameters, separately for each glucose concentration. In both glucose concentrations the frequency of slow oscillations was around 0.2 min–1 . In contrast, the frequency of fast oscillations depended significantly and with a very large effect size  on the stimulation level and was, on average, 4.8 and 8.4 min–1 in 8 and 12 mM glucose, respectively . Apparently, only the activity of the fast oscillatory component is modulated by stimulatory glucose levels. Finally, we characterized the beta cell responses to stimulation by calculating the average time lag until the cells in a given islet responded to stimulation, TS, and the average time required for the cells to reach the phase of sustained activity, i.e., the plateau phase, TP. Both of these parameters were significantly higher under lower stimulation levels and the effect sizes were very large . In 8 mM, values of TS and TP were approximately 5 and 8 min, whereas in 12 mM glucose, they shortened on average almost twofold  . On average, the correlation of the fast oscillatory activity was higher when compared to the slow component. For both dynamical components, the average correlation between cell pairs is a monotonically decreasing function of the intercellular distance, irrespective of the stimulatory glucose concentration  . On the  = 0.31) , which c = 0.31) . The difntration . In otheechanism . For tharonicity . Notablyronicity , 4, but 2+ traces from all cells were statistically compared in a pairwise manner to build correlation matrices . In both glucose concentrations, the fast component network layer exhibited higher clustering levels in comparison to the slow component network . Moreover, the network architecture of the fast component was found to be less cohesive . Under supraphysiological glucose levels the difference was not significant and characterized by a medium effect size . This can be attributed to the fact that high stimulatory conditions evoke high fractions of global waves in the domain of fast activity, which results in very integrated functional connectivity patterns . A higher dispersion of data in this case can probably be attributed to morphological heterogeneity of islets and the resulting inhomogeneous distribution of beta cells in tissue slices.To further characterize the collective beta cell activity of both dynamical components, we constructed functional connectivity profiles for both glucose concentrations. Both types of Camatrices . Functiopatterns . Modularpatterns   and supraphysiological (12 mM) stimulation. Both the frequency and the phase duration of the fast component increased with increasing glucose concentration , corrobo2+ ions on ion channels. A rise in the intracellular Ca2+ concentration activates the calcium-dependent K (KCa) channels, causing hyperpolarization and closure of the voltage-dependent Ca2+ channels. The latter decreases Ca2+ influx triggering a decrease in the Ca2+ concentration that ultimately removes the inhibitory drive of the KCa channels, and the cycle can repeat  or vice versa (metabolism-driven Ca2+ oscillations) . Moreover, the frequency of the fast component depended on the phase of the slow component. These facts, taken together, imply that the mechanism driving the slow activity of intracellular Ca2+ concentration is distinct from the one responsible for the fast component, that the slow component influences the fast, but that the presence of fast oscillations and their characteristics do not influence the slow oscillations. More specifically, the slow oscillations cause shifts in the frequency of the fast oscillations, but the average value of these fast oscillations is set by a glucose-dependent mechanism, distinct from the one responsible for slow oscillations. This corroborates the recent developments in computational models of beta cell activity, suggesting that the slow oscillations may originate from intrinsic mechanisms, in addition to Ca2+ effects on the enzymes involved in beta cell metabolism  . Experimomponent . On the llations . While sllations , others llations . In thistabolism . We wish2+ waves across the islets. Collective behavior of fast oscillations is receiving much more attention from the scientific community, especially because inherent beta cell heterogeneity and the existence of subpopulations lead to complex spatio-temporal activity patterns, characterized by heterogeneous and non-stationary intercellular waves, which are also accessible to experimental and modeling approaches .MSK, JD, and AS performed the experiments. JZ, RM, and MG developed the software for the analysis. JZ analyzed the data. JZ and RM prepared the figures. MSK, JD, AS, and MG wrote the manuscript. MM and AS provided resources. MG supervised the study. All authors conceived the idea, designed the study, and reviewed and approved the manuscript.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.


