P3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdInsP2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications.Improved versions of inositol-1,4,5-trisphosphate (Ins P3) plays a central role in calcium signaling. Its production is catalyzed by phospholipase C enzymes activated through receptor stimulation. Measurements of InsP3 kinetics for a long time relied upon isotope labeling of cell populations followed by extraction and HPLC-separation of the active  isomer from the inactive  one InsP3 (Amersham Biosciences) and 200 ng protein in an incubation volume of 50 μl.mRFP-tagged wild type and mutant Ins5 cells/dish density. After one day the culture medium was changed to 1 ml Opti-MEM (Gibco) medium, and then 200 μl transfection solution containing the indicated DNA constructs  and 2 μl/dish Lipofectamine 2000 was added. After 6 hours 1 ml DMEM containing serum and antibiotics was added. Measurements were performed 24–32 hours after the transfection. Before the measurements the coverslips were placed into Attofluor cell chambers (Invitrogen) and the medium was changed to 800 μl of a modified Krebs–Ringer buffer containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose, and 10 mM Na-HEPES, pH 7.4. Measurements were performed at room temperature using an inverted microscope  equipped with a 40x/1.3 oil-immersion objective  and a Cascade II camera . Excitation wavelengths (435 nm and 500 nm) were set by a monochromator connected to a 75 W Xenon lamp . The emitted light was separated by a dichroic beamsplitter (Chroma 69008bs), and then detected through the appropriate emission filters for Cerulean (470/24 nm) and Venus (535/30 nm). Images were acquired every 5 s. The indicated reagents were also dissolved in modified Krebs–Ringer buffer and were added manually in 200 μl, and mixed three times. The MetaFluor  software was used for data acquisition, whereas for further data analysis including background subtraction, bleed through correction and 535/470 emission ratio calculation the MetaMorph (Molecular Devices) software was applied.For FRET measurements HEK 293T cells were trypsinized and plated on poly-lysine-pretreated  No 1.5 glass coverslips in 35 mm plastic dishes at 3x105 cells/well together with the indicated DNA constructs  and the cell transfection reagent (0.5 μl/well Lipofectamine 2000 or 1.5 μl/well GeneCellin). After 6 hours 100 μl/well DMEM containing serum and antibiotics was added. Measurements were performed 24–27 h after transfection. Before measurements the medium of cells was changed to a medium (50 μl) containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose, and 10 mM Na-HEPES, pH 7.4. Measurements were performed at 37°C using a Mithras LB 940 multilabel reader . The measurements started with the addition of the cell permeable luciferase substrate, coelenterazine h , and counts were recorded using 485 and 530 nm emission filters. Detection time was 500 ms for each wavelength. The indicated reagents were also dissolved in modified Krebs–Ringer buffer and were added manually in 10 μl. For this, plates were unloaded, which resulted in an interruption in the recordings. All measurements were done in triplicates. BRET ratios were calculated by dividing the 530 nm and 485 nm intensities, and normalized to the baseline.For BRET measurements HEK 293T cells were trypsinized and plated on poly-lysine-pretreated  white 96-well plates at a density of 104 cells/well) and transfected with the indicated constructs  using 0.5 μl/well Lipofectamine 2000 for 24 h. Confocal measurements were performed at 35°C in a modified Krebs-Ringer buffer described above, using a Zeiss LSM 710 scanning confocal microscope and a 63x/1.4 oil-immersion objective. Post-acquisition picture analysis was performed using the Photoshop (Adobe) software to expand to the full dynamic range but only linear changes were allowed.COS-7 cells were cultured on IBIDI μ-Slide 8 Well dishes . τ values were then averaged and subjected to a t-test.To calculate the half-time values (τ) of the decay phase, a curve fitting procedure was applied to the values in each individual experiment using the 3 parametric exponential decay equation of (y = yP3 sensor first we decided to perform a moderate modification in the InsP3 binding domain (224–605 amino acids) of human type-I InsP3 receptor (IP3R-LBD) InsP3 as the tracer. As shown in P3 binders. Scatchard analysis of the curves also showed the lower affinity of the mutants: the KD value for the R265K, R269K and R568K mutants were 6.06 nM, 10.07 nM and 5.30 nM, respectively compared to 3.04 nM for the wild type LBD. Based on these results, the R265K and R269K mutants were selected for further analysis.To design an improved InsP3R-LBD)  by replag domain  we selecP3R-LBD with various N-terminal fluorescent tags, such as mRFP or Cerulean, in combination with a C-terminal Venus tag. Application of the protein as InsP3 sensor in BRET measurements requires that the N-terminal fluorescent protein tag be replaced with a luciferase enzyme. P3 sensors that contain either Cerulean  level was rapidly reduced by the rapamycin-inducible PM PtdInsP2-depletion system P2 depletion evoked by rapamycin addition. The decay of the InsP3 signal correlated well with the decline of the cytoplasmic Ca2+ level P but only slightly affected the prestimulatory PtdInsP2 level [P3 level was much slower than the decline in the Ca2+ levels after the pretreatment with PI4K inhibitors P2 as a cause of declining InsP3 and refilling of the endoplasmic reticulum (ER) Ca2+ stores. These results suggested that PI4K inhibition had an additional effect on Ca2+ signals, most likely on entry via the store-operated pathways.Next we compared Insnhibitor . Wortmannhibitor . As show2 level . Howeverhibitors . This suP3 sensor that allows kinetic measurement of cytoplasmic InsP3 concentration both in singe cells and in cell populations. The conformational change evoked by InsP3 binding to the LBD domain of the InsP3 receptor served as a rational basis for the development of various energy transfer-based InsP3 sensors, summarized in P3 binding affinity is a crucial factor in their suitability. While using the high affinity wild-type LBD domain in these applications makes the sensors very sensitive, their buffering effects and their slow off-rate has limited their use, especially in experiments where assessment of InsP3 decreases was important. Several approaches have been used to modify the binding affinity of the LBD. For example, keeping the N-terminal inhibitory domain or truncation of the C-terminus of the LBD both reduced the binding affinity  of the human type-I InsP3 receptor, which we previously used as a cytoplasmic InsP3 buffer [In vitro binding measurements performed with tritium-labelled InsP3 resulted in an IC50 value of 4 nM for the ligand binding affinity of the GFP-tagged version of this domain [P3 binding site with lysines, trying to mimic the affinity close to that of the endogenous receptor. Among several mutants tested, the R265K and R269K mutants has proven to be suitable candidates for the development of improved InsP3 sensors.The purpose of the present studies was to develop an Insaffinity . Mutatio3 buffer . In vitrs domain , which is domain , we atteP3 level elevates, and normalizing the data to the resting values. Replacement of Venus with a double Venus construct (Venus cp173-Venus) did not yield further benefits either in the FRET or BRET format. Our mutant InsP3 sensors tested in BRET applications showed a slightly reduced but still high sensitivity to agonist stimulation, while they performed better when the decay of InsP3 was followed after termination of InsP3 production. This was either achieved by terminating receptor stimulation or rapidly removing PtdInsP2, the precursor of InsP3. This allowed us to perform specific experiments to test the utility of these sensors to address questions regarding the connection between InsP3 and Ca2+ entry regulation.For FRET applications the wild type, R265K and R269K mutant LBDs were tagged with monomeric versions of Cerulean and Venus on their N- and C-termini, respectively. To make the sensor suitable for use in BRET measurements, we replaced Cerulean with Luciferase. The advantage of BRET measurement over FRET is that it can be carried out in simple plate readers and that it shows the average change in a cell population. Since it does not require excitation, there