Cardiac subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial subpopulations possess distinct biochemical properties and differ with respect to their protein and lipid compositions, capacities for respiration and protein synthesis, and sensitivity to metabolic challenge, yet their responsiveness to mitochondrially active cardioprotective therapeutics has not been characterized. This study assessed the differential responsiveness of the two mitochondrial subpopulations to diazoxide, a cardioprotective agent targeting mitochondria.2+-dependent mitochondrial permeability transition (MPT) pore and mitochondrial membrane potential were assessed using standard Ca2+ and TPP+ ion-selective electrodes.Mitochondrial subpopulations were freshly isolated from rat ventricles and their morphologies assessed by electron microscopy and enzymatic activities determined using standard biochemical protocols with a plate reader. Oxidative phosphorylation was assessed from State 3 respiration using succinate as a substrate. Calcium dynamics and the status of Ca2+ overload-mediated inhibition of adenosine triphosphate (ATP) synthesis with decreased ATP production . In addition, SSM exhibited reduced Ca2+-accumulating capacity as compared to IFM , suggestive of increased Ca2+ sensitivity of MPT pore opening. Despite enhanced susceptibility to stress, SSM were more responsive to the protective effect of diazoxide (100 μM) against Ca2+ overload-mediated inhibition of ATP synthesis .Compared to IFM, isolated SSM exhibited a higher sensitivity to Ca2+-dependent metabolic stress and the protective effect of diazoxide on mitochondrial energetics.These results provide evidence for the distinct sensitivity of cardiac SSM and IFM toward Ca These results thus provide evidence of distinct sensitivity of cardiac mitochondrial subpopulations toward the protective effect of diazoxide, indicating that SSM could be the preferred target for drug treatment.Two distinct mitochondrial subpopulations – subsarcolemmal (SSM), situated underneath the sarcolemmal membrane, and interfibrillar (IFM), distributed between myofibrils – have been previously identified in myocardium Guide for the Care and Use of Laboratory Animals, National Academic Press, Washington, D.C., 1996.The study was approved by the Mayo Clinic Institutional Animal Care and use Committee (Protocol # A28201), and all procedures were in accordance with recommendations published in 2PO4 5, EGTA 1, 0.2% BSA, MOPS 5 (pH  = 7.3) as described by Holmuhamedov et al. 2+ handling of isolated SSM in the presence or absence of Nagarse treatment. Protein concentration was determined using DC™ Protein Determination Kit .Mitochondria were isolated from the hearts of pentobarbital -anesthetized male adult rats . Following thoracotomy, the heart was rapidly removed from the chest and ventricles were trimmed of atria and connective tissue. The ventricles were placed in ice-cold media containing (in mmol/L): sucrose 50, mannitol 200, KHen bloc with 2% uranyl acetate for 30 min at 60°C, rinsed, dehydrated, and embedded in Spurr's resin. Thin sections were cut on an Ultracut E ultramicrotome , placed on copper grids and stained with lead citrate. Mitochondria were micrographed with a 1200 EX II electron microscope .SSM and IFM were fixed using Trump's buffer , rinsed and post-fixed in phosphate-buffered 1% osmium tetroxide The activity of citrate synthase (CS) in SSM and IFM was determined as described by Short et al. ™, Bio-Rad Laboratories) and then transferred to polyvinylidene fluoride membranes as described by Short et al. ™, Amersham) was used for detection. Images captured on Kodak Omat film  were then used for densitometry of bands using Kodak Image Station 1000.Aliquots of mitochondria solubilized in Laemmle sample buffer were separated on polyacyrlamide gels  equipped with oxygen-, tetraphenylphosphonium (TPP+)- and Ca2+-selective minielectrodes, as previously described 2HPO4 5, succinate 5, pyruvate 5, and MOPS 10 (pH  = 7.35) and respiration was measured using calibrated Clark-type O2 minielectrode. Mitochondrial membrane potential was measured simultaneously with respiration using TPP+-sensitive minielectrode, manufactured and calibrated as described by Kamo et al. + was 200 nM, and mitochondrial membrane potential was calculated as previously described 2+ uptake was measured from changes in the free Ca2+ concentration within the suspension using calibrated Ca2+-selective minielectrodes  as described 2+-accumulating capacity was determined as the total amount of Ca2+ accumulated into the matrix from a train of 50-μM Ca2+ pulses added at 1-min intervals until the load reached a threshold pulse after which mitochondria underwent irreversible and rapid Ca2+ release Respiration, membrane potential and Ca2CO3/MOPS-neutralized HClO4-soluble mitochondrial extracts by high-pressure liquid chromatography  as described by Holmuhamedov et al. 4, and precipitated proteins were removed by centrifugation . After neutralization of the supernatant with 80 μl of a mixture containing 2.5-M K2CO3 in 1-M HEPES, the precipitate was separated by centrifugation , and the concentration of ATP within the extract was determined in coupled enzymatic reactions Adenosine triphosphate (ATP) production in mitochondria was determined using KDiazoxide  was dissolved as a concentrated stock solution in dimethylsulfoxide (DMSO), and the maximal concentration of DMSO in the incubation medium was kept under 0.5%. All other chemicals were from Sigma Chemicals .Data are expressed as mean ± standard error of mean, and “n” represents the number of mitochondrial isolations. Comparison between groups was made using analysis of variance (ANOVA) with post-hoc test. ANOVA was performed for multiple comparisons between groups using two-way comparison of means by Tukey-Kramer HSD test, and p<0.05 was considered to be statistically significant.in situ and after isolation (top panels). Both subpopulations of isolated mitochondria demonstrated similar levels of expression of CS and ANT1 (top panels). In addition, the activity of CS in mitochondrial subpopulations was similar .Electron micrographs of the heart muscle demonstrate intracellular localization of mitochondrial subpopulations and morphological appearance of SSM and IFM solation . Intracesolation . The con2+-induced mitochondrial permeability transition (MPT) pore opening was determined from the number of Ca2+ pulses required to reach the threshold for rapid and spontaneous Ca2+ release 2+ in isolated SSM and IFM measured immediately after isolation . However, the maximal Ca2+-accumulating capacity of mitochondrial subpopulations  was significantly decreased in SSM and was 230±13 nmol Ca2+/mg protein as compared with 450±46 nmol Ca2+/mg protein in IFM . Although the rates of respiration upon completion of ADP-to-ATP conversion (State 4) were different in these populations , the resulting respiratory control ratio was not significantly different , demonstrating similar coupling of oxidative phosphorylation in SSM and IFM.The capacity for oxidative phosphorylation assessed from ADP-mediated increase in mitochondrial respiration and membrane depolarization was also different in SSM and IFM . On aver2+/mg protein inhibited oxidative phosphorylation in both SSM and IFM, but the extent of inhibition was significantly higher in SSM. The rate of ATP synthesis in the presence of Ca2+ was decreased from 375±25 to 83±15 nmol ATP/min/mg protein in SSM, and from 875±39 to 583±45 nmol ATP/min/mg protein in IFM -induced depolarization of the inner membrane (19±2 mV) compared with IFM  suppressed the rate of Ca2+ uptake preferentially in SSM (from 347±9 to 137±7 nmol Ca2+/min/mg protein) than in IFM (from 503±13 to 326±10 nmol Ca2+/min/mg protein), demonstrating a 61% vs. 35% inhibition of Ca2+ uptake in SSM and IFM, respectively (2+ during 20 min of observation (data not shown). However, diazoxide had a differential effect on Ca2+ release from Ca2+-loaded mitochondria. In mitochondria preloaded with 100 nmol Ca2+/mg protein, the same concentration of diazoxide (100 µM) induced faster release of accumulated Ca2+ from SSM than from IFM (2+ release was fivefold higher (10±2 vs. 2±1 nmol Ca2+/min/mg protein) in SSM and IFM, respectively  was estimated at 104±15 and 289±32 µM in SSM and IFM, respectively , whereas at 30 nmol Ca2+/mg protein, the protective effect of diazoxide was only by 12±3% and 4±0.9% in SSM and IFM, respectively (hatched bars). ATP synthesis in mitochondria, monitored using coupled enzymatic reactions 2+-inhibited ATP production in SSM than IFM. In mitochondria loaded with 150 nmol Ca2+/mg protein, diazoxide restored the rate of ATP production by 48±5% in SSM compared with 5±3% in IFM. Thus, diazoxide restores Ca2+-inhibited mitochondrial ATP synthesis in both SSM and IFM populations, but the magnitude of this protective effect is markedly higher in SSM compared to IFM.Excessive mitochondrial Carylation  and diazrylation . The resd to IFM  and was overload . In mito2+-dependent inhibition of oxidative phosphorylation, opening of MPT pore and sensitivity to the protective effect of diazoxide, a mitochondrially active cardioprotective agent. These results provide additional insights into the functional and pharmacological differences between the mitochondrial subpopulations previously shown to differ in their biochemical characteristics, protein and lipid composition, and susceptibility toward metabolic challenge 2+ overload and inhibition of oxidative phosphorylation when compared to IFM, in line with previous observations 2+-mediated inhibition of oxidative phosphorylation was more prominent in SSM compared to IFM 2+-inhibited oxidative phosphorylation in SSM than IFM  and membrane protein (adenine nucleotide transporter) were not different in the two subpopulations, suggesting that distinct properties were not introduced due to differences in the isolation protocol or the number of mitochondria but are intrinsic features of mitochondrial subpopulations. The salvaging effect of diazoxide on ATP production in Ca2+-loaded mitochondria was greater in mitochondria with a higher level of Ca2+ load, indicating that the effect of diazoxide is condition-selective, and it is rather the release of inhibited oxidative phosphorylation than the activation of mitochondrial ATP synthesis. In accordance with this notion is the fact that in the absence of Ca2+ loading, diazoxide had little effect on State 3 respiration in both populations, and may even slow the rate of ATP production as reported previously This study demonstrates that SSM and IFM, two subpopulations of cardiac mitochondria, exhibit differential susceptibility to Cathan IFM  and 4. Tin vivo remains unknown, 2+ load and metabolic stress. This is of significance in view of the different responsiveness of subsarcolemmal and interfibrillar mitochondria in intact cardiomyocytes 2+ loading demonstrated in various cellular microdomains 2+ injury is consistent with reports on the higher vulnerability and reduced tolerance of this mitochondrial population toward Ca2+-mediated functional and structural damages. Therefore, our observation indicates that by preferentially targeting SSM (the more vulnerable subpopulation of cardiac mitochondria) diazoxide will be protective against ischemia/reperfusion-mediated injury.The precise mechanism of diazoxide action on mitochondria + and K+ ions across the sarcolemmal membrane, resulting in increased cytosolic Ca2+ (2+ from the cytosol. By promoting MPT pore opening or inhibition of oxidative phosphorylation, this oxygen availability causes greater injury to the more vulnerable SSM than IFM, resulting in additional structural and functional derangements that limit the capacity of SSM, which are located in the close vicinity of plasma membrane ionic pumps 2+ overload by decreasing the rate of Ca2+ uptake or releasing accumulated Ca2+ or both, resulting in preservation of ATP production in the more vulnerable and strategically distributed SSM (Under normal conditions, both SSM and IFM are efficient in meeting demands of the cellular ATP-dependent processes and maintaining ionic homeostasis of cells . During lic Ca2+ . At repeuted SSM , thus re2+ uptake, reduction of mitochondrial Ca2+ loading and through release of excessively loaded Ca2+ and restoration of Ca2+-inhibited ATP production in postischemic heart muscle.In summary, SSM are a preferential target for the cardioprotective drug diazoxide in the setting of ischemia and heart reperfusion. Our data suggest that the mechanism of protective action of diazoxide could be through decreased Ca