NPM1) gene in acute myeloid leukaemia (AML). This will include analysis of the structure and normal cellular function of NPM1, the type of mutations commonly witnessed in NPM1, and the mechanism by which this influences the development and progression of AML. The importance of NPM1 mutation on prognosis and the treatment options available to patients will also be reviewed along with current guidelines recommending the rapid return of NPM1 mutational screening results and the importance of employing a suitable laboratory assay to achieve this. Finally, future developments in the field including research into new therapies targeting NPM1 mutated AML are considered.The aim of this literature review is to examine the significance of the nucleophosmin 1 ( Acute myeloid leukaemia (AML) is a rare haematological disorder characterised by excess proliferation of haematopoietic precursors of myeloid lineage. Primarily originating in the bone marrow, disease progression is often characterised by infiltration into the peripheral blood system and other tissues. Whilst presenting at all ages, incidences of AML increase with age with the median age at diagnosis in the UK being 70.6 years . In NortLeukaemia as a pathological disease was beginning to be discussed in the literature in the mid-19th century following observation of excess white blood cells in patients post-mortem with the term ‘leukaemia’ coined in 1847 ,6,7,8. ICEBPA) and AML with a mutated nucleophosmin 1 (NPM1) gene [The utilisation of such techniques in the study of haematological malignancies has highlighted the diverse nature of the disease at the genetic, cytogenetic and immunophenotypic level and led to a major reclassification by the World Health Organisation (WHO) in 2001, which accounted for recurrent genetic abnormalities known to be of significance in AML . FurtherM1) gene . Both siNPM1 and the presence of several nuclear import and export signals contribute to its shifting cellular localisation and associated functions  that facilitate movement from the nucleoplasm to cytoplasm in association with a nuclear exporter protein exportin 1/chromosomal region maintenance 1 XPO1/CRM1 and has a significant role in binding a wide range of proteins [The conserved N-terminus of proteins . Post trproteins ,19. Withproteins . Numerous functions of NPM1 have been described and are often facilitated by the ability to shuttle intracellularly between nucleoli, nucleoplasm, and cytoplasm.NPM1 plays a role in cell growth and proliferation by participating in ribosomal biogenesis in the nucleolus which is essential for protein translation. NPM1 has been shown to interact with ribosomal protein L5 facilitating export from the nucleolus of the larger ribosomal subunit 60 S containing ribosomal RNA (rRNA). The blocking of rRNA export via NPM1 inhibition has been shown to lead to cell cycle arrest . NPM1 is an important histone chaperone necessary for the organisation of chromatin as well as assisting in chromatin transcription. The association of NPM1 to histone proteins is thought to be due to an abundance of negatively charged amino acids in the acidic region which mimic the ionic bonding of histones with DNA/RNA . NPM1 chNPM1 ortholog Npm1 gene have been shown to have uncontrolled centromere duplication and are not viable embryonically past day 16 due to impaired organogenesis [NPM1 is closely associated with centrosome activity and helping to maintain faithful cell division. NPM1 associates with unduplicated centrosomes via its nuclear export signal . Centrosogenesis .Despite being a membrane-less organelle, the nucleolus is organised into three sub compartments: a fibrillar centre (FC) surrounded by a dense fibrillar component (DFC) which is encompassed by the granular component (GC) . The GC NPM1 plays an important role in DNA repair, helping to modulate apurinic/apyrimidinic endonuclease 1 (APE1) protein activity. APE1 plays a crucial role in the base excision repair (BER) pathway. NPM1 facilitates its function by influencing localisation in the nucleoli and promoting endonuclease activity . NPM1 diDue to such wide ranging and important cellular functions, it is not surprising that aberrant functioning of NPM1 has been found in a wide range of solid tumours such as prostate, ovarian, gastric and colon .NPM1 is the most commonly mutated gene in adult AML, present in approximately 25–35% of patients [NPM1 mutation [NPM1 mutated AML often presents with a high white blood cell and blast count with NPM1 mutations observed in all AML subtypes under the FAB classification system except acute promyelocytic leukaemia [NPM1-mutated AML typically has cytological characteristics of either myeloblastic leukaemia with/without differentiation or acute leukaemia with monocytic/monoblastic differentiation and cup-like nuclei often being observed in blast cells [NPM1 mutated cells are often positive for the surface antigens CD33, CD117 and MPO in myeloblastic like cases, whilst CD64 and CD14 positivity is typical in cells with monocytic differentiation. Over two thirds of cases have blasts that are CD34 negative whilst a third are negative for HLA-DR [NPM1-mutated AML early in