is no crosstalk between the emission channels. Therefore, energy transfer can be followed by simply calculating the emission ratio without any correction. Data processing is reduced only to taking the reciprocal of the ratio values to obtain an increase when Ins2+ signal by eliminating its sustained elevation [2+ influx through the capacitative Ca2+ entry pathway is regulated by the ER luminal Ca2+ concentration, which in turn, is controlled by the opening of the InsP3 receptor channels in the ER, it was logical to assume that the falling InsP3 levels in wortmannin-treated cells would allow ER Ca2+ pools to refill and shut down the store-operated Ca2+ entry process. In fact, it has been shown by early studies that Icrac (the current that corresponds to store-regulated Ca2+ entry) is inhibited by wortmannin [2+ entry route either by affecting the Orai channels or the ER-sensor STIM1 molecule [It has been shown previously that pretreatment of cells with concentrations of wortmannin, that inhibit PI3- and PI4-kinases, modifies the shape of the agonist-evoked Calevation , 26. Thilevation . Since Crtmannin . Subsequmolecule , 28–30.P3 and Ca2+ changes allowed us to address this question in a way that relied upon endogenous levels of STIM1 and Orai1.Our new ability of monitoring Ins2+ and InsP3 either after rapidly removing PtdInsP2 or limiting phosphoinositide supplies by wortmannin  pretreatment. We found a significant difference between the two manipulations: while the InsP3 and Ca2+ decreases were parallel after PtdInsP2 depletion, the Ca2+ decrease was substantially faster than that of InsP3 in the PI4K inhibitor pretreated cells. This suggested that PI4KIIIa inhibition exerted an additional effect on Ca2+ signaling. that was different from a simple PtdInsP2 depletion evoked by the PM-recruited 5-ptase.We compared the decay kinetics of CaP. While PtdInsP2 elimination by the 5-ptase increases PtdIns(4)P levels, A1 or wortmannin pretreatment selectively depletes PtdIns(4)P even before agonist stimulation and PtdInsP2 decreases only occur after agonist addition. All of these argue for a role for PtdIns(4)P or a mechanism sensitive to its level in playing a role in the control of store-operated Ca2+ entry. These conclusions were consistent with those of earlier reports using different approaches [The most important difference between the two manipulations is the change in the level of plasma membrane PtdIns(4)proaches , 27.2+ levels below the prestimulatory values when cells were stimulated after PI4K inhibition. While this may be a feature brought out by our genetically encoded Ca2+ sensor, it raised the possibility that Ca2+ extrusion is also stimulated by agonists and together with inhibition of Ca2+ entry due to the simultaneous elimination of both PtdIns(4)P and PtdInsP2 manifests in a Ca2+ decrease below basal. Further exploration of this possibility, however, was beyond the scope of this study.We also observed a peculiar decrease of the CaP3 sensor which is based on the ligand binding domain of human type-1 InsP3 receptor but contains the R265K mutation. While this sensor maintains its sensitivity and can be used to monitor small increases of cytoplasmic InsP3 concentration, it also has an improved off-rate to follow the decrease of the InsP3 level. While the sensor also works in FRET applications, an important new feature was its adaptation for BRET applications usable in plate readers to support high-throughput measurement of cytoplasmic InsP3 in live cells alone, or in combination with measurements of other signaling factors, such as cytoplasmic Ca2+ concentration.In summary, in this paper we described and characterized an improved Ins


Although second harmonic generation (SHG) microscopy provides unique imaging advantages for voltage imaging and other biological applications, genetically-encoded SHG chromophores remain relatively unexplored. SHG only arises from non-centrosymmetric media, so an anisotropic arrangement of chromophores is essential to provide strong SHG signals. Here, inspired by the mechanism by which K-Ras4B associates with plasma membranes, we sought to achieve asymmetric arrangements of chromophores at the membrane-cytoplasm interface using the fluorescent protein mVenus. After adding a farnesylation motif to the C-terminus of mVenus, nine amino acids composing its β-barrel surface were replaced by lysine, forming an electrostatic patch. This protein (mVe9Knus-CVIM) was efficiently targeted to the plasma membrane in a geometrically defined manner and exhibited SHG in HEK293 cells. In agreement with its design, mVe9Knus-CVIM hyperpolarizability was oriented at a small angle (~7.3°) from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential. Second harmonic generation (SHG), or frequency-doubling, is a second-order non-linear optical phenomena, in which light with twice the frequency of the incident light is generated upon the interaction of matter and intense light Shen, . Second-Taking advantage of its sensitivity to local centrosymmetry, SHIM has been used to distinguish juxtaposed regions of membrane vesicles from un-adhered region  interacts with plasma membranes . Transfection was performed using Lipofectamine 2000 reagent  according to the manufacturer's protocol. The crystal structure for Venus . The electrostatic surface potential maps for the mutant models were generated by the web-based software, eF-surf (http://ef-site.protein.osaka-u.ac.jp/eF-surf/top.do).We used a custom modified pCS2+ vector for heterologous expression experiments in mammalian cells. Site-directed mutagenesis was performed as described previously  equipped with a 75 W xenon lamp and a CMOS camera . Excitation, dichroic, and emission filters used were ex500/24, dm520, and em542/27 , respectively. A rotatable polarizer in the excitation light path  was used to study polarization angle dependency. A 40× objective lens with a moderately low numerical aperture  was used to minimize aperture effect. Intrinsic angle-dependency of the imaging system (g-factor) was calibrated using isotropic solution of fluorescein deposited between two coverslips.We used two setups of laser scanning microscope. One (Columbia University) was a custom-made two-photon laser scanning microscope based on the Olympus FV-300 system  and a Ti:sapphire laser . Details for the instrumentations have been described elsewhere , which acts as the farnesylation signal Figure . It has A previous study has revealed that the amount of basic amino acids in the polybasic region, rather than the specific sequence, is essential for efficient membrane localization  and the two-photon excited fluorescence band (480~550 nm) were separated by narrow band-pass filters. This required the fundamental wavelength to be shorter than 960 nm. SHG was extremely weak or undetectable at wavelengths below 900 nm, suggesting a resonant enhancement by electronic excitation. Under such constrains, the optimal wavelengths to obtain SHG signals were 940~950 nm. Typically, a field of view of 70 × 70 μm was scanned as a 320 × 320 pixel image at a pixel dwell time of 40 μs. Laser power as large as 30~40 mW at the sample was normally needed to detect clear second harmonic signals. Representative two-photon fluorescence and SHG image upon illumination at 940 nm are shown Figures . The posy Figure . The angIn this study we have successfully engineered a GFP-based SHG membrane chromophore, even though one may not in principle expect strong SHG from GFP-based membrane proteins, because the SHG intensity from a chromophore is proportional to the square of its density Shen,  and the 2, a voltage change ΔV of ~100 mV will associate change in the surface charge:FM4-64 and other organic dyes bound in cell membrane produce robust voltage dependent second harmonics  which are known to rely on the electrostatic mechanism similar to K-Ras4B. While all these proteins likewise possess apparent basic clusters in their primary structures, the present study clearly demonstrated that the basic amino acids are not necessarily clustered in the primary structure whenever they constitute a basic patch in the tertiary structure. Thus, might be more natural proteins which have not yet been recognized to interact electrostatically.Compared to the recent achievements building genetically-encoded probes basing on fluorescence (Miyawaki, 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.