Stimulation of cells with physiological concentrations of agonists trigger increases [Ca2+]i due to intracellular Ca2+ release and Ca2+ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca2+]i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca2+]i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca2+ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca2+ store depletion in these cells, it is not clear how the cells decode individual Ca2+ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca2+]i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca2+ entry generates [Ca2+]i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca2+ entry generates sustained [Ca2+]i elevation at high [CCh] and contributes to frequency of [Ca2+]i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca2+ dependent gene expression pathways, consistent with the different patterns of [Ca2+]i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca2+]i generated by Orai1, independent of global [Ca2+]i changes or TRPC1-mediated Ca2+ entry. In contrast, Ca2+ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca2+ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca2+-dependent gene expression pathways.Regulation of critical cellular functions, including Ca The temporal and spatial pattern of [Ca2+]i signals generated in response to agonist stimulation are utilized by the cells to regulate various critical functions, such as gene expression, ion channel activation and fluid secretion 2+]i, whereas lower [agonist] elicit oscillatory [Ca2+]i responses . Two types of oscillations are seen; baseline oscillations that are usually seen at very low [agonist] or oscillations over a sustained elevation in baseline [Ca2+]i at relatively higher [agonist]. Such oscillatory responses have been proposed to represent the physiological mode of signaling in many cell types and have been observed in almost all cell types, including cell lines as well as primary cell preparations from various tissues 2+ release from the ER stores via IP3Rs, inhibition of Ca2+ release, and reuptake into the store due to SERCA pump activity. In several cell types, sustained [Ca2+]i oscillations require extracellular Ca2+ influx to achieve refilling of the ER store after every release event, thus priming it for the next release cycle. Even in cells where the oscillations are sustained for longer periods in the absence of external Ca2+, intracellular Ca2+ stores are eventually depleted and there is a run-down of [Ca2+]i oscillations.Stimulation of cells with physiologically relevant agonists that target G protein- or tyrosine kinase-coupled receptors leads to increases in cytosolic [Ca2+ within the ER as a result of IP3-induced Ca2+ release following agonist stimulation of cells 2+]i oscillations in a number of cells. Removal of extracellular Ca2+, or inhibition of Ca2+ influx with La3+, induced cessation of [Ca2+]i oscillations 2+ binding protein that serves as a sensor for ER-[Ca2+] and regulates plasma membrane calcium channels mediating SOCE. 2+-selective Ca2+ release-activated Ca2+ (CRAC) channel has now been established as an essential component of SOCE 2+ influx in some cell types 2+ entry triggers recruitment of TRPC1 into the plasma membrane where it is activated by STIM1 2+ entry in salivary acinar cells is associated with loss of salivary fluid secretion as well as Ca2+-dependent K+ channel activation 2+]i signals that are specifically required for the activation of KCa channels in acinar cells, which cannot be achieved or are not compensated for by the residual Orai1 channel in these cells. A similar decrease in Ca2+-activated Cl- channel activity has been shown in pancreatic acini from TRPC1−/− mice Store-operated calcium entry (SOCE) is activated in response to depletion of Ca2+]i oscillations in RBL cells and T lymphocytes 2+]i oscillations have been shown to underlie regulation of Ca2+-dependent gene expression via activation of the transcription factor, nuclear factor activated T cells (NFAT). NFAT is translocated in an “all-or-none” manner following its activation which involves its complete dephosphorylation by calcineurin, a Ca2+-CaM dependent phosphatase 2+-dependent degradation of the inhibitor of NFκB (IκB), and is suggested to exhibit a strong dependence on peak amplitude, rather than duration, of a [Ca2+]i signal. In B cells, a high level of [Ca2+]i, exceeding that achieved by CRAC channel activity alone, is required for activation of NFκB. CRAC channel activity has been associated with i increases in human submandibular gland (HSG) cells, but only Orai1-mediated Ca2+ entry regulates the activation of NFAT, it is unclear how the cells decode individual Ca2+ signals generated by the two channels for the regulation of specific cellular functions. We hypothesized that the two channels induce distinct [Ca2+]i signals which determine their functional specificity in the regulation of Ca2+-dependent gene expression. To assess this, we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca2+]i signals and Ca2+-dependent gene expression in single cells. Herein we report that endogenous Orai1 and TRPC1 channels contribute to distinct local and global [Ca2+]i signals following agonist stimulation of salivary gland epithelial cells. Increasing concentrations of a physiologically relevant agonist, CCh (0.3 µM to 100 µM) induced baseline [Ca2+]i oscillations, oscillations over a sustained baseline elevation, or sustained baseline elevation without oscillations. While suppression of Orai1 expression or function completely eliminated Ca2+ influx at all [CCh], inhibition of TRPC1 reduced sustained [Ca2+]i elevation and decreased the frequency of residual [Ca2+]i oscillations. Thus, Orai1 channel function is the primary determinant of [Ca2+]i oscillations. Importantly, at any [agonist] and irrespective of TRPC1 contribution to [Ca2+]i, “all-or-none” activation of NFAT was solely dependent on Orai1 and did not reflect global [Ca2+]i. In contrast, TRPC1 had a substantial contribution to activation of NFκB-dependent gene expression. In aggregate, these novel findings demonstrate that Orai1 and TRPC1 mediate distinct and specific [Ca2+]i signals following agonist stimulation that determine their differential activation of Ca2+-dependent transcription factors in a single cell.Our previous study suggested that Orai1-mediated Ca2+]i elevation was seen, with an initial rapid increase followed by a sustained elevation above baseline that slowly declined over time , and lower than in control cells (0.193±0.014)  or in cells expressing siOrai1 or Orai1E106Q (both TRPC1 and Orai1 functions are attenuated). Suppression of both Orai1 and TRPC1 functions induced an overall reduction in [Ca2+]i elevation, similar to that seen with siSTIM1, with abrogation of both the oscillatory responses as well as sustained [Ca2+]i elevation . Interestingly in cells expressing STIM1-KK/EE, the proportion of cells with [Ca2+]i oscillations over an elevated baseline decreased to 10% (from 66% in control cells) and those with baseline oscillations increased to 63%, whereas only 6% of cells showed transient responses. Similar changes were seen in cells expressing shTRPC1. In the case of Orai1E106Q- and siOrai1-expressing cells, the proportion of cells with transient response was increased to 40% or 67%, respectively, as would be expected if the SOCE process was inhibited .The relative proportions of cells affected by these maneuvers are shown in 2+ entry via TRPC1 and Orai1 channels generate distinct cytosolic Ca2+ signals. Orai1 primarily controls oscillatory [Ca2+]i signals while TRPC1-mediated Ca2+ entry generates a more sustained elevation of [Ca2+]i. These contributions of Orai1 and TRPC1 to CCh-stimulated [Ca2+]i signals were confirmed in HEK293 cells. HEK293 cells displayed baseline oscillatory [Ca2+]i responses following stimulation with 1 µM CCh, which were primarily dependent on the STIM1-activated Orai1 channel , reaching a maximum at about 20 min which was maintained during the period of the experiment (60 min)  . NFAT ac1 µM CCh . At this2+]i signals. Thus, we measured [Ca2+]i changes elicited by relatively high [CCh] (100 µM) in cell populations, as well as in individual cells. In control cells, there was higher and more sustained [Ca2+]i elevation with <10% of the cells showing any type of oscillations  were slightly more prolonged . Importantly at this low level of stimulation, there was a detectable contribution of TRPC1 to the frequency of [Ca2+]i oscillations as cells expressing STIM1-KK/EE showed a rundown of oscillations with >75% decrease in the number of oscillations , only about 30% of the cells responded by displaying sustained baseline oscillations . Initial2+]i changes and NFAT activation seen in cells stimulated with 300 nM CCh. Despite detection of [Ca2+]i oscillations in 30% of the cells, only 4.8% of the cells exhibited nuclear translocation of NFAT . Since the number of oscillations at this very low [CCh] is about 40–50% less and amplitude of the [Ca2+]i signal is ∼75% lower than that at 1 µM CCh  was insufficient to induce activation of NFAT1, a second pulse of 120 nM LTC4  within 10 min of the first was sufficient to activate NFAT1. We have observed similar results when two pulses of 300 nM CCh within 3 min of each other were added to the cells. The number of cells showing nuclear translocation of NFAT increased from about 4.35% (n = 184) to 11.7% (n = 171). Furthermore, this was not altered by suppression of TRPC1 function in STIM1-KK/EE-expressing cells, where 14.4% of cells (n = 118) showed NFAT activation following two pulses of 300 nM CCh (c.f. 5.8% (n = 86) for a