laboratory investigations.patients . It is leukaemia . NPM1-must cells . Immunopr HLA-DR . The immNPM1 locus is involved in translocations causing hematologic malignancies such as acute promyelocytic leukaemia where t leads to NPM1-RARα, anaplastic large cell lymphoma where t leads to NPM1-ALK, and myeloid neoplasms where t leads to NPM1-MLF1 [The PM1-MLF1 ,40,41. RPM1-MLF1 .NPM1 mutations in AML followed on from the study of the chimeric fusion protein NPM-ALK which is present in 30–50% of advanced anaplastic large-cell lymphoma cases [NPM1 [Initial investigations into the role of ma cases . Immunohma cases . An expaes [NPM1 . NPM1 mutations are found in exon 12 and approximately 50 different NPM1 mutation types have been described with the most common being Type A, which involves a TCTG tetranucleotide duplication and accounts for 75–80% of all NPM1 mutations in AML [NPM1 mutations  as being responsible for the duplication witnessed in utations . TdT is utations ,49. BorrThe different mutations mentioned result in the same defining feature of mutated NPM1, permanent cytoplasmic localisation which appears to be the driving force for leukemogenesis. Losing one or more tryptophan residues at the C-terminal leads to a loss of the NoLS whilst simultaneously gaining a leucine rich NES. Loss of tryptophan at 290 alone results in a stronger NES than the loss at positions 288 and 290 . Export NPM1 mutated AML the loss of all NPM1 nucleolar localisation is perhaps surprising as NPM1 mutations are always heterozygous in nature due to the non-viability of homozygous NPM1 mutations. The localisation of wild type NPM1 in the cytoplasm is believed to be due to the formation of heterodimers of NPM1wt/NPM1mut which are relocated to the cytoplasm [In ytoplasm .NPM1 mutated myeloid cells? One set of genes known to be highly upregulated in AML are the class I homeobox (HOX) genes which play a crucial role in proliferation, differentiation, and self-renewal in haematopoietic stem cells [HOXA and HOXB genes leads to a stem-cell like state [HOXA and HOXB genes as witnessed by epigenetic modification of Histone 3 (H3). H3 is acetylated at leucine position 27 (H3K27ac) to enhance transcription of HOX genes in NPM1mut AML [HOX gene overexpression is facilitated by NPM1mut. The exact mechanism by which NPM1mut upregulates HOX expression is not yet known, although research has pointed to involvement of the histone methyltransferase mixed lineage leukaemia 1 (MLL1) in association with its cofactor menin in the upregulation of Meis homeobox 1 (MEIS1). Transcriptionally active MEIS1 is believed to be a cofactor in the expression of HOX genes in early leukaemic development in AML and NPM1mut AML is believed to be dependent on the MLL1-menin interaction for increased HOX gene expression [This raises the question of what is the mechanism by which cytoplasmic localisation triggers the leukaemic state in em cells . It is bke state . Recent 1mut AML . To provpression ,56.Due to the wide range of functions of NPM1wt it has also been suggested that NPM1mut might also contribute to leukaemic phenotype by chaperoning other molecules to the cytoplasm, altering their function. The N-terminal of NPM1mut appears to be unaffected in its interactions with other molecules, associating with them as normal and then transporting them to the cytoplasm. The irregular cytoplasmic localisation of associated proteins involved in DNA repair pathways, apoptosis, and differentiation such as alternate reading frame (ARF), apurinic/apyrimidinic endonuclease 1 (APE1) and mouse double minute 2 (MDM2) may be involved in NPM1mut leukaemia, but there is not yet any definite proof that this plays a significant role.It had been reported that the transcriptional factor PU.1/spi-1 responsible for monocytic-granulocytic differentiation is translocated to the cytoplasm in association with NPM1mut and possibly contributes to arrest of differentiation, conceivably resulting in the monocytic features common in NPM1mut AML . Recent bcl-2 and oncogene c-MYC [A similar effect of cytoplasmic relocation altering function has been reported in the interaction of NPM1mut with bromo-domain protein 4 (BRD4) which is an epigenetic regulator of transcription. NPM1wt inhibits BRD4 in the nucleus and cytoplasmic relocation of NPM1 leads to a reduction in this inhibition with BRD4 increasing transcription in its target genes, the anti-apoptotic ne c-MYC .NPM1 mutations occurring in a cell population with evidence of clonal haematopoiesis appears to be sufficient to progress the clone to development of AML. The absence of NPM1 mutations in cases of clonal haematopoiesis is further evidence that NPM1 is a ‘driver’ or ‘gateway’ mutation needed for progression to AML in NPM1mut cases.Like most cancers, AML does not result from a single gene mutation and usually requires the sequential acquisition of numerous genetic mutations in a single lineage over time for disease evolution. One of the precursors to AML is clonal haematopoiesis, where a number of somatic