However, the study of therapeutic blockades of Ca2+ signaling induced by dust particles in human bronchial epithelial cells is poorly understood. We investigated how to modulate dust particles-induced Ca2+ signaling and proinflammatory cytokine IL-8 expression. Bronchial epithelial BEAS-2B cells were exposed to PM10 dust particles and subsequent mediated intracellular Ca2+ signaling and reactive oxygen species signal. Our results show that exposure to several inhibitors of Ca2+ pathway attenuated the PM10-induced Ca2+ response and subsequent IL-8 mRNA expression. PM10-mediated Ca2+ signal and IL-8 expression were attenuated by several pharmacological blockades such as antioxidants, IP3-PLC blockers, and TRPM2 inhibitors. Our results show that blockades of PLC or TRPM2 reduced both of PM10-mediated Ca2+ signal and IL-8 expression, suggesting that treatment with these blockades should be considered for potential therapeutic trials in pulmonary epithelium for inflammation caused by environmental events such as seasonal dust storm.Bronchial epithelial cells are the first barrier of defense against respiratory pathogens. Dust particles as extracellular stimuli are associated with inflammatory reactions after inhalation. It has been reported that dust particles induce intracellular Ca Meteorological and seasonal dust events in eastern Asia negatively impact humans and the ecosystem , 2. ThesBronchial epithelial cells are the first physical barrier of defense against exogenous stimuli, such as dust, allergens, pollen, and osmotic molecules. Thus, they are an important defense protecting the airway from respiratory pathogens. There are several evidences to address the airway pathology that repetitive exposure of mice to airborne dust particles induces lung inflammation , 11. Fur10, particulate matter with a diameter of less than 10 μm, promotes fibrosis and intracellular reactive oxygen species (ROS) : 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 D-glucose, titrated to pH 7.4. For the 0 Ca2+ extracellular solution, CaCl2 was replaced with 10 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). Change in intracellular Ca2+ was measured by the intensity of fluorescence with excitation wavelengths of 340 and 380 nm, respectively, and an emission wavelength of 510 nm. All results are reported as the fluorescence ratio, calculated as the ratio of F340/F380. Fluorescence was monitored with a charge-coupled device camera  attached to an inverted microscope  and analyzed with a MetaFluor system . Fluorescence images were obtained at 1-second intervals. Background fluorescence was subtracted from the raw background signals at each excitation wavelength. The number of cells used in ΔCa2+ analysis was counted by Integrated Morphometry Analysis of MetaMorph software (Molecular Devices).BEAS-2B cells were cultured on cover glass, incubated with 4 μg/mL PM10 in PSS containing 10 μg/mL ROS fluorescence probe 5-(and-6)-choloromethyl-2′,7′-dicholorodihydrofluorescin diacetate  for 5 min and washed for 5 min in PBS. H2DCFDA fluorescence was measured using a confocal laser-scanning microscope  by excitation at 488 nm and measuring the emitted light at 525 nm. H2DCFDA fluorescence was collected for six different regions in each image, and the signal was normalized to that at the beginning of the experiment.BEAS-2B cells were incubated on a cover glass in the presence or absence of 50 Total RNA was extracted from BEAS-2B cells using the TRIzol Purification System (Invitrogen) according to the manufacturer's instructions. Total RNA was amplified according to the manufacturer's protocol using AccuPower RT PreMix . cDNA was amplified by PCR with HiPi Thermostable DNA polymerase  and nested primers. The forward and reverse GAPDH primers were GTCGGAGTCAACGGATT and GCCATGGGTGGAATCATA, respectively. The forward and reverse IL-8 primers were ATGACTTCCAAGCTGGCCGTGGCT and TCTCAGCCCTCTTCAAAAACTTCT, respectively. cDNA PCR began with a denaturation step at 95°C for 5 min, followed by 35 cycles of 1 min at 95°C, 1 min at 56°C (GAPDH) and 58°C (IL-8), 1 min at 72°C, and a final step at 72°C for 10 min. The PCR products were electrophoresed on 1% agarose gels. Expression levels of all PCR products were subtracted from those of a GAPDH loading control by measuring the intensity of PCR products with MetaMorph software (Molecular Devices).μg heated protein samples were subjected to SDS/PAGE and subsequently transferred to methanol-soaked polyvinylidene difluoride (PVDF) membranes. Transferred proteins on PVDF membranes were visualized with TRPM2 and β-actin antibodies by enhanced luminescent solution (Thermo scientific).Cells were cultured and cell lysates were prepared in lysis buffer  by passing 10–12 times through a 27-gauge needle after sonication. The lysates were centrifuged at 11,000 ×g for 20 min at 4°C, and protein concentration in the supernatants was determined. Proteins were denatured by heating in SDS sample buffer at 37°C for 30 min. The 30 t-test.Data are reported as mean ± SE. Statistical significance was determined by analysis of variance in each experiment using the paired Student's μm and characterized as PM10, which included submicroparticles less than 900 nm in size as well as fine nanoparticles less than 100 nm in size . To characterize the source of Ca2+, cells were stimulated by PM10 in the presence or absence of extracellular Ca2+. The PM10-induced Ca2+ signal was dramatically reduced in the absence of extracellular Ca2+ (n = 140 cells). Number of the responding cells increased PM10 concentration-dependently  even though this oscillation did not show periodic pattern. Because 50 μg/mL PM10 did not induce cell death (data not shown) and generally produced reliable Ca2+ oscillations, we selected this concentration to analyze the mechanism by which PM10-stimulated Ca2+ signaling occurred in subsequent experiments. Cells were loaded with H2DCFDA to determine the extent of dust particles-induced ROS signal. Fluorescence was increased in a time-dependent manner after the application of dust particles . These observations indicated that PM10 induced an intracellular Ca2+ signal which was mediated by the availability of Ca2+ influx and ROS signal increase in bronchial epithelial cells.Particles were filtered to 10  in size . For the10-induced Ca2+ signal was modulated by store-operated Ca2+ influx machinery, cells were treated with cyclopiazonic acid (CPA) in the absence of extracellular Ca2+ to deplete Ca2+ stores in the endoplasmic reticulum, and 2 mM extracellular Ca2+ was then applied. PM10-induced Ca2+ was additive effect on the store-depleted Ca2+ influx signal  with a sustained Ca2+ level , a nonselective cation inhibitor. La3+ prevented the increase in intracellular Ca2+ (n = 86 cells). In addition, nifedipine, an inhibitor of voltage dependent L-type Ca2+ channels [n = 59 cells). These results indicate that PM10 spontaneously triggers Ca2+ influx into the cytosol from the extracellular media, independent of voltage dependent L-type Ca2+ channels.To determine whether the PM2+ level . To verichannels , has no 3) pathway in PM10-induced Ca2+ influx, cells were pretreated with U73122, a specific blocker of PLC, or its inactive analog U73343, as a control. U73122 blocked PM10-induced Ca2+ increases (n = 135 cells), whereas U73343 did not prevent Ca2+ increases (n = 81 cells). After removal of U73122, the PM10-mediated signal increased. The PM10-induced Ca2+ signal was measured after treatment with 20 mM caffeine as an IP3 receptor (IP3R) antagonist [2+ induced by PM10 (n = 162 cells). When treated with BAPTA, AM, the PM10-induced Ca2+ signal was blocked by the chelation of basal and increased Ca2+ (n = 117 cells). These results suggest that PM10-induced Ca2+ increases were associated with the PLC-IP3-IP3R pathway.To determine the role of the phospholipase C (PLC)/inositol 1,4,5-trisphosphate . PM10-induced Ca2+ signals were modestly attenuated by the antioxidant NAC (n = 174 cells), indicating that ROS signal is involved in the Ca2+ response. Moreover, PM10-induced Ca2+ increases were blocked by NADPH oxidase inhibitor DPI [n = 141 cells). Although NAC and DPI attenuated the PM10-induced Ca2+ increases, the removal of NAC in the continued presence of PM10 again increased Ca2+. DPI-treated cells exhibited an increase in their basal Ca2+ level even though no transient increase in Ca2+ occurred, suggesting that NAC and DPI differ only in their ability to antagonize ROS-mediated Ca2+ release. To determine whether the PM10-induced response is involved in PARP signaling, we applied a well-characterized PARP-1 inhibitor, 3-AB [10-mediated