single pulse). We suggest that the first pulse of CCh was not sufficient to completely dephosphorylate NFAT, while the second pulse of agonist achieved the extra [Ca2+]i increase needed to fully dephosphorylate NFAT, a requirement for its nuclear translocation. Collectively, the data presented in 2+]i responses at very low [agonist], nuclear translocation of NFAT is solely dependent on local [Ca2+]i signals generated by Orai1. Further, the lack of NFAT translocation at low levels of stimuli when compared to the relatively higher levels, suggests that local [Ca2+]i required to drive this process is likely to depend on the number of Orai1 channels activated at any given [agonist] and that NFAT regulation is mediated by an “all-or-none” mechanism in which complete dephosphorylation of the transcription factor is required for its nuclear translocation. What is more important is that TRPC1-mediated Ca2+ entry does not modulate the local [Ca2+]i near Orai1, although it increases global [Ca2+]i and contributes to the frequency of [Ca2+]i oscillations.A recent study showed that there is a threshold for local [Ca2+]i signal “memory” is retained even after the initial [Ca2+]i elevation has declined. Both NFAT- and NFκB-driven luciferase  activities were clearly detected following 100 µM CCh treatment of cells . Consistent with the findings shown in 2+ entry is required for the assembly of functional TRPC1-STIM1 channels in the plasma membrane 2+-dependent gene expression.To further establish whether the functional specificity of TRPC1 and Orai1 seen in short-term responses (i.e. nuclear translocation of NFAT) is also retained for long-term effects at the level of gene expression in the nucleus, we measured NFAT- or NFκB-driven luciferase activities. Such long-term effects would indicate that a [Ca2+]i signals. It is not fully understood how cells decode [Ca2+]i signals originating from multiple sources for the regulation of specific Ca2+-dependent functions. Variations in the pattern of individual [Ca2+]i signals generated by the two channel types is most likely the primary determinant of the functional specificity of the channels in regulation of cell function. Here we have studied the contributions of endogenous TRPC1 and Orai1 to agonist-stimulated [Ca2+]i signals in a single HSG cell. We show that Ca2+ entry via each channel generates a specific pattern of [Ca2+]i elevation, with Orai1 controlling the generation of [Ca2+]i oscillations and TRPC1 mediating sustained [Ca2+]i elevation at higher [agonist] and contributing to the frequency of baseline [Ca2+]i oscillations. Even more significant is the finding that the channels display functional specificity in the activation of Ca2+-dependent transcription factors and gene expression. Consistent with the oscillatory [Ca2+]i signals generated by Orai1, NFAT translocation and NFAT-dependent gene expression were exclusively dependent on Orai1-mediated Ca2+ entry, without any contribution of TRPC1. Our data suggest that NFAT is strictly regulated by the [Ca2+]i achieved locally near the Orai1 channel, likely due to localization of calmodulin-calcineurin-NFAT within the Orai1-associated microdomain, such that the Ca2+ entering via Orai1 can be locally sensed by the calcium sensor. Moreover, since Ca2+ entering into this microdomain via Orai1 rapidly rises to concentrations that exceed a threshold level required for activation, NFAT activation did not reflect global [Ca2+]i changes achieved at the various stimulus intensities. We also show that NFAT activation follows an “all-or-none” mode of activation which is strictly dependent on Orai1; if an insufficient number of Orai1 channels is activated, NFAT dephosphorylation is not completed and nuclear translocation does not occur. However, we cannot rule out the possibility that at even higher level of stimulus (e.g. a more potent agonist or involving different receptor pathways) or if more channels were expressed, this pattern could vary and sustained [Ca2+]i elevations could be induced by Orai1 (e.g. in lymphocytes or in HEK293 cells overexpressing Orai1+STIM1). In contrast to the regulation of NFAT, we show that NFκB is primarily regulated by TRPC1. Furthermore, we previously reported that Ca2+ entry via TRPC1, but not Orai1, is required for sustained activation of KCa in HSG cells as well as acinar cells isolated from mouse salivary glands 2+]i achieved near the TRPC1 channel or global [Ca2+]i changes mediated by TRPC1 are involved in the activation of NFκB and KCa channel. In aggregate, these findings provide conclusive evidence that Orai1 and TRPC1 generate functionally specific local and global [Ca2+]i signals.In some cell types, including salivary gland cells, more than one channel contributes to agonist stimulated [Ca2+]i signals is the clustering of the channels within the signaling microdomain 2+ signaling components, such as Ca2+ pumps in the ER and plasma membrane or mitochondria, could also affect the amplitude as well as temporal and spatial aspects of [Ca2+]i signals 2+]i signals that are decoded to induce NFAT-driven gene expression in RBL cells 2+ signaling are triggered by Cav1 and Cav2 channels within the same neurons, which are differentially used for regulating gene expression v1 and Cav2 channels are activated following plasma membrane depolarization and employed the same calmodulin kinase (CaMK)-dependent pathway to activate CREB-dependent gene expression. Nonetheless, Ca2+ entry via both channels contributed to different pools of Ca2+ within a single neuron with Cav1 contributing to the local [Ca2+]i but Cav2 to the global [Ca2+]i increases. Interestingly, Cav1 was generally clustered close to the puncta of βCaMKII, the predominant CaMKII isoform in neurons, while Cav2 clusters were located supramicrons away. Another important point was that Ca2+ entry via Cav2 channels was preferentially buffered by the ER and mitochondria. Hence, Cav2-mediated Ca2+ signaling was dampened to a greater degree than Cav1 and requires a greater depolarizing stimulus for channel activation 2+ signaling microdomain associated with TRPC1 and Orai1 needs to be further investigated. It is important to note that while the present study has revealed the Orai1-dependent [Ca2+]i changes that underlie CCh-stimulated [Ca2+]i increases, we have not yet resolved the [Ca2+]i signals generated by TRPC1 alone. We have identified the contributions of TRPC1 to the overall [Ca2+]i increases, but since TRPC1 function obligatorily depends on Orai1, we currently do not have conditions where Orai1 function can be selectively inhibited following activation of both channels. Efforts are underway to sort out this rather complicated issue.An important determinant in the generation of local i signals, it does not have any impact on the local Ca2+ signals driven by Orai1. This was demonstrated by the exclusive role of Orai1 in regulation of NFAT, without any effect of global [Ca2+]i increases or contributions due to TRPC1-mediated Ca2+ entry. Together our findings strongly demonstrate that distinct global and local [Ca2+]i signals are generated by Orai1 and TRPC1 in a single cell, which are specifically decoded to activate different gene expression pathways. Moreover, stimulus intensity determines the number of each channel that is activated and consequently the magnitude of [Ca2+]i achieved locally near the pore of either channel. Further studies will be required to measure the local [Ca2+]i within each channel microdomain and identify the components involved in decoding the specific [Ca2+]i signals generated by them, as well as the mechanisms utilized for acute and long-term regulation of cellular functions.In conclusion, agonist stimulation of cells leads to activation of SOCE with contributions from both TRPC1 and Orai1 channels. While CaHSG and HEK293 cells were cultured as described previously t−F0).Fura-2 fluorescence was measured in single HSG cells as described previously 2 camera  and the MetaMorph imaging software (Molecular Devices). MetaMorph was also used to measure the fluorescence intensity in the nucleus and cytoplasm before and after stimulation with CCh. Briefly, regions of interest were selected to obtain the values for their fluorescence intensities during a time course experiment. These values were then plotted using the Origin 8 software . Due to the low responsiveness of HSG cells to stimulation with 300 nM CCh, a 20× fluorescence objective was used to screen larger numbers of cells.Translocation of NFAT in transfected HSG cells was observed using an Olympus IX81 motorized inverted microscope  and a TIRF-optimized Olympus Plan APO 60× (1.45 NA) oil immersion objective. Excitation was achieved 3 per well in a 96-well plate one day prior transfection. The shTRPC1 (0.25 µg/well) and siOrai1 (200 nM/well) constructs were transiently transfected into cells using Lipofectamine 2000 or RNAiMAX (Invitrogen), respectively, following manufacturer’s protocol. After 24 h, the firefly luciferase reporter constructs for NFAT (pGL4.30[luc2P/NFAT-RE/Hygro]) or NFκB (pGL4.32[luc2P/NF-κB-RE/Hygro]) were transfected with the renilla luciferase reporter construct  into HSG cells using Lipofectamine 2000 for another 24 h. All three luciferase constructs were obtained from Promega . Cells were then left untreated or treated with CCh at various concentrations for 6 h at 37°C. For NFAT luciferase activity, cells were stimulated with CCh alone. For NFκB luciferase activity, cells were stimulated with CCh in the presence of PMA (10 ng/ml). Luciferase activities were determined using Dual-Glo Luciferase Assay, as per manufacturer’s instructions (Promega). Luminescence intensity was monitored in using the FLUOStar OMEGA microplate reader . At least 3 separate experiments were performed using samples in triplicates. The firefly luciferase values were normalized to renilla luciferase values. All data were presented as fold-change relative to the vector control.HSG cells were seeded at 15×10p<0.001.Data analysis was performed using Origin 7.0 (OriginLab). Statistical comparisons were made using Student’s t-test. Experimental values are expressed as mean±SEM. Differences in the mean values were considered to be significant at Figure S12+i responses in HEK293 cells.Effect of expressing TRPC1 and STIM1 on low [CCh]-induced Ca Baseline Ca2+i oscillations in HEK293 cells following 1 µM CCh stimulation in control cells (A), cells expressing TRPC1+STIM1 (B) or STIM1-KK/EE (C), and control cells without Ca2+ present in the extracellular medium (D). Each trace is representative of ≥20 cells in at least 2 separate experiments.(TIF)Click here for additional data file.Figure S22+]i increases in HSG cells stimulated with high [CCh].SOCE-driven [Ca [Ca2+]i responses induced by high [CCh] (100 µM) in control HSG cells (A) and cells expressing siSTIM1 (B), Orai1E106Q (C), siOrai1 (D), shTRPC1 (E), or STIM1-KK/EE (F). Each trace is representative of ≥50 cells in at least 3 separate experiments (G). Average data showing amplitude of [Ca2+]i increase at t = 250 s (Ft−F0). *** indicates a significant difference .(TIF)Click here for additional data file.