mutations exist in haematopoietic cells without evidence of malignancy . NPM1 muDNMT3A) and FMS like tyrosine kinase 3 (FLT3) genes.Many genes with diverse roles in cellular activities such as signalling, epigenetic modulation, chromatin modification, RNA splicing, and tumour suppression are commonly mutated in AML . PatternDNMT3A appears to be a preleukaemic mutation commonly existing in a clonal population before additional mutations drive the development of AML. DNMT3A is often still present in remission indicating DNMT3A mutations are not a key driver of AML development [DNA methyltransferase 3 alpha (DNMT3A) is a protein responsible for DNA methylation, mutations in which are found in approximately 20% of all AML cases and as a co-mutation with NPM1 in 50% of cases ,63. DNMTelopment .FLT3) gene encodes a transmembrane receptor tyrosine kinase normally expressed in haematopoietic stem cells that contributes to signalling in P13K, RAS and JAK-STAT pathways [FLT3 mutation [FLT3 mutated AML. NPM1, DNMT3A and FLT3 are the three most mutated genes in AML with some research showing them as the most commonly occurring three gene co-mutations, present in up to 6% of patients and conferring a poor prognosis as has also been demonstrated in murine studies [The FMS like tyrosine kinase 3  guidelines released in 2017 stratified risk into the categories of favourable, intermediate, and adverse depending on the genetic abnormalities present  at first remission. Whilst being a crucially important treatment in cases where relapse risk is expected to be high, the associated risk of potentially fatal infection due to immunosuppression and graft vs. host disease is thought to be greater than any benefit in patients with a relapse rate of less than 35% such as in NPM1mut AML falling into the favourable risk category.One of the most significant consequences of the ELN guidelines is the recommendation that patients with an NPM1, it is hoped that knowledge of the mutational profile in individuals will help allow clinicians to treat these patients with therapies targeted at specific mutations present. The main treatment for AML has remained relatively unchanged for over 40 years comprising the ‘7 + 3’ chemotherapy regimen involving infusion of the cytotoxic drugs cytarabine and daunorubicin for 7 and 3 days, respectively [In addition to helping categorise the risk for AML patients based on the presence or absence of mutations such as ectively . Unsurprectively ,76. The ectively ,77. The ectively . More reectively .NPM1 mutation [NPM1 and FLT3 mutation testing be returned within 24–48 h of diagnosis to facilitate intensive chemotherapy for all eligible patients.What is the benefit of having molecular information at hand when it comes to treatment options? Research has shown that a high dose of daunorubicin, twice that of the standard dose, is beneficial in improving survival in patients with an mutation . Presentmutation . Whilst mutation . The curFLT3 mutated AML showed a survival benefit in the midostaurin treated group with a hazard ratio of 0.78 in comparison to the placebo group [Similarly, the RATIFY clinical trial, showed the advantage in returning genetic testing results in a short period of time, in this instance 48 h. RATIFY, a phase 3 trial testing the addition of the tyrosine kinase inhibitor midostaurin to the standard 7 + 3 treatment regimen in bo group .With increasing knowledge of the genetic heterogeneity in AML and the specific mutations influencing the course of disease, the need for rapid detection of relevant mutations to guide clinical decision making has become crucial. Large scale, parallel DNA sequencing responsible for the discovery of many of the previously discussed genes has advanced to a level where it is economically viable and practical to be used more routinely, being available to most patients in UK during their course of treatment . The creNPM1 and FLT3. Current guidance for return of NGS panel results from NHS England shows a turnaround time of 21 days for NGS haemato-oncology referrals [Whilst NGS is a vital tool in understanding the overall mutational landscape in individuals, it is unlikely to yet be suited in providing results in the required turnaround time of 24–48 h as recommended by the ELN for determining mutational status of eferrals . It may FLT3-ITD mutations has led to fragment analysis by capillary electrophoresis being routinely used and this was the method of testing in the RATIFY trial. This relatively straight forward assay involves the separation of sections of a gene of choice based on size following amplification by polymerase chain reaction (PCR) and detection via fluorescence. This assay is suited to detecting FLT-ITD mutations as the mutation results in a fragment size larger than the wild type FLT3 gene. Similarly, it has been suggested that this method would be ideal in testing for NPM1 mutations as approximately 95% of mutations result in a 4-base pair insertion that can be separated from the wild type using capillary