Ca2+ increases markedly and protractedly (n = 182 cells). These results indicate that PM10-induced increases in Ca2+ in bronchial epithelial cells are mediated by oxidation and are dependent on PARP-1 signaling.Having established that ROS signal by PMng agent . Most ofitor DPI  , a well-characterized TRPM2 inhibitor, also significantly decreased PM10-induced Ca2+ influx in bronchial epithelial cells . To evaluate the combined effect of an antioxidant and TRPM2 blocker, cells were exposed to the antioxidant NAC and the TRPM2 inhibitor CLZ (CLZ + NAC∗) . PM10-induced Ca2+ increases were diminished by the combination treatment . We checked that BEAS-2B cells were expressed TRPM2 protein endogenously . Next, we assessed whether the blockades of Ca2+ signaling attenuate the expression of IL-8 mRNA level; cells were treated with PM10 after pretreatment with one of the following: the PLC inhibitor U73122, its inactive analog U73343, a NADPH oxidase inhibitor (DPI), a PARP-1 inhibitor (3-AB), a TRPM2 inhibitor (CLZ), an antioxidant (NAC), an intracellular Ca2+-selective chelator , a Ca2+-CaM inhibitor (CLP), an anthranilic acid-derived TRPM2 inhibitor (ACA), a sulfhydryl-reducing agent (DTT), or a combined low dose of CLZ and NAC (CLZ + NAC∗). IL-8 mRNA levels were markedly decreased by U73122, 3-AB, CLZ, CLP, and the combination of CLZ and NAC . U73343, which served as a positive control for U73122, did not prevent mRNA expression of IL-8. DTT and DPI did not affect PM10-induced expression of IL-8 mRNA, suggesting that sulfhydryl oxidation and NADPH oxidase-mediated effects may only partially affect PM10-induced signaling in bronchial epithelial cells, since PM10-induced Ca2+ signaling was attenuated by these compounds in Figures 10-induced IL-8 mRNA expression (n = 4). These data indicate that PM10 associated with the TRPM2-mediated Ca2+ increase, which, in turn, affected IL-8 mRNA expression in bronchial epithelial cells.To determine whether PMith PM10 , n = 4.  Figures , n = 4. pression , n = 4. 2+ signaling blockades on dust particles PM10-mediated Ca2+ signaling and proinflammatory cytokine IL-8 mRNA expression in human bronchial epithelial cells. An increased ROS signal as well as plasma membrane channel activation caused by dust particles PM10 application can trigger intracellular Ca2+ and ROS signaling and the subsequent expression of proinflammatory cytokines. The enhanced ROS level by dust particles has been implicated in the pathology of several lung diseases, including asthma, lung fibrosis, and chronic obstructive pulmonary disease (COPD) [2+ signaling are closely involved in various stress responses and the inflammatory response through activation of IL-8 production. It is therefore likely that the PM-induced Ca2+ signaling activates similar pathways to regulate the release of proinflammatory cytokines and may progress the immune response seen in asthma and COPD.In this study, we demonstrate the effect of several Cae (COPD) . Moreove2+ and other ions [2+ is known to be critical for TRPM2 activation [2+-saturated calmodulin (CaM) [2+ influx and provide the evidence that several blockades attenuate the signal through the activation of TRPM2, which regulates the expression of proinflammatory cytokines and may induce pathological responses beyond physiological homeostasis. Oxidative stress is associated with many pathologic events, including pulmonary fibrosis [β1/Smad2/3 exerts an antifibrotic activity in lung tissue [10-induced Ca2+ response and IL-8 expression will provide experimental relevance to apply in oxidative inflammatory lung diseases. Thus, the precise role of TRPM2 will be elucidated in pulmonary fibrotic tissues in the near future. Although neither NAC nor DPI are solely selective for ROS-dependent Ca2+ release and IL-8 mRNA expression induced by dust particles  . Howeverin (CaM) . Recentlin (CaM) . Our stufibrosis . Indeed,g tissue . Collectg tissue . Howeverarticles , these p10 was filtered with mesh which has pore size of 10 μm, we observed that dust particles were also contained between 3 μm and 100 nm in size (Although dust particles PM in size . This si2+ signaling and proinflammatory cytokine IL-8 expression in human lung airway epithelial cells. Collectively, we suggest that the Ca2+ signaling by dust particles was attenuated by antioxidants, inhibitors of Ca2+ signaling pathway, and TRPM2 inhibitors and provides the evidence that treatment with blockades of Ca2+ signal should be considered for therapeutic trials in bronchial epithelia for inflammatory signaling caused by environmental dust events.Dust particles induced intracellular Ca


IDH1/NPM1/NRAS mutations. In conclusion, asparaginase-containing regimens, which are approved for treatment of acute lymphoblastic leukemia (ALL) but not AML, can be used to treat patients with AML who do not accept blood transfusion.Treatment of patients with acute myeloid leukemia (AML) who do not wish to accept blood product transfusion, including Jehovah’s Witnesses, is extremely challenging. The use of conventional chemotherapy for induction of complete remission (CR) results in profound anemia and thrombocytopenia requiring frequent transfusions of blood products, without which such treatment will be life-threatening. Finding a well tolerable, minimally myelosuppressive induction regimen for such patients with AML is a clear example of area of unmet medical need. Here, we report a successful treatment of a 52-year-old Jehovah’s Witness with newly diagnosed AML with peg-asparaginase, vincristine and methylprednisolone. The AML was characterized with normal karyotype, and mutations in isocitrate dehydrogenase 1 (IDH1-Arg132Ser), nucleophosmin 1 (NPM1-Trp289Cysfs*12) and neuroblastoma RAS viral oncogene homolog (NRAS-G1y12Va1). After one 28-day cycle of treatment, the patient achieved complete remission with incomplete count recovery (CRi) and after the second cycle, he achieved CR with full blood count recovery. The patient has never received any blood products. Notwithstanding that myeloperoxidase-induced oxidative degradation of vincristine results in its lack of activity as monotherapy in AML, its combination with corticosteroid and asparaginase has resulted in a robust remission in this patient. Diminished steroid clearance by asparaginase activity as well as reduction in serum glutamine level induced by glutaminase enzymatic activity of asparaginase may have contributed to effective killing of the myeloblasts that carry  Treatment of individuals who do not accept transfusion of blood products, including Jehovah’s Witnesses, with acute myeloid leukemia (AML) is exceptionally challenging ,2,3. ConAsparaginase products, which are approved for treatment of acute lymphoblastic leukemia (ALL) ,8,9, but9/L with 57% peripheral blast, hemoglobin 6.2 g/dL, platelet 64 × 109/L. Bone marrow aspiration and biopsy confirmed the diagnosis of AML  7.3 × 10s of AML A,B.IDH1-Arg132Ser, nucleotide change: c.394C > A, mutation level: 50%), nucleophosmin 1  and neuroblastoma RAS viral oncogene homolog . Due to religious belief, the patient did not accept transfusion of red blood cells, platelets or other blood products but wished to proceed with treatment. After a comprehensive discussion of risks and benefits with the patient and his family, a minimally myelosuppressive treatment with peg-asparaginase (2500 units/m2 intravenously on day 8), vincristine  and methylprednisolone (20 mg/m2 twice daily on days 1–28) was initiated [Immunophenotypic studies by flow cytometry on the bone marrow specimen showed 60%–70% blasts expressing CD117 (heterogeneous), CD33, CD13, CD15 , CD11b (heterogeneous), CD11c, CD38, CD64 , CD71 , and myeloperoxidase. The blasts were negative for CD34, HLA-DR, CD14, CD16, CD56, TdT, and all T- and B-lymphoid antigens. Chromosome analysis revealed 46XY in 20 metaphase cells. Mutational analysis of the DNA from bone marrow blasts revealed mutations in isocitrate dehydrogenase 1  as well as left lower extremity edema. The duplex ultrasound revealed acute thrombus in the right distal subclavian vein, right axillary vein and right brachial vein as well as acute, occlusive deep venous thrombosis (DVT) of the paired left peroneal veins  without extension proximally into the left popliteal vein or the more-proximal deep veins of the left leg. Since at this point patient had adequate platelet recovery to 142 × 109/L with 54% granulocytes, no blasts, hemoglobin 10.4 g/dL, and platelets 255 × 109/L  as an outpatient. Patient remained on anticoagulation and tolerated the chemotherapy well except for development of Grade 2 vincristine-induced peripheral neuropathy. The