Introduction: Genetic forms of the Short QT Syndrome (SQTS) arise due to cardiac ion channel mutations leading to accelerated ventricular repolarization, arrhythmias and sudden cardiac death. Results from experimental and simulation studies suggest that changes to refractoriness and tissue vulnerability produce a substrate favorable to re-entry. Potential electromechanical consequences of the SQTS are less well-understood. The aim of this study was to utilize electromechanically coupled human ventricle models to explore electromechanical consequences of the SQTS.Methods and Results: The Rice et al. mechanical model was coupled to the ten Tusscher et al. ventricular cell model. Previously validated K+ channel formulations for SQT variants 1 and 3 were incorporated. Functional effects of the SQTS mutations on [Ca2+]i transients, sarcomere length shortening and contractile force at the single cell level were evaluated with and without the consideration of stretch-activated channel current (Isac). Without Isac, at a stimulation frequency of 1Hz, the SQTS mutations produced dramatic reductions in the amplitude of [Ca2+]i transients, sarcomere length shortening and contractile force. When Isac was incorporated, there was a considerable attenuation of the effects of SQTS-associated action potential shortening on Ca2+ transients, sarcomere shortening and contractile force. Single cell models were then incorporated into 3D human ventricular tissue models. The timing of maximum deformation was delayed in the SQTS setting compared to control.Conclusion: The incorporation of Isac appears to be an important consideration in modeling functional effects of SQT 1 and 3 mutations on cardiac electro-mechanical coupling. Whilst there is little evidence of profoundly impaired cardiac contractile function in SQTS patients, our 3D simulations correlate qualitatively with reported evidence for dissociation between ventricular repolarization and the end of mechanical systole. C interval of ~320 ms or less, tall and peaked T-waves, and increased Tpeak − Tend width  was first recognized as a distinct clinical entity in 2000  gene encoding the α-subunit of the rapidly-activating delayed rectifier K+ channel IKr . SQT1-3 result from + channel openers to left ventricular wedge preparations  and (2) to evaluate the relationship between ventricular repolarization and mechanical systole in the setting of the SQTS. In order to address these aims, established models of the SQT1 and SQT3 K+-channel-linked SQTS variants  and N588K-mutated hERG/For electrophysiology, we utilized the ten Tusscher and Panfilov (TP) human ventricular single cell model  was used as the coupling link to the myofilament mechanics model (MM). [Ca2+]i produced as dynamic output from the EP model during the time course of the AP served as input to the MM model from which the amount bound to troponin is calculated. The formulation of the myoplasmic Ca2+ concentration in the EP model is:Caibufc is the total cytoplasmic buffer concentration, Vsr is the sarcoplasmic reticulum (SR) volume, Vc is the cytoplasmic volume, Ileak is the SR Ca2+ leak current, Iup is the SR Ca2+ pump current, Ixfer is the diffusive Ca2+ current current between dyadic Ca2+ subspace and bulk cytoplasm, Cm is the membrane cell capacitance per unit surface area, IbCa is the background Ca2+ current, IpCa is the plateau Ca2+ current, INaCa is the Na+/Ca2+ exchanger and F is the Faraday constant.The intracellular calcium concentration i transient, SL shortening and the active force and—notably—SR calcium content (CaSR). These effects are similar to the results shown in Figure 2+]i, SL shortening, CaSR and the active force can be attributed solely to consequences of application of the shorter AP waveform. This suggests that the key reason for the reduced active force in the SQTS setting .The observed reduction in the active force under the mutation conditions in Figure g Figure  is the i2+]i transient than that when conditioning trains of shorter duration APs were applied. Results are shown in Figure ICaL  to the WT electromechanics model; the conditioning train was followed by an identical single square voltage command to +10 mV for 300 ms Figure . With thL Figure , but a se Figure  and actie Figure  for the e Figure . This fuIsac. Figures +, K+ and Ca2+ in the ratio 1:1:0  and to −79 mV (Isac at 1:1:1 permeability ratio) for the WT and SQT1 conditions respectively. Depolarization of the membrane potential is an effect of SACs, which has been observed experimentally  produced the greater effect, with contractility across the ventricular wall being approximately 85% of control under the SQT1 mutation and 92% of control under the SQT3 mutation. In contrast, with Isac incorporated at a permeability ratio of 1:1:0, on average across the ventricular wall, the contractile force was 62% of control under the SQT1 condition and 82% of control under the SQT3 condition.We then performed comparable simulations with the incorporation of 0 Figure  and 1:1:0 Figure . The res Figures , 7Ai–Ci. Figures , SLs  on incorporation of Isac, which consequently led to greater cell contractility in the SQT1 and SQT3 mutations particularly with a permeability ratio of 1:1:1  in the WT, SQT1 and SQT2 conditions. Under the WT, SQT1 and SQT3 conditions, it was shown that incorporation of Isac did not produce a noticeable change in the amplitude of ICaL, but elevated [Na+]i, [Ca2+]i, and CaSR. These changes in the intracellular Na+ and Ca2+ concentrations were associated with an altered INaCa as shown in Figures Isac was absent, during the initial depolarization phase of the AP, INaCa operated briefly in its reverse-mode that brought Ca2+ into the cytoplasmic space, producing an outward INaCa. During the plateau and early repolarization phases, INaCa remained almost zero for a period before switching to a forward mode to extrude Ca2+ out of cell cytoplasmic space, producing an inward INaCa current in the late repolarization phase. However, in the case with Isac, the activation of Isac brought more Na+ into the cell cytoplasmic space (as it is permeable to Na+) producing an elevated level of [Na+]i  into our single cell models to investigate the consequences of its inclusion under WT and SQTS mutation conditions. Our simulations suggest that: (i) at least in silico, abbreviated repolarization in the SQTS has the potential to reduce ventricular mechanical function; (ii) the inclusion of (Isac) in the model acts to maintain the normal amplitude of the contractile force   of the force reduction in the SQTS settings was observed for the maximal SOC channel conductance varying from 0.2 to 20 pS/pF. Whilst it is important that these potential limitations are stated, they do not fundamentally alter the principal conclusions of this study.In addition to acknowledged limitations of both the TP electrophysiology model (Ten Tusscher and Panfilov, Isac offsets a negative inotropic effect of ventricular action potential abbreviation that might otherwise occur for K+-channel linked SQTS. If, in vivo, Isac does not execute such a role, then it is possible that other compensatory changes exist in SQTS patients as accelerated repolarization might otherwise result in altered SR Ca2+ loading and a reduction in contractile activity.Our tissue simulations qualitatively reproduce and provide a possible explanation for dissociation between the end of mechanical systole and ventricular repolarization (Schimpf et al., 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.


The bundle Ca2+ signal reported the physiological state of the bundle and cell; extruding cells had very high bundle Fluo-4 fluorescence, cells with intact bundles and tip links had intermediate fluorescence, and damaged cells with broken tip links had low fluorescence. Moreover, Fluo-4 fluorescence in the bundle correlated with Ca2+ entry through transduction channels; mechanically activating transduction channels increased the Fluo-4 signal, while breaking tip links with Ca2+ chelators or blocking Ca2+ entry through transduction channels each caused bundle and cell-body Fluo-4 fluorescence to decrease. These results show that when tip links break, bundle and soma Ca2+ decrease, which could serve to stimulate the hair cell’s tip-link regeneration process. Measurement of bundle Ca2+ with Fluo-4 AM is therefore a simple method for assessing mechanotransduction in hair cells and permits an increased understanding of the interplay of tip links, transduction channels, and Ca2+ signaling in the hair cell.We optimized Fluo-4 AM loading of chicken cochlea to report hair-bundle Ca In addition to the typical roles that Ca2+ plays in other neuronal cell types, such as modulating neurotransmitter release and synaptic transmission 2+ also influences three components of mechanotransduction. First, Ca2+ sets the resting open probability of the mechanotransduction channel 2+ modulates slow adaptation by changing the activity of the adaptation motor 2+ controls fast adaptation, perhaps by binding directly to the mechanotransduction channel 2+ may influence the regeneration of broken tip links 2+-permeable 2+ in the mechanically sensitive hair bundle that may modulate these processes; indeed, in one model, the predicted–but never before measured–decrease in resting bundle Ca2+ after tip links break is the signal that triggers tip-link regeneration Hair cells of the inner ear are specialized sensory cells that rely on intracellular Ca2+ imaging experiments use a whole-cell recording electrode to deliver a fluorescent Ca2+ indicator 2+ dynamics. However, only one cell is examined at a time, and surrounding hair cells are damaged to gain access to the cell of interest. Because one must individually dialyze each cell, studying a population of cells is time-consuming. Furthermore, the intracellular environment may become compromised during whole-cell recordings; for example, Ca2+ buffering by the pipette solution may not replicate that of the unperturbed hair cell.Typical hair-cell Ca2+ indicators in hair cells. To examine defects in the hair-bundle Ca2+ pump, Bortolozzi and colleagues used the cell-permeable Ca2+ indicator Fluo-4 AM to monitor clearance rates in the bundle after uncaging intracellular Ca2+2+ dye, Oregon Green BAPTA 488 AM, in a semi-intact mouse cochlear preparation, reporting modest changes in cell body fluorescence in response to stimulation of the stapes as an indicator of mechanotransduction 2+ loading of the cells A handful of previous studies have reported the use of cell-permeable Ca2+ simultaneously in a population of hair cells, we have optimized loading of hair cells with the cell-permeable Ca2+ indicator Fluo-4 AM. This minimally-invasive protocol labeled most hair cells in the chicken cochlea while maintaining excellent tissue and hair-bundle morphology. By combining live-cell imaging with scanning electron microscopy (SEM), we correlated Fluo-4 bundle fluorescence with the presence of tip links. Direct displacement of the bundle by a fluid jet verified that the Fluo-4 bundle signal was due to Ca2+ entry through functional transduction channels. Both breaking tip links and blocking the transduction channel lowered the intracellular Fluo-4 fluorescence, which confirms that when tip links break, bundle Ca2+ decreases.To examine intracellular Ca2+ dye that shows a >100-fold increase in fluorescence when bound to Ca2+d of 350 nM, similar to the buffering capacity of BAPTA (136 nM), Fluo-4 is sensitive enough to detect and report low concentrations of free intracellular Ca2+. Modification of the dye with an acetoxymethyl (AM) ester moiety allows this dye