electrophoresis giving a rapid, qualitative result with high sensitivity, being able to detect down to 2% mutant allele [NPM1 mutation.The requirement for a rapid assay with suitable sensitivity for detecting t allele . Figure FLT3-ITD mutated AML.Another benefit in using fragment analysis is in determining the mutational burden, essentially the proportion of mutant alleles in comparison to the wild type of the gene in question. This is commonly done by calculating the allelic ratio (AR) of the mutant present by dividing the area under the curve of the mutant allele fragment by the area under the wild type allele . AllelicFLT3 mutation reporting, the benefits of reporting mutational burden in NPM1mut cases has yet to be fully demonstrated and is currently debated, with some research indicating that an increased mutational burden in NPM1mut AML leads to a significant reduction in overall and event free survival [NPM1 mutated AML is still being debated and may arguably be of importance in rapid diagnostic testing as knowledge improves.Whilst AR is commonly used in line with international guidance in survival . This clsurvival . The utiNPM1 mutations do not drive clonal haematopoiesis and are highly expressed, the presence of detectable levels of NPM1mut transcripts at various points in treatment can be determined using a highly sensitive assay such as real time quantitative PCR (RT-qPCR) or droplet digital PCR (dPCR) [FLT3-ITD status with FLT3-ITD positive patients shown to be high risk and FLT3-ITD negative patients shown to be low risk, respectively. Conversely, the detection of NPM mut MRD prior to HSCT is associated with inferior survival [NPM1 in AML will have to be a consideration for laboratories in large testing centres with standardisation and access to external quality assessment programmes being required.In assessing disease status of patients through their course of treatment, NPM1mut also provides a useful target for minimal residual disease (MRD) monitoring. As R (dPCR) ,92. The R (dPCR) . MRD staR (dPCR) . The rissurvival . Having NPM1 have provided researchers with challenges and opportunities. Challenges in the numerous ways NPM1mut may contribute to the development of AML that are still not fully understood and opportunities in the multiple therapeutic targets that exist and are being actively investigated.The multiple, diverse functions of HOX gene expression has proven to be another target with inhibitors of MLL1-menin shown to have anti-leukaemic activity in NPM1 mutated leukaemia [NPM1 mutated AML with recent research in mice showing the possibility of using chimeric antigen receptor T cell (CAR-T) therapy in the future [Some therapeutic targets include inhibitors of the nuclear exporter XPO1 such as Selinexor with the aim of correcting the aberrant cytoplasmic localisation of NPM1mut . The sigeukaemia . There ieukaemia . As preve future .One of the most promising treatments for older individuals is in the use of the bcl-2 inhibitor Venetoclax, commonly used in B-cell malignancies . Recent FLT3-ITD status. Such research highlights that there are likely to be further discoveries in this area in the near future and that laboratories may have to adapt at relatively short notice to facilitate such testing.With rapidly expanding molecular diagnostic techniques and new technological methods, further insights into AML are likely to be elucidated. Recent research using such techniques as RNA sequencing and chromatin accessibility analysis suggests there are two distinct NPM1mut subtypes classified as primitive and committed, differing in their expression profiles . The priNPM1 in the development of such an aggressive and difficult to treat disease like AML has led to more accurate prognoses and several potential treatment options where there has been few before, particularly for elderly patients. The speed of change gives rise to difficulties in translating such knowledge to the clinical area with laboratories having to be ever more adept at providing fast, reliable results to help guide clinical decisions. With rapid advancements and potential therapeutic options being developed, the utility in providing clinicians with NPM1 and FLT3 status in a matter of days as opposed to weeks is evident, with the hope that such results can guide early decisions regarding treatment and help patients receive the most appropriate, personalised treatment at the most appropriate time which will hopefully lead to improved outcomes.Rapidly expanding knowledge of the role of genetic mutations such as