CBC upon completion of the second cycle showed WBC 9.2 × 10 × 109/L . His bon × 109/L E,F. The E. coli and Erwinia-derived asparaginase products including long acting pegasparaginase (polyethylene glycol L-asparaginase) possess a dual asparaginase and glutaminase enzymatic activity that diminishes both serum asparagine and glutamine levels by deamidating them to aspartate and glutamate, respectively [Both ectively . It is bectively ,14,15,16IDH1 and IDH2 have been reported in approximately 20% of de novo AML [IDH mutations confer adverse prognostic effect in patients with AML whose myeloblasts lack NPM1 mutation [13C and isotope-labeling experiments, it has been shown that the primary source for α-KG in AML cells with IDH mutation is glutamine [in vitro interruption of glutamine metabolism preferentially slows the growth of primary AML cells with mutant IDH versus wild type IDH [IDH mutations are ongoing (NCT02283190 and NCT01810705). Mutations in novo AML ,19,20,21novo AML ,24,25,26mutation . Wild-tymutation . Alteredmutation ,20. By 1lutamine . Subsequtype IDH ,30. ClinThe major adverse events of asparaginase products, reported from clinical trials involving patients with ALL, include anaphylaxis and serious allergic reactions, thrombosis, pancreatitis, glucose intolerance, coagulopathy, and hepatotoxicity. Interestingly, myelosuppression does not occur frequently and seriously after asparaginase administration. Also the myelosuppressive effect of vincristine compared to other conventional chemotherapeutic agents is minimal.The fact that asparaginase and vincristine are minimally myelosuppressive is important for patients such as Jehovah’s Witnesses that do not accept blood product transfusion; as it is well known that the use of conventional chemotherapy for remission induction in AML results in life-threatening anemia and thrombocytopenia, requiring blood product transfusions with an average of 10.8 and 8.5 units of red blood cell and platelet transfusions, respectively . We combIDH1/NPM1/NRAS AML who did not want to receive blood product transfusions. Moving forward, we plan to continue using non-myelosuppressive mono- or combination-therapies as consolidation regimens for this patient. We also plan to prospectively test the effect of this combination for similar patients with prospective measurement of serum biomarkers.A minimally myelosuppressive regimen including pegasparaginase, vincristine and steroid was used to induce durable complete remission in a patient with mutant


But because the topography of VGCCs and their proximity to vesicles is unknown, a quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local [Ca2+] imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca2+ buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca2+ sensors for vesicular release are located at the perimeter of VGCC clusters (<30 nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This “perimeter release model” provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course.Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca •2+ channels form clusters with highly variable numbers of channelsCa•EGTA sensitivity suggests that synaptic vesicles are tightly coupled to clusters•2+ channel number per cluster alters synaptic efficacy, but not precisionCa•A perimeter model accounts for synaptic efficacy and precision during development 2+ channels and synaptic vesicles critically influences synaptic transmission. Nakamura et al. find that action potentials evoke release of vesicles coupled to the perimeter of Ca2+ channel clusters, explaining synaptic efficacy and precision during development.Nanoscale topography of presynaptic Ca However “loose”  or “tigh “loose” . Detaile 5–10 nm . However2+ channel antibodies and freeze-fracture replica labeling electron microscopy (EM) have established that VGCCs form clusters at the AZ of central mammalian synapses . In another set of labeling samples using a different batch of antibodies (La2), a higher particle density was observed (8.6/μm2 at P7 and 21.7/μm2 at P14).The calyx of Held synapse undergoes both morphological and functional changes during the second postnatal week, when rodents start to hear sounds . To exampe VGCCs . Large cred face A. Specif3, Because the gold particles appeared to form clusters, we tested this possibility by comparing the gold particle distribution with a randomly distributed particle model C–S1F. Tov2.1 labeling followed by RIM staining revealed that the majority of Cav2.1 clusters were closely associated with RIM particles  between clusters was similar, but not identical, across ages (La1): 899 ± 60 nm for P7, 779 ± 32 nm for P14, and 880 ± 38 nm for P21  to P14  and then remained constant for P21 . These data indicate that the density of Cav2.1 remains similar from P7 to P21, but the number of Cav2.1 per cluster and the cluster area increase with development from P7 to P14.The number of particles per cluster in La1 samples varied over a wide range, from 2 to 27 D with an2+] changes was altered during the hearing acquisition period, we recorded local Ca2+ transients in response to single APs using a high-speed confocal spot detection method and low-affinity Ca2+ indicator Oregon green BAPTA-5N (XY axis) and 650 nm (Z axis)  and slow (>10 ms) component, consistent with localized detection of a [Ca2+] domain  . In P7 c] domain . In conttic face B and S4,tic face .2+ transients were also observed for confocal spot locations along the synaptic face, albeit less frequently than in P7 calyces , and exhibited a similar spatial dependence  to the Ca2+ entry site , thus mimicking the AP measured at P7 (2+ transient amplitude (7) or P14 (AP14) calyces. The shorter duration AP14 waveform reduced the time to peak of the Ca2+ current (ICa) by 51% ± 1%  as compared to using AP7 waveforms. Ca2+ transients recorded from the same spot location were 56% smaller when elicited using AP14 than when elicited by AP7  with either 0.1 mM or 2 mM EGTA-containing pipette solutions  matched that of the experimental data  and a Cav2.3-specific blocker SNX-482 , suggesting the VGCC-sensor distance is similar among VGCC subtypes. Consistent with these results, we observed no significant difference in the spatial distribution of Ca2+ entry estimated with confocal line-scans with and without these subtype-specific VGCC blockers (v2.2 and Cav2.3 have a similar distribution to Cav2.1 before hearing onset.The sensitivity of excitatory postsynaptic currents (EPSCs) to presynaptic EGTA has been used to assay the VGCC-sensor distance at the calyx of Held . We reviblockers , suggest2+ buffers remained intact. Bath application of EGTA-AM (10 μM) for 15 min reduced the EPSC amplitude by 35% ± 11%  following termination of Ca2+ entry  across the entire synaptic face  at different locations with respect to the VGCC cluster, we found Pv = 1 within those voxels containing a VGCC, 0.8–1 for voxels within the cluster center, and 0.01 for voxels 55 and 92 nm from the edge of a cluster with 4 and 12 open VGCC, respectively. Increasing the intraterminal [EGTA] from 0.1 to 10 mM inhibited Pv by 20% at the cluster center. Only when Ca2+ sensors were positioned outside the cluster .To simulate vesicular release, we drove a five-state Case model  using thproteins . Computi2+ sensor for vesicular release is located within a short distance from the edge of VGCC clusters. We call this topography the perimeter release model and refer to the distance between the closest open VGCC and the Ca2+ sensor as the perimeter coupling distance (PCD). Simulations using an AP7 waveform and a Ca2+ sensor with higher Ca2+ affinity for P8–P10 rats  is based on single-channel conductance (3.3 pS in 2 mM [Ca2+]) measured for Cav2.1 at the calyx of Held (v2.2 (2.7 pS) at an autonomic preganglionic synapse  and P14 were performed using APeach age . We usedeach age D. Surprieach age E. In coneach age  predicteeach age C. The sheach age B can be each age F and 8A.each age G, similaeach age B and 8C.2+ sensor . This finding could account for spontaneous EPSCs (Whether vesicular release requires opening of one  or multius EPSCs  but not us EPSCs . Our res2+ current measurements (see v2.1 channels per cluster was 18 (=11.1/0.62) on average and varied between 3 and 73 across clusters. At P7 we estimate that there are 10 Cav2.1 channels per cluster, but because Cav2.1 channels comprise 53% of the VGCCs at P7  are located 15–30 nm from the outer perimeter of VGCC clusters . SDS-FRLthan AZs  and 2. Tthan AZs H did notthan AZs , unless v2.1 channels also form clusters within AZs at hippocampal  amplifier (Molecular Devices). Presynaptic internal solution exchange was performed using pipette perfusion, as described previously (2+] changes with Oregon green BAPTA-5N added to presynaptic pipette solution. All experiments were performed at room temperature (22°C–24°C). Data analysis was performed with IgorPro 6.3 (WaveMetrics) using NeuroMatic. Deconvolution of EPSCs was performed as describe previously (Whole-cell patch-clamp recordings were made from calyces of Held and MNTB neurons of acute brainstem slices prepared from P7–P22 Wistar rats. Presynaptic AP, Ieviously . Confocaeviously . All val2+ diffusion and binding with Oregon green BAPTA-5N and buffers in the vicinity of VGCC clusters was simulated using a finite-difference method (2+ transients. Nanoscale simulations were performed without the Ca2+ indicator and then used to drive a five-state Ca2+-dependent release model (Cae method  in orderse model . All sim