to cross the cell membrane, whereupon endogenous esterases cleave the AM group to liberate the active dye. Fluo-4 AM is virtually non-fluorescent, ensuring that the indicator fluoresces only after the AM group is cleaved and Ca2+ binds Fluo-4 intracellularly.Fluo-4 is a high-affinity CaTo load hair cells with Fluo-4 AM, post-hatch chicken cochleae (P0) were removed from the skull and acutely dissected at room temperature, with removal of the tectorial membrane after incubation with the protease subtilisin. Careful preparation of the Fluo-4 AM dye, as well as optimization of incubation time and temperature, were necessary to ensure the dye stayed in solution and was taken up by hair cells with minimum entry through endocytosis ; Ref. 172+ indicator signal using Fluo-3 was greater at the stereocilia tips, where transduction channels are located 2+ at some stereocilia tips, thought to represent Ca2+ entry through functional transduction channels, in 9 out of 13 cells analyzed. Bundle fluorescence was influenced by the concentration of extracellular Ca2+, with 0.5 mM Ca2+ paradoxically yielding a brighter Fluo-4 signal in the bundle compared to 1.26 mM Ca2+ (data not shown). Consistent with this result, high extracellular Ca2+ is known to partially block the Ca2+ current through the transduction channel 2+ affinity, cells loaded with this dye also had the brightest signal in the bundle at rest. We therefore used this dye for our experiments.In transducing hair cells, Lumpkin and Hudspeth reported that the Ca2+ imaging, organs were fixed and processed for SEM. A modified sample-preparation method that included dehydration with progressive cooling was used to preserve tip links To examine the ultrastructure of the hair bundle and overall epithelium after Ca2+ entry through functional mechanotransduction channels. Tip links are disrupted by exposure to extracellular Ca2+ chelators The presence and number of tip links was further examined by high-resolution SEM. We examined regions of epithelia loaded with Fluo-4 that showed intermediate levels of bundle fluorescence; in these undamaged regions, we found tip links in 92±5% of the positions where tip links were expected  axis of the epithelium, to maximally stimulate bundles along their axis of sensitivity ; the approval number was A684. All experiments began with euthanasia of the animal, which was carried out using methods approved by American Veterinary Medical Association Panel on Euthanasia.Gallus domesticus chickens were dissected as described previously 2, 1.25 NaH2PO4, 2 ascorbate, 2 creatine, 6 Na-pyruvate, 75 sucrose, 25 D-glucose, 10 HEPES . Fluo-4 AM dye was prepared as described in P0 2 immediately prior to use. Organs were incubated in EGTA solution for 5 min prior to Fluo-4 AM loading, then returned to normal Ca2+ solution for all subsequent steps.Stock solutions of (+)-tubocurarine chloride (Sigma) at 50 mM were stored at −20°C and diluted to 100 µM in extracellular solution a few hours before use. Tubocurarine was added to organs prior to Fluo-4 AM loading and was included in all washing and imaging solutions. A stock solution of 0.5 M EGTA was stored at room temperature and diluted to 5 mM in extracellular solution lacking CaCl2+ signals, organs were imaged on an inverted Zeiss LSM710 confocal microscope (40xW/1.1 NA objective), bundles facing down. For the fluid jet experiments, an upright Olympus FV-1000 confocal microscope was used (60xW/1.0 NA or 40xW/0.8 NA dipping objectives). Dental floss attached to an electrophysiology harp was used to pin the organ down at the lagena and basal ends. Images were acquired using 1.0–2.0 µm Z-steps through the hair bundles and cell bodies. Z-stacks were processed with Imaris 3D software, where individual 2 µm XZ- or YZ-reslice images were taken through the field of hair cells. Fiji software (http://fiji.sc/wiki/index.php/Fiji) was used to select and quantify hair-bundle and cell body ROIs; integrated density was calculated and normalized to the control mean for each data set. In some experiments, organs were immediately placed into SEM fixative following imaging, fixed overnight at 4°C, and processed for SEM.For measuring control, EGTA, and tubocurarine Fluo-4 CaOrgans were fixed in the dark at 4°C overnight using 2% gluteraldehyde, 1% tannic acid in 0.1 M cacodylate buffer (Electron Microscopy Sciences), washed 3 times in PBS, and stored for less than 2 days at 4°C. Organs were post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences) in 0.1 M cacodylate buffer for 15 min, followed by 4–5 washes in PBS. To preserve tip links, organs were dehydrated in the following acetone series with sequential cooling, using 15 minutes for each step: 30% at 4°C, 50% at −20°C, 70% at −35°C, 90% at −35°C, 100% at −35°C, 100% at −35°C, and 100% at 4°C. Samples were kept on ice and immediately critical point dried in 100% cold ethanol. Organs were mounted in silver paint on carbon-coated stubs, sputter coated in gold/palladium to yield a ∼10 nm coat, and imaged on an FEI Sirion field-emission scanning electron microscope with 5 kV accelerating voltage.Borosilicate glass pipettes with long, thin shafts were pulled and broken to yield 10–12 µm diameter tips. An open flame was used to bend the pipette along the shaft to a 30° angle, which allowed the pipette to be lowered close enough to the tissue without bumping the sidewall at the basal end of the cochlea. Pipettes were filled with extracellular solution. A Pico-Spritzer III controlled the pipette, which was mounted on a micromanipulator on the microscope stage and positioned underneath the objective. Olympus FV-1000 software was used to deliver 10 sec, 0.2 psi pulses; an in-line manometer was used to accurately measure pressure in mm Hg. By isolating a single hair bundle region of interest (ROI) of 12×20 pixels, two-color images were acquired at 70 milliseconds per frame. Data was analyzed using IgorPro software, and ΔG/R was calculated by normalizing Fluo-4 to CT-Red, then normalizing to the average response from the first 40 seconds of imaging.


Therefore, we tested the hypothesis that dietary n-3 PUFAs alter the vulnerability of ventricular myocytes to cellular arrhythmia by stabilizing intracellular Ca2+ cycling. To test this hypothesis, we used a canine model of post-MI ventricular fibrillation (VF) and assigned the animals to either placebo (1 g/day corn oil) or n-3 PUFAs (1-4 g/day) groups. Using Ca2+ imaging techniques, we examined the intracellular Ca2+ handling in myocytes isolated from post-MI hearts resistant (VF-) and susceptible (VF+) to VF. Frequency of occurrence of diastolic Ca2+ waves (DCWs) in VF+ myocytes from placebo group was significantly higher than in placebo-treated VF- myocytes. n-3 PUFA treatment did not decrease frequency of DCWs in VF+ myocytes. In contrast, VF- myocytes from the n-3 PUFA group had a significantly higher frequency of DCWs than myocytes from the placebo group. In addition, n-3 PUFA treatment increased beat-to-beat alterations in the amplitude of Ca2+ transients  in VF- myocytes. These n-3 PUFAs effects in VF- myocytes were associated with an increased Ca2+ spark frequency and reduced sarcoplasmic reticulum Ca2+ content, indicative of increased activity of ryanodine receptors. Thus, dietary n-3 PUFAs do not alleviate intracellular Ca2+ cycling remodeling in myocytes isolated from post-MI VF+ hearts. Furthermore, dietary n-3 PUFAs increase vulnerability of ventricular myocytes to cellular arrhythmia in post-MI VF- hearts by destabilizing intracellular Ca2+ handling.It has been proposed that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) can reduce the risk of ventricular arrhythmias in post-MI patients. Abnormal Ca Cardiac arrhythmias are recognized as a major factor contributing to morbidity and mortality in patients with healed myocardial infarction (MI). The search for an effective anti-arrhythmic therapy remains a major unmet challenge. Initial observational and interventional studies indicated that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) may be effective in preventing cardiac arrhythmias –3. Howev2+ handling observed in various cardiac disease settings, including post-MI hearts, has been implicated in the genesis of both triggered and reentrant arrhythmias SR was significantly lower in n-3 PUFA-treated VF- myocytes compared to untreated control and VF- myocytes, respectively. More frequent Ca2+ sparks at lower [Ca2+]SR indicate increased RyR2 functional activity in VF- myocytes from n-3 PUFA-treated group.We further characterized the effect of dietary n-3 PUFAs on properties of intracellular Caed group ; table 12+ cycling occur following chronic dietary supplementation with n-3 PUFAs. Dietary n-3 PUFAs did not significantly affect expression of RyR2, SR Ca2+ ATPase, phospholamban (PLB), alpha subunit of cardiac L-type Ca2+ channels, and Na+/Ca2+ exchanger either in control or in VF- ventricular preparations  dietary n-3 PUFAs were not effective in inhibiting DCWs in ventricular myocytes isolated from VF+ animals; b) dietary n-3 PUFAs caused marked destabilization of intracellular Ca2+ cycling in myocytes from VF- animals manifested as an increased rate of occurrence of DCWs and increased amplitude of Ca2+ alternans; c) effects of dietary n-3 PUFAs observed in VF- myocytes were associated with enhanced RyR2 activity. These cellular findings may explain our previous in vivo observation that dietary n-3 PUFA not only failed to reduce the risk for ventricular tachyarrhythmias in VF+ dogs but actually increased arrhythmia formation in VF- dogs [In the present study we tested the hypothesis that dietary n-3 PUFAs would stabilize intracellular CaVF- dogs .2+ dysregulation is recognized as an important factor contributing to the genesis of various forms of cardiac arrhythmias. Remodeling of intracellular Ca2+ cycling leading to increased occurrences of spontaneous Ca2+ releases and diastolic Ca2+ waves is typically associated with triggered arrhythmias [2+ handling resulting in beat-to-beat variations in the amplitude of Ca2+ transient  are believed to contribute to reentrant excitation, providing an additional form of proarrhythmic dysregulation [2+ alternans [2+ cycling [2+ alternans  in contrast to physiological temperature (37°C) and O2 tension (~5%). Therefore, our experimental conditions could influence dynamics of intracellular Ca2+ cycling, fluidity of the membrane and potentially could alter the intracellular effects of incorporated n-3 PUFAs. Finally, efficacy of incorporated n-3 PUFAs may be different in canine and human hearts. We acknowledge that present study has some limitations that could affect the interpretation of the results. Due to technical reasons all cellular experiments were performed at room temperature (22-24°C) and ambient O2+ sparks in myocytes from post-MI (VF-) hearts. The increased frequency of Ca2+ sparks along with the reduced SR Ca2+ content observed in VF- myocytes suggest that incorporated n-3 PUFAs increased sensitivity of ryanodine receptors to SR Ca2+ in diseased hearts. We further demonstrated that dietary n-3 PUFA supplements were associated with a high predisposition of both VF- and VF+ myocytes to DCWs in response to β-adrenergic receptor stimulation. Thus, we conclude that incorporated n-3 PUFAs produce disturbances in Ca2+ cycling that would increase rather than decrease the risk for ventricular tachyarrhythmias in post-MI hearts. In the present study, we have demonstrated that increases in left ventricle n-3 PUFA content mediated by dietary intake of EPA +DHA ethyl esters similar to those noted in patients ,40 were