Microelectrode recordings revealed that the sinus node of the mouse, which had a higher beating rate, had a steeper slope of the pacemaker depolarization than that of the guinea pig. BAPTA and ryanodine, agents that interfere with intracellular Ca2+, significantly decreased the slope of the pacemaker depolarization in both species. In contrast, SEA0400, a specific inhibitor of the Na+-Ca2+ exchanger (NCX), as well as change to low Na+ extracellular solution, significantly decreased the slope in the mouse, but not in the guinea pig. Niflumic acid, a blocker of the Ca2+ activated Cl− channel, decreased the slope in both species. Confocal microscopy revealed the presence of spontaneous Ca2+ oscillations during the interval between Ca2+ transients; such phenomenon was more pronounced in the mouse than in the guinea pig. Thus, although intracellular Ca2+-mediated mechanisms were involved in the pacemaker depolarization of the sinus node in both species, the NCX current was involved in the mouse but not in the guinea pig.Intracellular Ca The action potential of the sinus node has a characteristic pacemaker depolarization phase (phase 4 depolarization) in the membrane voltage range of −60–−40 mV which leads the membrane potential to the threshold level for the rapid action potential upstroke (phase 0 depolarization). The pacemaker depolarization is formed by multiple inward membrane currents including the hyperpolarization-activated current (Int (Ist) ,2,3,4,5. animals . 2+. The Ca2+ released from the sarcoplasmic reticulum (SR) causes the Na+-Ca2+ exchanger (NCX) to generate a depolarizing current and accelerate the pacemaker depolarization. This mechanism was referred to as the Ca2+ clock and was considered to function in coordination with the ion channels of the cell membrane (membrane clock) to form the pacemaker depolarization [2+-mediated mechanisms or the precise pacemaking mechanisms in each animal species. For example, in the guinea pig sinus node, some researchers emphasize the involvement of the Ca2+-clock while others reported negative results [It has been postulated that the pacemaker depolarization is also influenced by intracellular Carization ,9. Howev results ,9,10.2+-mediated mechanisms in the pacemaker depolarization of the mouse and guinea pig sinus node using tissue preparations. We performed standard microelectrode measurements of action potentials with sinus node tissue preparations and applied selective pharmacological agents.One of the possible reasons for this discrepancy is the use of isolated sinus node cells. The sinus node consists of cardiomyocytes with different electrophysiological properties ,11, and 2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 24.9, and glucose 11.1 (pH 7.4), and the solution was gassed with 95% O2-5% CO2 and maintained at 36 ± 0.5 °C. The low Na+ extracellular solution was prepared with the equimolar substitution of NaCl with LiCl so that the final Na+ concentration was 70 mM. Change to low Na+ solution and return to normal solution were performed with a flow-switching device which enabled change of the extracellular solution within approximately 1 s. To chelate intracellular Ca2+ with O,O’-Bis (2-aminophenyl) ethyleneglycol-N,N,N′,N′-tetraacetic acid (BAPTA), its cell-permeable tetraacetoxymethyl ester (BAPTA-AM) was applied to the preparations at a final concentration of 300 μM.Standard glass microelectrode experiments were performed with isolated sinus node tissue from the mouse and the guinea pig as described previously ,3. Micromax), peak potential, and duration at 50% repolarization (APD50). To obtain the slope value, the middle portion of the pacemaker depolarization phase was fitted by a straight line; the curved regions close to the maximum diastolic potential and threshold potential were not included in the fitting. The spontaneous firing of the sinus node tissue preparations was well maintained; the change in firing rate at 10 and 30 min after the addition of vehicle was less than 1% and 2% of the basal value, respectively.The action potential parameters measured were firing rate, cycle length, maximum diastolic potential, threshold potential, the slope of the pacemaker depolarization (slope), maximum rate of rise of the action potential upstroke 2+ movements, the sinus node tissue preparations were incubated with a Ca2+ indicator  at 10 μM for 1 h at 37 °C in the following composition: NaCl 143, KCl 4.7, MgCl2 1.0, NaH2PO4 0.33, glucose 5.5, and HEPES 5 (mM). The recording chamber, the bottom of which was a coverslip, was placed on the stage of an inverted microscope. Preparations were placed at the bottom of the recording chamber epicardial surface down. The normal extracellular solution  maintained at 32 °C) was continuously perfused. Confocal microscopic analyses of the Ca2+ oscillations in the myocardial layer were performed with a rapid scanning confocal microscope A1R . The emission 570–620 nm on excitation at 561 nm was detected for Cal-590 fluorescence, and the scanning was performed at a speed of frame/16.9–33.8 ms. The fluorescent intensity of Cal-590 at each time point was normalized against the basal intensity.For the analysis of intracellular Cat-test. A p value less than 0.05 was considered statistically significant.BAPTA-AM , ryanodine , SEA0400 (synthesized in our faculty), niflumic acid , and Cal-590 AM  were dissolved in dimethyl sulfoxide (DMSO). All data were expressed as the mean ± standard error of the mean (S.E.M). Data were analyzed by the paired