Channel inactivation mostly follows the so-called ball-and-chain mechanism, in which the N-terminal structures of either the K+ channel’s α or β subunits occlude the channel pore entry facing the cytosol. Inactivation of Kv1.1 and Kv1.4 channels induced by Kvβ1.1 subunits is profoundly decelerated in response to a rise in the intracellular Ca2+ concentration, thus making the affected channel complexes negative feedback regulators to limit neuronal overexcitation. With electrophysiological and biochemical experiments we show that the Ca2+ dependence is gained by binding of calmodulin to the “chain” segment of Kvβ1.1 thereby compromising the mobility of the inactivation particle. Furthermore, inactivation regulation via Ca2+/calmodulin does not interfere with the β subunit’s enzymatic activity as an NADPH-dependent oxidoreductase, thus rendering the Kvβ1.1 subunit a multifunctional receptor that integrates cytosolic signals to be transduced to altered electrical cellular activity.A-type K To precisely match the need of such counteraction with the shape and frequency of action potentials, some K+ channels, often referred to as A-type channels, undergo rapid voltage-dependent inactivation; they are responsible for regulating the action potential width and afterhyperpolarization, and thus Ca2+ entry and neurotransmitter release1+-selective pore of the channels, which are formed of four α subunits with each six transmembrane segments and cytosolic N and C termini, is occluded by one of the four cytosolic N termini that block the internal mouth of the channel4+ channel α subunits, only a few are capable of inducing N-type inactivation 7811The activity of voltage-dependent K+ channels into inactivating A-type channels not only expand the diversity of K+ current inactivation kinetics found in vivo, they also substantially contribute to various means of regulation by which cells fine-tune K+ channel inactivation and thereby regulate electrical excitation. For example, cysteine residues in the “ball” domain make N(β)-type inactivation sensitive to changes in the intracellular redox milieu as with Kv1.4 and Kv3.4 channels67131516+, i.e. enzymatic activity and electrophysiological function are coupled21Such Kvβ subunits capable of transforming non-inactivating delayed rectifier K2+ concentration slows down inactivation induced by Kvβ1.1 subunits, and the N terminus of these subunits appeared to be necessary for that function+ channels that undergo inactivation by means of Kvβ1.1 subunits provide a negative feedback regulation, as they tend to limit excitation in response to Ca2+ overload. This phenomenon is not universal because inactivation induced by the N-terminal splice variant Kvβ1.3 does not depend on [Ca2+]i2+ dependence of Kvβ1.1-mediated inactivation remained to be elucidated. Knowledge of this mechanism would furthermore allow addressing the question of a potential crosstalk between Ca2+ sensitivity on the one hand and dependence of Kvβ-induced inactivation on the enzymatic activity on the other.Most importantly, an increase in the intracellular Ca2+ sensor protein responsible for the Ca2+ sensitivity of Kvβ1.1–induced K+ channel inactivation. This Ca2+/CaM dependent inactivation modulation was independent of cellular oxidation and of the intrinsic enzymatic activity of Kvβ1.1.Here, we identified calmodulin (CaM) as the Ca2+232+-buffered with 100 μM EGTA. Under this condition, Kvβ1.1 induced rapid inactivation when channels were activated by a depolarizing step  and the peak outward current was increased about twofold . While the “Calmodulation database and meta-analysis predictor” algorithm according to Mruk et al. (2014)2+ dependence of Kvβ1.1-induced inactivation, asparagine was replaced for the arginines yielding the Kvβ1.1 mutant RRR and serine for phenylalanines to yield the mutant Kvβ1.1-FF. Upon coexpression with Kv1.1 in HEK 293T cells, both constructs induced rapid inactivation; inactivation by RRR was slightly slower than that of the wild type (13.5 ± 0.4 ms), while inactivation induced by FF was about 2.5-fold faster (4.0 ± 0.6 ms)  . Applica 0.6 ms) . The pea 0.6 ms) .2+ dependence of Kvβ1.1-induced inactivation. The impact of the mutations on inactivation was also observed when currents were recorded in the whole-cell mode with defined intracellular solutions containing either no free Ca2+ (buffered with 10 mM EGTA) or 1 μM Ca2+ -terminal inactivation is not Ca2+ dependent either , thus rendering the N-type inactivation endogenous to Kv1.4 channels insensitive to acute increases in [Ca2+]i. However, in a physiological setting Kv1.4 often coassembles with Kvβ1.1 subunits, thus yielding Kv channel complexes with eight N-terminal inactivation domainsP < 0.05; center, P = 0.12; right, 2+]i also increased the peak outward current in the combination of Kv1.4/Kvβ1.1-C7S but not with Kv1.4 alone or when coexpressed with Kvβ1.1-C7S-RRR 2+ dependence of the Kvβ1.1-induced inactivation time constant in inside-out patches without CaM application. Although we cannot offer an unequivocal explanation for that observation, we noticed that the amount of cytosol that sticks to the membrane patch upon establishment of the inside-out configuration has a strong influence on how much endogenous CaM is available to facilitate loss of inactivation. As illustrated in 2+, however, channels do not inactivate anymore; yet inactivation is restored by transfer of the patch into Ca2+-free solution and subsequently inactivation is preserved even in 1 μM free Ca2+.The lack of a Caet al. (2011)+; in this state, the chain of the inactivation domain binds to the Kvβ1.1 core domain such that the distal ball domain cannot reach its receptor anymore and, hence, is unable to induce inactivation. Key players for the electrostatic coupling of “chain” and “core” domain are residues R37/R48 and E265/E349, respectively. Since the arginine residues R37 and R48 are apparently taking part in binding CaM to the Kvβ1.1 subunit, it is plausible that the regulation of Kvβ1.1-induced inactivation by NADPH/NADP+ and Ca2+/CaM are coupled.Kvβ subunits exhibit an enzymatic activity as aldoketoreductases using NADPH as a cofactor2+/CaM dependence. Since the enzymatic activity requires oxidizing conditions, which would eliminate inactivation of Kvβ1.1 by means of the regulatory N-terminal cysteine (C7)+ currents. During the course of substrate diffusing into the cytosol, the inactivation time course became progressively slower for Kvβ1.1-C7S (left). As expected, this did not happen for mutant RRR (center). However, 4CY also potently slowed down inactivation induced by Kvβ1.1-FF (right). As a control, we also measured mutant Kvβ1.1-E349K, which did not show a significant response to 4CY 2+ applied to the cytosolic face of clean membrane patches of Xenopus oocytes does not eliminate Kvβ1.1-mediated inactivation, while a combination of CaM and Ca2+ (1 μM each) does  does . Furtherch) does , and mutg motifs . Thus, Kctance2+ , and prece2+  and rat Kv1.4 ; (b) Kvβ subunit human Kvβ1.1 . An overlap-extension mutagenesis approach as described previouslyE. coli BL21 cells and purified using glutathione sepharose 4 fast flow . Recombinant human calmodulin (CaM) was purified as described previously2+-free buffer (PBS-EGTA), or with Ca2+-containing buffer (PBS-Ca). Each 20 μl beads were mixed with 20 μl purified CaM (1 μg/μl) and 400 μl PBS-EGTA or PBS-Ca, respectively, and incubated with shaking for 1 h at 4 °C. The beads were centrifuged for 1 min at 4000 g and washed three times with the respective buffer. Subsequently, the resin-bound proteins were eluted with SDS-PAGE loading buffer, separated on 12% acrylamide gels and stained with Coomassie blue. The PBS buffers (pH 7.4) contained (in mM): 