The ALDHbright compartment contains, besides normal HSC, more differentiated, normal CD34+CD38+ progenitors. Furthermore, we show that in CD34-negative AML, containing solely normal CD34+ cells, LSC are CD34– and ALDHlow. In CD34-positive AML, LSC are also ALDHlow but can be either CD34+ or CD34–. In conclusion, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that LSC have lower ALDH activity than the CD34+CD38– HSC that co-exist with these LSC in the AML bone marrow. Our findings form the basis for combined functionally and immunophenotypically based identification and purification of LSC and HSC within the AML bone marrow, aiming at development of highly specific anti-LSC therapy.Persistence of leukemic stem cells (LSC) after chemotherapy is thought to be responsible for relapse and prevents the curative treatment of acute myeloid leukemia (AML) patients. LSC and normal hematopoietic stem cells (HSC) share many characteristics and co-exist in the bone marrow of AML patients. For the development of successful LSC-targeted therapy, enabling eradication of LSC while sparing HSC, the identification of differences between LSC and HSC residing within the AML bone marrow is crucial. For identification of these LSC targets, as well as for AML LSC characterization, discrimination between LSC and HSC within the AML bone marrow is imperative. Here we show that normal CD34+CD38– HSC present in AML bone marrow, identified by their lack of aberrant immunophenotypic and molecular marker expression and low scatter properties, are a distinct sub-population of cells with high ALDH activity (ALDH Only a small subpopulation of cells within acute myeloid leukemia (AML) is responsible for sustaining the leukemia LSC capable of initiating human AML in NOD/SCID mice were thought to be solely of the CD34+CD38– phenotype, similar to the normal HSC Discrimination and purification of CD34+CD38– LSC and CD34+CD38– HSC have been performed by using leukemia-associated proteins identified by us and others low. LSC values are more than 1.4 times (FSC) and 1.7 times (SSC) that of the lymphocytes and defined as FSC/SSChigh. However, still in a considerable part of AML cases neither marker expression nor the combination with scatter properties is able to distinguish LSC from HSC. Therefore, we searched for a functional difference between normal HSC and LSC within the AML BM.Recently, we discovered additional differences between HSC and LSC in both AML and chronic myeloid leukemia (CML) Aldehyde dehydrogenases (ALDHs) are cytosolic enzymes involved in the conversion of retinol (Vitamin A) to retinoic acids and important in HSC maturation, differentiation and loss of quiescence low and of the CD34– immunophenotype. In CD34-positive AML, CD34+CD38– HSC are, like in CD34-negative AML, ALDHbright while the LSC are ALDHlow and either of the CD34+ or CD34– immunophenotype. This marked difference in ALDH activity between HSC and LSC offers an opportunity for identification and purification of LSC and CD34+CD38– HSC in every AML case.We investigated the ALDH activity in LSC as compared to normal CD34+CD38– HSC, both present in the BM of AML patients. We used discriminative aberrant cell surface markers and scatter aberrancies for the identification of HSC and LSC and analysed AML samples both from CD34-positive and CD34-negative AML. In this paper, we have demonstrated that, in both AML subtypes, high ALDH activity is an unique marker distinguishing CD34+CD38– HSC from CD34+ and CD34– LSC present within the BM of all the AML cases studied. In CD34-negative AML, CD34+CD38– stem cells are all normal (HSC) and have high ALDH activity. The LSC within this AML subtype are ALDHWe have defined two groups of AML cases based on frequency and pattern of CD34 expression in the blast compartment. One of the subtypes, which we call CD34-negative AML, has a small, usually less than 1%, population of CD34+ cells which are cytogenetically and molecularly normal, as assessed by FISH analyses bright. The CD34– cell population within normal BM is ALDHlow (low (bright cells comprised the complete (>99%) CD34+CD38– population of cells (n = 5).BM from a healthy donor contains a small population of cells with high ALDH activity . This AL ALDHlow , blue. Tbright and ALDHlow  had no expression of immunophenotypic leukemia-associated markers , strongly suggesting that ALDHbright cells are CD34+CD38– HSC. The level of ALDH activity corresponds with that of normal BM CD34+CD38– HSC  two separate ALDH activity populations, which we refer to as ALDH ALDHlow , were sebright, cell population of 7 of these 19 CD34-positive AML cases was essentially devoid of cells with the leukemia-specific cytogenetic abnormalities FLT3-ITD and/or mutated NPM1 . CD34+CD38– AML cells can be divided in an ALDHbright and ALDHlow compartment , the CD34+CD38– compartment can not be segregated into HSC and LSC by expression of aberrant immunophenotypic markers or by scatter properties. In such cases, nevertheless ALDH activity identified two separate populations within the CD34+CD38– compartment . The ALDlow and CLL-1+, FSC/SSChigh., respectively. These stem cell fractions were analysed for ALDH isoform expression by RNA-sequencing. The putative HSC contained within the AML BM have high expression of the ALDH1A1 enzyme as compared to almost no expression in the LSC (Different isoforms of ALDH might contribute to the ALDH activity profile seen. To search for the ALDH enzyme(s) responsible for the high ALDH activity seen in HSC as compared to LSC, the HSC from healthy donor BM and CD34+CD38– HSC and LSC from the AML BM were purified. HSC and LSC purified from the AML BM were defined as CLL-1-, FSC/SSC the LSC , AML-598 the LSC . In HSC bright CD34+CD38– cells by DEAB was not in all cases till the same level as the LSC present in the same sample  level of the population. We standardized the ALDH-MFI values of normal and neoplastic stem cell candidates by dividing these by the ALDH-MFI value of lymphocytes present within the same sample. Lymphocytes are negative for ALDH, resulting in MFI values that can be considered as background signal and as a stable characteristic of the individual sample. Treatment with DEAB was used in 9/19 samples as an extra control and showed similar basement ALDH activity levels in most samples used . From thbright compartment while there were essentially no CD34+CD38– cells in the ALDHlow compartment . The bulk of the AML (CD34– cells) were ALDHlow  and have low FSC/SSC . The ALDHlow compartment contained cells with various levels of ALDH activity within one AML patient but also inter-individual ALDH activity differences, relative to the ALDHbright compartment. These differences were also seen in earlier studies In AML, ALDH activity in leukemic blasts has been shown to define a subgroup with adverse prognosis and superior NOD/SCID engrafting potential In the present study, we have identified the activity of ALDH enzymes as a consistent functional discrimination between LSC and HSC concomitantly present in the BM of both CD34-negative and CD34-positive AML patients. Although HSC and LSC can, in a considerable part of AML cases, be distinguished using aberrancies of marker expression First, the relapse that occurs in 50% of AML patients after a seemingly successful chemotherapeutic treatment is likely caused by persistent LSC. This is reflected by the fact that frequency of LSC both at diagnosis and after treatment as well as expression of LSC signatures are tightly linked to the survival of AML patients Second, it is thought that LSC need to be eradicated in order to prevent relapse and to cure AML. The optimal anti-LSC therapy will spare the normal HSC. ALDH activity as a strong discriminative marker allows purification of LSC and HSC paving the way for LSC-specific target identification.Third, it could be hypothesized that firm differences in ALDH activity between HSC and LSC in the BM of AML patients might result in consequent resistance of HSC for particular drugs subjected to ALDH-dependent detoxification. The high ALDH activity in HSC, compared to LSC, provided to be effective for LSC, predicts lower toxicity of alkylating agents, such as cyclophosphamide. The difference between the ALDH activity in LSC and HSC defines here the therapeutic window. We are currently testing drugs, known to be dependent on low ALDH activity for proper activity, i.e. LSC-specific killing. Besides this, we are using drug libraries to identify novel drugs that can be detoxified via high ALDH activity. We hypothesize that AML patients with a large difference in ALDH activity between HSC and LSC might benefit from ALDH activity dependent treatment strategies.low compartment (defined by the inhibition with DEAB) which might represent LSC populations with different levels of ALDH activity. In the study by Pearce et al Lately, ALDH has received considerable attention as a functional marker for identification of cells with enhanced tumorigenic/metastatic potential and elevated therapeutic resistance in several cancers of epithelial origin The difference in ALDH activity level as seen between normal and neoplastic stem cells may also add to the discussion and controversies regarding the cell of origin for AML, i.e. derived from transformation of HSC or from more committed progenitors Overall, we show a marked difference between ALDH activity of HSC and LSC within the AML BM indicating the importance of ALDH activity as a functional stem cell biomarker and its potential usefulness in identification and purification of HSC and LSC, with the aim of treatment decision making, relapse prediction and development of LSC-specific therapies.BM samples were collected after informed consent from 32 AML patients at diagnosis. Normal BM was obtained after informed consent from healthy donors or patients undergoing cardiac surgery. The patients we used in this study gave informed consent following the procedure that was approved by the Medical Ethical Committee of our institute, the METC-VUmc. We (the Hematology department of the VUMC) have formal approval from the METC-VUmc for all our studies in acute myeloid leukemia patients. The patients gave written informed consent and the material of patients that did not gave this consent is not used for research purposes. AML phenotyping, cytogenetic, and molecular genetic analysis were carried out in the context of routine diagnostics at our facility. Cells were analyzed freshly or after thawing of samples frozen in liquid nitrogen. Mononuclear cells were isolated by Ficoll gradient . Red blood cells were lysed by 10 minutes incubation on ice, using 10 ml lysing solution containing 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA pH7.4 applied directly to the cell pellet. AML cells were frozen in RPMI  with 20% heat-inactivated fetal bovine serum  and 10% dimethylsulfoxide  in isopropanol-filled containers and subsequently stored in liquid nitrogen. When needed for analysis, cells were thawed and suspended in pre-warmed RPMI with 40% FBS at 37°C. Cells were washed in PBS with 0.1% HSA.6/ml) were resuspended in the aldefluor assay buffer at a concentration of 2×106 cells/ml. Cells were incubated with the ALDH-substrate BAAA  with or without the ALDH inhibitor, diethylamino-benzaldehyde  and incubated at 37°C for 60 minutes according to the manufacturer protocol. After performing the Aldefluor assay, cells were resuspended in icecold PBS/0.1%HSA and washed once. The BAAA substrate can be detected in the FITC channel of the flow cytometer.Primary AML cells  combinations consisting of phycoerythrin (PE), allophycocyanin (APC), R-phycoerythrin-cyanine 