Microelectrode recordings showed that the sinus node of the mouse, which had a higher firing rate, had a steeper slope of the pacemaker depolarization and a shorter action potential duration than that of the guinea pig. There was no difference in the maximum diastolic potential and threshold potential between the mouse and guinea pig; the maximum diastolic potential was about −60 mV, and the threshold potential was about −40 mV in both species.2+ with BAPTA (300 μM) induced significant decreases in the firing rate and the slope of the pacemaker depolarization  significantly decreased the firing rate and the slope of the pacemaker depolarization . Ryanodin = 6), respectively.In the mouse sinus node, SEA0400 (1 and 10 μM) decreased the firing rate and the slope of the pacemaker depolarization . SEA0400n = 5), respectively.In the guinea pig sinus node, SEA0400 had no effect on the action potential waveform . Further+ extracellular solution. In the mouse sinus node, changing the extracellular solution to low Na+ solution significantly decreased the firing rate and the slope of the pacemaker depolarization , a blocker of the Carization . Niflumirization . The act2+ movements, the sinus node tissue preparations loaded with the Ca2+ sensitive fluoroprobe, Cal-590, were observed with a rapid scanning confocal microscope. In both species, Ca2+ transients, synchronized elevations of Ca2+ fluorescence throughout the myocardial tissue, were observed at a constant frequency. In mouse sinus node, spontaneous Ca2+ oscillations, non-synchronized local rises in Ca2+ fluorescence, were observed in roughly one third of the cells , respectively. SEA0400 caused an increase in basal Ca2+ concentration; the basal Ca2+ fluorescence at 10 min after the addition of 1 μM SEA0400 was 110.3 ± 8.7% (n = 5) of the value before application. SEA0400 had no apparent effect on the local Ca2+ oscillations.In the mouse sinus node, SEA0400 (1 μM) markedly reduced the firing frequency of spontaneous Caansients ; the fre2+ transients (n = 5), respectively. SEA0400 caused an increase in basal Ca2+ concentration; the basal Ca2+ fluorescence at 10 min after the addition of 1 μM SEA0400 was 112.0 ± 4.5% (n = 5) of the value before application.In the guinea pig sinus node, SEA0400 had no effect on the firing frequency of spontaneous Caansients ; the fre2+-mediated mechanisms in the pacemaker depolarization of the mouse and guinea pig sinus node. We applied standard microelectrode techniques on tissue preparations and used selective pharmacological agents. Compared to isolated cardiomyocytes, myocardial tissue preparations may have disadvantages such as less efficient delivery of oxygen and pharmacological agents to the cells of interest. On the other hand, they have advantages such as absence of cell isolation damage and cell selection bias. Above all, tissue preparations enable the observation of the sinus node as a whole working as a functional syncytium.In the present study, we intended to clarify the involvement of intracellular Ca2+-mediated mechanisms are involved in the pacemaking of the sinus node. Treatment of the sinus node tissue preparations with either BAPTA or ryanodine, agents that interfere with intracellular Ca2+, decreased the firing rate and the slope of the pacemaker depolarization both in the mouse and guinea pig. These results indicated that Ca2+ released from the SR through the ryanodine receptor contributes to the formation of the pacemaker depolarization of the sinus node in both species. As both BAPTA and ryanodine may have a broad effect on intracellular Ca2+-mediated events, their effects may also reflect changes other than the instantaneous reduction of intracellular Ca2+ [2+ into the pacemaker depolarization.First of all, we examined whether or not intracellular Calar Ca2+ . Thus, w+/Ca2+ exchanger (NCX) is the major pathway for transsarcolemal Ca2+ extrusion from the cytoplasm; it pumps out one Ca2+ ion in exchange for three Na+ ions (forward mode NCX). It has been postulated that the net inward current that occurs in this process contributes to the pacemaker depolarization of the sinus node. To examine this hypothesis in sinus node tissue preparations, we used SEA0400, a potent and selective inhibitor of NCX. In voltage-clamped guinea pig ventricular myocytes, 1 μM SEA0400 inhibited the NCX current by more than 80%, with no effect on the sodium current, L-type calcium current, delayed rectifier potassium current, and the inwardly rectifying potassium current [The Na current . The eff current ,14,15.2+ transients, was decreased by SEA0400. Similar results were obtained with low Na+ extracellular solution. Thus, the NCX current activated by the Ca2+ released from the SR contributes to the pacemaker depolarization in mouse sinus node. These results were consistent with the observation that the firing frequency of the sinus node was lower in atrial-specific NCX knock out mice than in wild type mice [+ extracellular solution, affected neither the firing rate nor the slope of the