137 NaCl, 2.7 KCl, 10 Na2HPO4, 1.8 KH2PO4, plus 10 EGTA (PBS-EGTA) or 2 CaCl2 (PBS-Ca), respectively.Full-length cDNA of human Kvβ1.1 and point mutants thereof  were cloned into the pGEX-5X vector  to encode GST fusion proteins. The recombinant fusion proteins were expressed in et al. (2014)The N-terminal sequences of various Kvβ subunits were subjected to an analysis searching for potential binding sites for calmodulin according to Mruk in vitro using the mMessage mMachine kit . Oocytes were surgically removed from the ovarian tissue of Xenopus laevis that had been anesthetized by immersion in ice water⁄tricaine according to the local animal care program. The oocytes were defolliculated, and healthy stage V and VI oocytes were isolated and microinjected with 50 nl of a solution containing channel wild-type or mutant mRNA. In co-expression experiments, the ratio of mRNA coding for α and β subunits was 1:3. Electrophysiological measurements were performed 2–4 days after mRNA injection.Capped mRNA was synthesized HEK 293T cells were transiently transfected using the Roti-Fect transfection kit . Dynabeads  were used for visual identification of individual cells, cotransfected with CD8. The ratio of DNA coding for α and β subunits was 1:3. Electrophysiological recordings were performed 2–3 days after transfection.Xenopus oocytes; macroscopic currents were measured using aluminum silicate glass pipettes with resistances of about 1 MΩ. The intracellular solutions contained (in mM): “0 Ca2+”, 100 K-aspartate, 15 KCl, 10 EGTA, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or “1 μM Ca2+”, 100 K-aspartate, 15 KCl, 5.4 CaCl2, 10 HEDTA, 10 HEPES, both (pH 8.0 with KOH). The extracellular solution contained (in mM): 103.6 Na-aspartate, 11.4 KCl, 1.8 CaCl2, 10 HEPES (pH 7.2 with NaOH). Free Ca2+ concentrations were estimated with WEBMAXC software ( http://maxchelator.stanford.edu). Solution changes in patch-clamp experiments were performed using a multi-channel perfusion system in which the patch was placed directly in the center of streaming solution. Calmodulin was applied to inside-out patches with the intracellular solution (either 0 or 1 μM free Ca2+).Ionic currents were recorded using the inside-out or whole-cell configuration at room temperature using an EPC-9 patch-clamp amplifier operated with PatchMaster software . Inside-out patch-clamp experiments were carried out with 2+”,140 KCl, 10 EGTA, 10 HEPES (pH 7.3 with KOH); or “1 μM Ca2+”,140 KCl, 10 HEDTA, 1.7 CaCl2, 10 HEPES (pH 7.3 with KOH); bath solution (in mM): 146 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES (pH 7.4 with NaOH). To study the effect of intracellular Ca2+ in the whole-cell patch-clamp mode, the intracellular solution was buffered with 100 μM EGTA to remove cell free Ca2+, and ionomycin  was applied via the bath solution to elevate intracellular [Ca2+]. Cells with series resistance above 5 MΩ were discarded; the series resistance was compensated electronically for by more than 70%. Leak and capacitance currents were subtracted using a p/6 correction method.Whole-cell voltage-clamp experiments were carried out with transiently transfected HEK 293T cells. Patch pipettes from borosilicate glass with resistances of 0.9–2.0 MΩ were used. The pipette solution contained (in mM): “0 CaP values are specified.Data were analyzed with FitMaster (HEKA Elektronik) and IgorPro . Averaged data are presented as means ± s.e.m. (n = number of independent measurements) unless specified otherwise. Averaged data were compared with a two-sided Student’s t-test or Wilcoxon signed rank test as applicable. The resulting How to cite this article: Swain, S. M. et al. Ca2+/calmodulin regulates Kvb1.1-mediated inactivation of voltage-gated K+ channels. Sci. Rep.5, 15509; doi: 10.1038/srep15509 (2015).


However, maximal ADP-stimulated O2 consumption was specifically reduced in interfibrillar mitochondria from aged hearts. Second generation proteomics disclosed an increased mitochondrial protein oxidation in advanced age. Because energy production and oxidative status are regulated by mitochondrial Ca2+, we investigated the effect of age on mitochondrial Ca2+ uptake. Although no age-dependent differences were found in Ca2+ uptake kinetics in isolated mitochondria, mitochondrial Ca2+ uptake secondary to SR Ca2+ release was significantly reduced in cardiomyocytes from old hearts, and this effect was associated with decreased NAD(P)H regeneration and increased mitochondrial ROS upon increased contractile activity. Immunofluorescence and proximity ligation assay identified the defective communication between mitochondrial voltage-dependent anion channel and SR ryanodine receptor (RyR) in cardiomyocytes from aged hearts associated with altered Ca2+ handling. Age-dependent alterations in SR Ca2+ transfer to mitochondria and in Ca2+ handling could be reproduced in cardiomyoctes from young hearts after interorganelle disruption with colchicine, at concentrations that had no effect in aged cardiomyocytes or isolated mitochondria. Thus, defective SR–mitochondria communication underlies inefficient interorganelle Ca2+ exchange that contributes to energy demand/supply mistmach and oxidative stress in the aged heart.Mitochondrial alterations are critically involved in increased vulnerability to disease during aging. We investigated the contribution of mitochondria–sarcoplasmic reticulum (SR) communication in cardiomyocyte functional alterations during aging. Heart function (echocardiography) and ATP/phosphocreatine (NMR spectroscopy) were preserved in hearts from old mice (>20 months) with respect to young mice (5–6 months). Mitochondrial membrane potential and resting O These subcellular alterations likely contribute to the reduced adaptive capacity to stress  and increased vulnerability to disease of the aged hearts.Age is the main independent risk factor for cardiovascular morbidity and mortality.8 This privileged interorganelle communication facilitates mitochondrial ATP transport for SR Ca2+ cycling and ensures energy replenishment by reciprocal Ca2+ and ADP exchange. Ca2+ is taken up by mitochondria using a low-affinity uniporter whose activity is driven by the elevated Ca2+ concentration in the microenvironment present around ryanodine receptors (RyR).9 Indeed, the kinetics of mitochondrial Ca2+ uptake is more dependent on the concentration of Ca2+ at the SR–mitochondria contact points than on bulk cytosolic Ca2+ concentration.8 Mitochondrial Ca2+ uptake allows energy supply–demand matching through the activation of Krebs cycle dehydrogenases and electron transport chain activity, and at the same time it regulates the regeneration of Krebs-coupled antioxidative defenses (NAD(P)H).10In cardiac cells, electrochemical coupling and metabolic adaptations are based upon the coordination between sarcoplasmic reticulum (SR) and mitochondria tightly interconnected forming an interface to support local ionic exchange and signal transduction in a beat-to-beat basis.2+ uptake in failing hearts and may underlie their increased oxidative stress.11 Also, in diabetic cardiomyopathy, intracellular Ca2+ overload and depletion of energy stores appear to develop as a consequence of sequential SR–mitochondria dysfunction.12 Atrial fibrillation has been associated with an increased fusion of mitochondria and a subsequent increased colocalization of giant mitochondria with SR, a subcellular remodeling process that contributes to the perpetuation of the arrhythmia.13 Because mitochondria are highly dynamic structures, some molecular links have been proposed to provide a stable physical interorganelle bridge15 while others appear to facilitate direct tunneling of Ca2+ and other signaling mediators.16 In the present study, we hypothesized that aging may negatively impact on mitochondria–SR communication by mechanisms involving defective Ca2+ transmission, and we identified reduced physical interaction between RyR and mitochondrial voltage-dependent anion channel (VDAC) as the main responsible of this effect.Defective SR–mitochondria cross talk has been causally linked to the abnormal mitochondrial Caβ-galactosidase  activity, as hallmarks of cell senescence  and after ADP-stimulation (state-3) were largely preserved in subsarcolemmal mitochondria from aged mouse hearts, independently of the substrates used to feed the respiratory complexes, except a slight decrease in complex 1-mediated state-3 respiration, without changes in respiratory control rate H regeneration), in cardiomyocytes of aged hearts, despite normal mitochondrial Ca2+ uniporter activity.All together, these data suggest a defective CaTotal