7 (PC7), peridinin chlorophyll protein (PERCP) labeled MoAbs, anti-CD45 PerCp, Anti-CD34 PC7, Anti-CD38 APC, and anti-CD7 PE, Anti-CD56 PE, Anti-CLL1 PE, Anti-CD19 PE, Anti-CD33 PE, Anti-CD22 PE, Anti-CD123 PE. The antibodies were all from BD Biosciences . After antibody staining, cells were washed with icecold PBS/0.1%HSA, resuspended in 250 µl cold PBS/0.1%HSA and stained for 5 minutes with Sytox Blue  enabling to exclude dead cells. Cells were kept on ice before FACS analysis. Labeled samples were analyzed and cells were sorted using a flow cytometer . Prior to molecular analysis, subfractions were sorted. Data acquisition was performed using either a FACS Calibur or a FACS CantoII, both from BD Biosciences. Analysis was performed using Cellquest and FACS Diva software (BD Biosciences). For analysis of the presence molecular aberrancies, cells were sorted directed into cold culture medium. Purity of sorted population was in most cases >98%.FLT3/ITD and NPM1. Direct PCR was applied to cell sorted subfractions under similar reaction conditions, except for the use of lower reaction volumes . Mutations in NPM1exon12 were analyzed with PCR with the following primers: NPM1forward: 5′-TTAACTCTCTGGTGGTAGAATGA-3′; NPM1 reverse: 5-CTGACCACCGCTACTATGT-3, located in intron 11 and exon12 of the NPM1 genomic DNA, respectively. FLT3 was amplified using the primers spanning the entire transmembrane and JM domains of FLT3. Subsequent fragment analysis was performed with a tetrachlorofluorescein phosphoramidite-labeled forward primer . For both FLT3 and NPM1 analysis, lymphocytes served as an internal negative control.To determine mutational status of flow cytometer-sorted subpopulations, only cryo-preserved cells were used. Genomic DNA (gDNA) was isolated from pellets of cells according to manufacturer’s instructions using the Quiagen Allprep kit . Mutation profiling was performed on isolated gDNA as previously described for Total RNA from cells  was extracted with TRIzol reagent  according to the manufacturer’s protocol. The quality of RNA samples was measured on a 2100 Bioanalyzer . The sequencing libraries, each with individual Illumina indexes, were constructed using the TruSeq™ RNA Sample Prep Kit v2  according to the manufacturer’s instructions. The resulting library DNA concentration and molarity was checked on a 2100 Bioanalyzer. Next, a mixture of three 10 pM libraries were pooled equimolar and the resulting DNA was clustered onto a V3 flowcell lane using a c-Bot cluster station and subsequently sequenced in single read fashion for 50 bp using the Illumina HiSeq2000 and V3 SBS Chemistry. Sequence reads were aligned to the human reference genome. In order to determine whether the aldehyde dehydrogenases are differentially expressed between samples, the R package DEGseq was used Figure S1bright and ALDHlow compartments.CD34-positive AML cases; variation in the difference in ALDH activity between ALDH ALDH versus SCC of the CD34+CD38– compartment of three CD34-positive AML cases. ALDH activity segregates the CD34+CD38– compartment in CD34+CD38– ALDHbright and CD34+CD38– ALDHlow cells. One AML case, AML-1048, with a relatively small difference between HSC and LSC ALDH activity levels (panel 1). An AML case, AML-1036, with a large difference between ALDH activity levels of HSC and LSC (panel 2). One AML case, AML-1013, with mainly HSC (panel 3).(TIF)Click here for additional data file.Figure S2Defining the ALDH compartments as has been done by others, amount of ALDH positive cells and pattern . The amount of ALDH positive cells is determined  and the samples are defined as ALDH-  or ALDH+  With this method used by Cheung et al. (28) AML patients are classified based on percentage of ALDH positive cells defined by DEAB treatment. Our classification shows that 18% of AML patients are positive for ALDH (more than 5% of cells are ALDH+). The pattern of ALDH activity is determined by the shape, level and scatters properties of the ALDH activity as defined by Pearce et al. (33) The “rare” pattern ALDH activity is seen in (A) and the numerous pattern of ALDH activity is seen in (B). All the positive AML cases have the numerous pattern.(TIF)Click here for additional data file.Table S1Quantification of ALDH activity in marker defined CD34+CD38– HSC and CD34+CD38– LSC compartments in CD34 positive AML. MFI values of CD34+CD38–marker- and CD34+CD38–marker+ were standardized by dividing them with the ALDH-MFI values from lymphocytes present in the same sample. AML samples were treated with diethylamino-benaldehyde (DEAB) to compare background MFI values in each cell population. Comparison of the median of the ALDH-MFI values of two populations; CD34+CD38– marker- cells and CD34+CD38– marker+ cells from 9 (9/19 CD34-positive AML cases were treated with DEAB) CD34-positive AML samples  was done. In all CD34-positive AML cases, the MFI of CD34+CD38–marker- cells was divided with the MFI of the CD34+CD38–marker+ cells to obtain fold induction of ALDH activity in HSC compared to LSC within the AML. MFI is mean fluorescent intensity.(TIF)Click here for additional data file.Table S2Quantification of ALDH activity in marker defined CD34+CD38– HSC and CD34– compartments in CD34 negative AML cases. *MFI is mean fluorescent intensity. MFI values of CD34+CD38–marker- cells and CD34– cells were standardized by dividing them with the ALDH-MFI values from lymphocytes present in the same sample. AML samples were treated with diethylamino-benaldehyde (DEAB) to compare background MFI values in each cell population. Comparison of the median of the ALDH-MFI values of two populations; CD34+CD38– marker- cells and CD34– cells from 8 (8/14 CD34-negative AML cases were treated with DEAB) CD34-negative AML samples  was done. In all CD34-negative AML cases, the MFI of CD34+CD38–marker- cells was divided by the MFI of the CD34– cells to obtain fold induction of ALDH activity in HSC compared to the bulk of the AML. *MFI is mean fluorescent intensity.(TIF)Click here for additional data file.Table S3bright CD34+CD38dim/+ progenitor populations.The presence of FLT3-ITD and mutated NPM1 in ALDH (A) CD34-negative AML cases. 4/4 CD34+CD38+/dim cells were negative. (B) CD34-positive AML cases. # In 3/5 CD34-positive AML cases, the CD34+CD38+ progenitor population has a tiny population of mutated cells present (likely contamination with ALDHlow cells). ne: not evaluable because CLL-1 (and sometimes CD33) is not a reliable aberrant marker for malignancy of CD34+CD38+ progenitors since part of normal progenitors and more mature CD34– cells can have CLL-1 and lineage marker expression (12). na: not applicable because no leukemia-associated aberrant marker present or expression to low.(TIF)Click here for additional data file.Table S4Classification of ALDH defined AML cases. Percentage of ALDH positivity defined by DEAB treatment in the total AML (Panel 2). Percentage of ALDH positivity defined by DEAB treatment in the CD45dim blast population of cells (panel 3). AML cases that are defined as positive  are indicated as plus. AML cases that are defined as negative  are indicated as min . AML cases were defined as rare or numerous . In our cohort there are no AML cases with the negative pattern. The percentage of CD45dim blasts in the total AML (panel 6), the frequency of CD34+ cells within the total CD45dim population (panel 7), the total amount of CD34+CD38– cells within the CD34+ compartment (panel 8) and the total amount of CD34+CD38– cells within the CD45dim population of cells (panel 9) is indicated in this table.(TIF)Click here for additional data file.


Cytosolic Ca2+ ([Ca2+]cyto) can be excellently quantified with the ratiometric Ca2+ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca2+ sensors, the cameleons, are efficiently used to specifically measure Ca2+ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca2+]cyto and mitochondrial Ca2+ ([Ca2+]mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca2+ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca2+]cyto and [Ca2+]mito within same individual cells. Our data indicate that depending on the kinetics of [Ca2+]cyto rises there is a significant lag between onset of [Ca2+]cyto and [Ca2+]mito signals, pointing to a certain threshold of [Ca2+]cyto necessary to activate mitochondrial Ca2+ uptake. The temporal correlation between [Ca2+]mito and [Ca2+]cyto as well as the efficiency of the transfer of Ca2+ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca2+ extrusion and a desensitization of mitochondrial Ca2+ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca2+ oscillations of pancreatic beta-cells in response to D-glucose.The transfer of Ca A rise of the Ca2+ concentration in the lumen of mitochondria stimulates ATP generation 2+, these organelles have also been recognized to locally clear cytosolic Ca2+ elevations 2+ buffer function impacts the spatiotemporal pattern of cellular Ca2+ signals and, thus, significantly influences Ca2+ sensitive processes within a cell 2+ signaling, excessive mitochondrial Ca2+ loads trigger cell death 2+ homeostasis can determine life and death of a cell Mitochondrial uptake of Ca2+ signals in intact cells requires sophisticated methods to specifically quantify changes of the free mitochondrial Ca2+ concentration ([Ca2+]mito) 2+ indicator is its correct targeting to the mitochondrial matrix. Out of many different chemical fluorescent Ca2+ indicators, only the positively charged dye Rhod-2 accumulates within respiring mitochondria 2+ sensor making it difficult to quantify Ca2+ signals and under most conditions significant amounts of Rhod-2 is also found in the cytosol and other cellular compartments. In contrast, genetically encoded Ca2+ indicators that additionally contain mitochondria-targeting peptides are selectively targeted to the mitochondrial matrix 2+ sensing bioluminescent protein aequorin was successfully targeted to mitochondria, which was primarily used to demonstrate that mitochondria definitely sequester Ca2+ under physiological conditions of cell stimulation 2+]mito on the single (sub)cellular level. This limitation does not persist for the genetically encoded fluorescent mitochondrial Ca2+ sensors. Principally, these Ca2+ indicators, which consist of one or two fluorescent proteins (FPs), sense Ca2+ via a calmodulin (CaM) domain and the Ca2+-CaM interacting peptide from the myosin light chain kinase, M13 2+ results in conformational changes of these domains, which alter the spectral properties of the adjacent FPs and accordingly [Ca2+]mito can be assessed directly 2+ sensors that is frequently used to measure [Ca2+]mito is the mitochondrial targeted ratiometric pericam (mtRP). The mtRP consists of a circularly permutated yellow FP that is flanked by CaM and M13 2+2+ sensors are the so-called cameleons. In the cameleons the CaM and the M13 peptide are fused in tandem between a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). Binding of Ca2+ to cameleons narrows the distance between FPs yielding an increase of the Förster resonance energy transfer (FRET) from CFP (FRET-donor) to YFP (FRET-acceptor) 2+ indicators were further improved by engineering CaM and M13, which minimized the interaction with endogenous CaM and increased the range of the measurable Ca2+ concentrations The investigation of mitochondrial Ca2+]cyto and [Ca2+]mito the measurement of both signals within same individual cells is desirable. However, co-imaging of most of the cytosolic fluorescent chemical Ca2+ indicators with genetically encoded fluorescent Ca2+ probes is problematic due to spectral overlaps of the excitation and/or emission