pacemaker depolarization. SEA0400 caused a small increase in the basal Ca2+ concentration. These results indicated that NCX contributes to transarcolemmal Ca2+ efflux but does not play an essential role in the pacemaker depolarization of the guinea pig sinus node. This lack of SEA0400 effect in the guinea pig sinus node was not due to its lower firing rate because SEA0400 was ineffective in the guinea pig right atria even after its beating rate was increased by noradrenaline to a level similar to that of the mouse, and effective in the mouse right atria even after its beating rate was decreased by carbachol to a level similar to that of the guinea pig.In the mouse sinus node, the slope of the pacemaker depolarization, as well as the firing rate of action potentials and Caype mice . In cont+ on the maximum rate of rise and the peak potential could be explained by their effect on the pacemaker depolarization. SEA0400 and low Na+ markedly reduced the slope of pacemaker depolarization in the mouse sinus node. This means that the membrane potential is kept in a partially depolarized potential range close to the threshold potential for a longer time, which may cause increased inactivation of the L-type Ca2+ channel leading to a reduced maximum rate of rise and peak potential. In contrast, in the guinea pig sinus node, in which the slope of the pacemaker depolarization was unchanged, neither the maximum rate of rise nor the peak potential was affected.The effects of SEA0400 and low Naf), L-type and T-type Ca2+ currents, and the sustained inward current (Ist) [K is dominant in the early phase of pacemaker depolarization in the guinea pig sinus node [2+ to the NCX, it is possible that some difference in intracellular Ca2+ handling properties causes a difference in NCX current density in the spontaneously firing sinus node. In fact, spontaneous Ca2+ oscillations were observed during the interval between Ca2+ transients in sinus node tissue preparations, which was more pronounced in the mouse than in the guinea pig. This appears to be consistent with the observation in isolated sinus node cardiomyocytes that the number of Ca2+ sparks occurring during the pacemaker depolarization was larger in the mouse than in the guinea pig [The pacemaker depolarization is considered to be formed by several inward currents such as the hyperpolarization-activated current (Int (Ist) ,2,3,4,5.nus node . The NCXnus node ,18. Sincinea pig ,19.2+ sparks occurred during the interval between Ca2+ transients, and the frequency of Ca2+ spark firing increased abruptly just before the onset of Ca2+ transients [2+ released from the SR was involved in the pacemaker depolarization of the pulmonary vein myocardium. Thus, the contribution of NCX to myocardial pacemaking varies among animal species and type of myocardium.Concerning the sinus node of other animal species, it was reported that SEA0400 had no effect on the heart rate in the isolated rabbit heart  and in aansients . These r2+-mediated pacemaking mechanisms of the sinus node, we examined the effect of niflumic acid, a blocker of the Ca2+-activated Cl− channel, which was identified in sinus node cells [− ions in cardiomyocytes was about −30 mV [2+-activated Cl− channel could generate a depolarizing current during the pacemaker depolarization. Niflumic acid reduced the firing rate and the slope of pacemaker depolarization in the sinus node of both mouse and guinea pig. These results indicated that the inward current through the Cl− channel activated by Ca2+ released from the SR contributes to the pacemaker depolarization of the sinus node both in the mouse and guinea pig.To obtain further information on the Cade cells ,25. As tt −30 mV , activat2+-activated Cl− current (IClCa) was involved in the pacemaker depolarization of both mouse and guinea pig, but the NCX current contributed to cardiac pacemaking only in the mouse. Using similar experimental techniques, we previously showed that the contribution of the T-type Ca2+ channel to cardiac pacemaking differs among animal species; the effects of R-(−)-efonidipine, which is a selective T-type Ca2+ channel inhibitor, was prominent in the mouse, small but significant in the guinea pig, and was not observed in the rabbit [+ channels, which are considered not to play a role in pacemaker activity in the majority of mammalian species including the guinea pig [CaL, Ist, If, and IClCa, but not INCX for its pacemaking [CaT, INCX, and INa also contribute to pacemaking [The mouse has a high heart rate 400 bpm>), and its sinus node has a steeper slope of pacemaker depolarization among experimental animal species. The present study showed that the Cainea pig , was repinea pig . The pre00 bpm>, cemaking ,11. In tcemaking ,16,27. T2+-mediated mechanisms were involved in the pacemaker depolarization of the sinus node both in the mouse and guinea pig. The NCX current was involved in the mouse but not in the guinea pig. This difference could partly explain the higher firing rate of the mouse sinus node.Intracellular Ca