wavelengths 2+ dye, fura-2/AM and CFP/YFP-based cameleons can be minimized by narrowing the bandwidth of emission filters 2+ indicators are reduced, yielding poor signal-to-noise ratios.For the spatiotemporal correlation between [Ca2+]mito and [Ca2+]cyto within same individual cells. Applying simultaneous cytosolic and mitochondrial Ca2+ measurements using fura-2 and the novel mitochondria-targeted D1GO-Cam, respectively, a significant lag between increase of [Ca2+]cyto and [Ca2+]mito, could be demonstrated, which depended on the mode of Ca2+ mobilization and the cell type used. Furthermore, a slow mitochondrial Ca2+ extrusion and a desensitization of mitochondrial Ca2+ uptake counteracted the synchronous transfer of cytosolic Ca2+ oscillations into mitochondria of pancreatic beta-cells, thus, partially uncoupling mitochondrial Ca2+ signal from cytosolic Ca2+ spiking.In this study we developed a novel red-shifted cameleon, which we named D1GO-Cam. In comparison to CFP/YFP-based cameleons the spectral crosstalk between fura-2 and D1GO-Cam was significantly diminished. D1GO-Cam could be targeted successfully to mitochondria and was used together with fura-2 to co-image ex) resulting in Ca2+ entry via voltage-gated Ca2+ channels +]ex was briefly increased from 5 mM to 130 mM the fura-2 signal transiently rose, while the respective changes in [Ca2+]mito were significantly delayed and remained elevated despite the decrease in [Ca2+]cyto , respectively, made the novel red-shifted genetically encoded Ca2+ sensor fully compatible with fura-2  -excitable fura-2. Miyawaki and colleagues constructed several red-shifted cameleons using a red fluorescent protein from a Discosoma species (DsRed) as a FRET acceptor 2+ sensors. The property of DsRed as a FRET acceptor was improved by using a tandem dimer mutant of this fluorescent protein (TDimer2), which was used together with an enhanced GFP (EGFP) to construct a red shifted cameleon 2+]cyto. A variety of FRET pairs have been tested for the development of genetically encoded FRET probes, including far red-shifted ones based on orange and red fluorescent proteins κ was successfully used in combination with fura-2 to correlate [Ca2+]cyto with changes of cytosolic or mitochondrial ATP levels Exchanging the most used FRET pair CFP/YFP in cameleons by GFP (cp173-mEGFP) and OFP  and Ca2+ affinities have been developed: D1 with two dissociation constants (Kds) of 0.6 µM and 56.5 µM appropriate to measure Ca2+ ranging from <1 µM up to >300 µM, D2 with Kds ranging from 0.1 to 7.7 µM, D3 with a Kd of 0.8 µM, and D4 with a Kd of 49.7 µM 2+ over a wide range of Ca2+ concentrations. Surprisingly, the Ca2+ affinity of the D1GO-Cam was enhanced compared to the reported Kds of the respective CFP/YFP based cameleon, D1cpv. Particularly, the D1 containing red-shifted cameleon exhibited just one Kd of 1.53 µM and was already saturated at Ca2+ concentrations >100 µM, when we calibrated the sensor in situ  2+ uniporter (MCU) 2+ into mitochondria. However, further studies are necessary to understand the exact functional and physiological roles of these proteins for the mitochondrial Ca2+ homeostasis in different cell types 2+]mito and [Ca2+]cyto in a ratiometric manner might help to better cope with these tasks.Interestingly, the speed of the transfer of cytosolic Caype used . These f2+ signals 2+ uptake in the different cell types we have shown in this study might also point to differences in the polyphosphate content and, thus, the mitochondrial Ca2+ buffer capacity of the different cell models used. This is possible particularly because of differences in the rate of ATP generating metabolic pathways within the different cells. In INS-1 832/13 cells most of the ATP is produced by oxidative phosphorylation It has been shown that the content of polyphosphate within mitochondria clearly affects mitochondrial Ca2+ from mitochondria was also diverse in the three different cell lines used in this study  by Ca2+ was also established in other cell types Testing the 4mtD1GO-Cam in fura-2/AM loaded INS-1 832/13 cells revealed several interesting aspects regarding the coupling of mitochondrial Ca high K+  points t2+]mito and [Ca2+]cyto was finally tested in INS-1 832/13 cells that exhibited complex and heterogeneous Ca2+ oscillations in response to D-glucose beta-cells the transfer of Ca2+ into mitochondria is of utmost importance as mitochondrial Ca2+ sequestration facilitates glucose-induced insulin secretion 2+ homeostasis is highly relevant for better understanding the physiology and pathology of pancreatic beta-cells. Our measurements revealed that in contrast to cell stimulation with high K+ the glucose-induced robust cytosolic Ca2+ signals are instantly transferred into mitochondria in INS-1 832/13 cells . Other media supplements, RPMI-1640, fetal calf serum (FCS) and all plastic ware were from PAA laboratories . Dulbecco’s modified eagle’s medium (DMEM), histamine dihydrochloride (Histamine), carbamylcholin chloride , adenosine 5′-triphosphate disodium salt (ATP), oligomycin and antimycin A were obtained from Sigma–Aldrich . Ionomycin (free acid) was from abcamBiochemicals  and fura-2/AM from Teflabs . Transfast™ transfection reagent, restriction enzymes and Taq polymerase used for subcloning were purchased from Promega . All other chemicals were obtained from Roth .2. INS-1 832/13 cells (INS-1) were kindly provided by C. B. Newgard (Duke University School of Medicine) and cultured with RPMI-1640 culture medium containing 2 mM L-glutamate and 11.1 mM D-glucose supplemented with 10% FCS, 1 mM sodium pyruvate, 5 µM mercaptoethanol, 100 U/ml penicillin and 100 µg/ml streptomycin. The human umbilical vein endothelial cell line EA.hy926 2, 95% air) for 16–20 hours before changing the medium back to normal culture medium. Experiments were performed 24–48 hours after transfection. Cells were loaded at RT with 2 µM fura-2/AM for 20 minutes in a HEPES buffered solution containing in mM: 135 NaCl, KCl, 2 CaCl2, 1 MgCl2, 1 HEPES, 2.6 NaHCO3, 0.44 KH2PO4, 0.34 Na2HPO4, 10 D-glucose, 0.1% vitamins, 0.2% essential amino acids and 1% penicillin/streptomycin pH 7.4. Prior to experiments cells were washed and stored in the same HEPES buffered solution without fura-2/AM.In this study all 3 different cell lines used were cultured and transfected in a humidified atmosphere at 37°C and 5% CO2+ sensitive calmodulin/M13 sequence (D1) from the D1ER probe ATCGATATGCATGACCAACTGACAGAA-3′5′-GG, 5′-GGCCATCACCGTCAATATCT-3′, 5′-GGAAGCAGATATTGACGGTG-3′ and GAATTCCATGAGCTCCAGTGCCCCG-3′5′-AA. D1 was then subcloned via its overhanging ClaI and EcoRI recognition sites into the newly developed red-shifted mitochondrial or cytosolic ATP sensor (4mtGO-ATeam or GO-ATeam) oF1-ATP synthase sequence. Accordingly, exchanging the ATP sensing ε subunit to the calmodulin/M13 sequences of D1 resulted in 2 new plasmids coding for Ca2+ sensitive probes: the cytosolic D1GO-Cam and the mitochondrial targeted variant, 4mtD1GO-Cam, respectively.The Ca2+ measurements in Ea.hy926 and HeLa cells were performed by stimulating the cells in a Ca2+ containing environment by perfusing the cells in a calcium containing buffer (CB), which was composed of (in mM): 138 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose and 10 HEPES, pH adjusted to 7.4 with NaOH. Stimulation of INS-1 with IP3-generating agonists was done using 100 µM CCh and 200 µM ATP under Ca2+ free conditions in an EGTA containing buffer (EB) that was composed like the CB, but contained 1 mM EGTA instead of 2 mM Ca2+. In order to trigger K+ induced depolarization in this cell line we used the CB and enhanced the K+ concentration from 5 mM to 30 mM or 130 mM K+, respectively. The concentration of NaCl was reduced accordingly in high K+ buffers to maintain osmolarity. Moreover, the impact of mitochondrial depolarization on [Ca2+]mito and [Ca2+]cyto was simultaneously observed using 2 µM oligomycin and 10 µM antimycin under this condition. For glucose-stimulated Ca2+ measurements INS-1 cells were preincubated in HBSS with (in mM) 114 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 2 HEPES, 1.2 KH2PO4, 25 NaHCO3, 0.2% bovine serum albumin (BSA) and 3 D-glucose for 30 minutes and then washed and equilibrated in CB (without glucose) for 10–15 minutes before imaging. Glucose-stimulated Ca2+ signals were then observed by increasing glucose from 0 mM to 16 mM in CB. For simultaneous measurements cells transfected with one of the GO-cam plasmids were loaded with fura-2/AM as described. Single cells containing both, the fura-2 and the GO-cam biosensor were alternately excited at 340, 380 or 477 nm and emissions were recorded at 510 nm for fura-2 and GFP or at 560 nm for FRET channel.CaCo-imaging of fura-2 and the red-shifted cameleons was performed on a digital wide field imaging system, the Till iMIC  using a 40× objective . For illumination of fura-2 and the cameleons an ultra fast switching monochromator, the Polychrome V (Till Photonics) was used. Fura-2 was excited alternatively at 340 nm and 380 nm and the red-shifted cameleons were excited at 477 nm, respectively. To avoid contamination of the emission channels with excitation light an excitation filter  and a dichroic filter (495dcxru) were installed. Emission light was simultaneously collected at 510 nm (fura-2 and GFP of GO-Cams) and at 560 nm (FRET-channel of GO-Cams) using a single beam splitter design  that was equipped with a dual band emission filter  and a second dichroic filter . The light path and spectra for the filters used for the co-imaging experiments are illustrated in Figure S1κ of D1GO-Cam, respectively.Characterization of spectral properties and photobleaching of the fluorescence proteins cp173-mEGFP and mKO (A) Representative images of HeLa cells expressing either D1GO-Cam (upper panel) or cp173-mEGFP alone (middle panel) or mKOκ alone (lower panel) showing the FRET (acceptor) channel (emission at ∼ 560 nm) on the left images and the GFP (donor) channel (emmsion at ∼ 510 nM) on the right images, respectively. The white scale bar in the upper left image represents 20 µm. (B) Statistical analysis of the contribution of the fluorescence signals of the D1GO-Cam  under resting conditions (i.e. low Ca2+ levels) in the FRET (acceptor) channel (left orange column) and the GFP (donor) channel (left green column) and the contribution of the fluorescence signals of cp173-mEGFP alone  in the FRET (acceptor) channel (right orange column) and the GFP (donor) channel (right green column). (C) Quantitative comparison of the photobleaching of D3cpv (containing the CFP/YFP FRET pair) relative to the D1GO-Cam (containing the GFP/OFP FRET pair). HeLa cells expressing the D3cpv (n = 15) were illuminated with excitation light at 430 nm with an exposure time of 400 ms and a camera binning of 4. Emission light was collected simultaneously at 480 nM (CFP donor fluorescence) and at 535 nm (FRET acceptor fluorescence) using the beam splitter device. With the same settings cells expressing D1GO-Cam (n = 12) were illuminated at 477 nm and emission light was collected at 510 nm  and 560 nm (FRET acceptor fluorescence), respectively. For both sensors the ratio FRET/Fdonor was plotted over time.(TIF)Click here for additional data file.Figure S2Imaging setup for the simultaneous recording of fura-2 and the novel red-shifted cameleons. Schematic representation of the imaging system with light paths of the excitation -  and emission light (green  = 510 nM and orange  = 560 nM) and the optical filters used to simultaneously image fura-2 and the novel red-shifted cameleons.(TIF)Click here for additional data file.