glutathione (GSH) levels (GSHtot) quantified in myocardial tissue were significantly decreased in aging hearts  in permeabilized cells, but it had only little nonsignificant effect in cardiomyocytes from old mice  overlaps with mitochondrial VDAC in young cardiomyocytes, this interaction is reduced to <60% in old cardiomyocytes. PLA, specifically addressed to detect those SR–mitochondria native clusters in which the spatial distance between RyR and VDAC is ⩽40 nm, confirmed the dissociating effect of age at this subcellar level.In cardiac myocytes, SR is surrounded by a dynamic network of mitochondria extending along myofibril length.20 Our proteomics data point to an increased overall oxidation of the MAM fraction, the subcellular proteinaceous SR–mitochondria tethering structure involved in intracellular Ca2+ signaling and bioenergetics regulation,21 as well as an increased oxidation of mitochondrial VDAC. However, the efficiency of our proteomics approach to specifically obtain Cys-containing peptides within RyR molecules is too low to get a reliable quantification of their redox state, probably due to the deep localization of the thiol sites in the receptor. RyR channels contain multiple, potentially redox-sensitive cysteine residues, and cysteine thiol oxidation appears to increase RyR channel activity.22 Our functional data on SR Ca2+ handling are fully consistent with an oxidation-induced alteration of the RyR gating properties, manifested by a substantial increase in spontaneous Ca2+ spark frequency and altered morphology (reduced Ca2+ diffusion pattern) and decreased Ca2+ transient amplitude in the aged cardiomyocytes, despite no significant alteration in total SR Ca2+ content. These SR Ca2+ abnormalities have been attributed to increased frequency of RyR opening events of less unitary duration and have been described in oxidative environments like those present in failing and aging hearts.24 It remains to be elucidated what type of conformational changes may occur in RyR proteins exposed to oxidative stress and how these changes may potentially modify their interaction with other proteins.Altered RyR–VDAC interaction in old hearts cannot be explained by a decrease in the expression of these proteins, because the amount of RyR and VDAC, as quantified by immunofluorescence labeling, western blot and proteomics, was similar in both groups of ages. The expression of other proteins potentially involved in SR–mitochondria bridging, like mitofusin-2 and the cytosolic chaperone Grp75, also remained preserved in old hearts at the different subcellular fractions. Because mitochondria are the major producers of superoxide, it is reasonable to expect that sustained mitochondrial ROS production derived from respiratory activity locally impacts on redox-sensitive proteins at the subcellular microdomain. Indeed, our experiments show increased mitochondrial ROS production in aging cardiomyocytes submitted to increased contractile activity and redox microdomains have been described at the interface between SR and mitochondria, in which NADPH oxidases contribute to ROS generation.2+ uptake that is secondary to reduced Ca2+ transfer from SR. This concept is supported by several observations: (1) it is specifically manifested in response to RyR stimulation with caffeine; (2) it takes place in digitonin-permeabilized cardiomyocytes, in which the contribution of sarcolemma and cytosol to mitochondrial Ca2+ handling is absent; (3) mitochondrial Ca2+ uptake is strictly normal when exposing isolated mitochondria to external Ca2+in vitro. Our data also indicate that reduced SR Ca2+ transfer in aged cardiomyocytes results in defective NAD(P)H regeneration when cells are submitted to increased contractile activity (high-rate pacing), indicating an altered bioenergetic feedback response. Moreover, impaired mitochondrial Ca2+ uptake in aged cardiomyocytes can be reproduced in young cells after pharmacological disruption of SR–mitochondrial physical interaction with colchicine. Of note, this disrupting maneuvre does not have any effect on mitochondrial Ca2+ uniporter activity or O2 consumption, which could reduce mitochondrial Ca2+ uptake by mechanisms independent of interorganelle communication.Our experiments demonstrate that old cardiomyocytes exhibit a depressed mitochondrial Ca2+ transfer from SR to mitochondria,25 and that altered SR–mitochondria Ca2+ exchange may underlie the pathophysiology of several cardiac diseases.26 In an acute setting, like the first phase of myocardial reperfusion, rapid and cyclic SR-induced Ca2+ oscillations – in a hyperoxidative cellular milieu – trigger mitochondrial permeabilization, hypercontracture and cell death.28 In this context, genetic or pharmacological inhibition of SR–mitochondria Ca2+ transfer have been demonstrated to have beneficial effects on cell survival.29 By contrast, chronically insufficient mitochondrial Ca2+ uptake due to defective SR–mitochondrial spatial organization contributes to oxidative stress and energy deficiency.15 This is because Ca2+ released by RyR is partially transferred to adjacent mitochondria, where it activates Ca2+-sensitive Krebs dehydrogenases and other downstream enzymes involved in energy production, like F1–F0 ATPase and adenine nucleotide translocase, as well as antioxidant pool regeneration.32 Therefore, ablation of mitofusin-2 not only disrupts SR–mitochondrial cytoarchitecture but has been demonstrated to alter Ca2+ transients and mitochondrial Ca2+ uptake and to induce bioenergetic–redox mismatch in cardiac myocytes submitted to β-adrenergic stimulation.15 In a recent three-dimensional integrated cardiomyocyte computational model,33 increasing SR–mitochondria distance from 50 nm to 200 nm depressed mitochondrial Ca2+ uptake by 17% and NADH generation by 11% when pacing rates were switched from 0.5 to 2 Hz.33 In our experiments, depressed NAD(P)H regeneration in response to pacing stress occurred only in cardiomyocytes from aged hearts, indicating impaired bioenergetic feedback response secondary to inadequate mitochondrial Ca2+ uptake, and was associated with increased ROS production at the mitochondrial compartment. This mechanism may aggravate the age-associated excess of toxic ROS in a vicious cycle. Importantly, the impact of aging on mitochondrial maximal O2 consumption capacity and RCR was specifically manifested in IFM. This finding is consistent with previous observations suggesting a greater susceptibility of IFM to aging 35 and should be put in the context that this mitochondrial population represents the most important fraction of total cell mitochondria closely juxtaposed with SR, although other mitochondria may contact with RyR2.36 Remarkably, resting O2 consumption, ATP/PCr and ΔΨm are preserved in old hearts, indicating that age-dependent mitochondrial energy deficiency develops only under stressful conditions  and involves the mitochondrial bioenergetics feedback response (NADPH regeneration and RCR). This bioenergetic inefficiency cannot be attributed to reduced mitochondrial mass, which seems to remain preserverd in aging mouse cardiomyocytes as quantified by specific staining with Mitotracker red, total cardiac mitochondrial yield quantification and CS activity measurement.An increasing number of evidences support the view that the coordination between mitochondrial ATP supply and mechanical activity demand is highly dependent on an efficient Ca37 An insidious increase in the oxidation status has been consistently observed during senescence.4 Our data confirm that mitochondrial respiratory complexes are overoxidized in the aged cardiomyocytes. This post-translational damage can reflect excessive ROS generation, decreased antioxidant capacity or reduced degradation of oxidized proteins. Recent evidences have suggested the defective mitochondrial Ca2+ uptake is one of the mechanisms involved in impaired antioxidant regeneration in failing cardiomyocytes.38Aging is characterized by augmented entropy, in which random and stochastic episodes not genetically controlled result in biologically defective molecules. Some covalent modifications – that is, oxidation, glycosilation, phosphorylation, conformational changes and genomic mutations – eventually exceed cell repair capability.2+ handling may, however, amplify cell damage in a positive feedback, as deficient Ca2+ transfer from RyR to adjacent mitochondria further increases oxidative damage (impairing NADPH regeneration) and ultimately leads to inefficient energy production.In conclusion, we propose a pathophysiological mechanism summarized in in situ functional analysis (echochardiography) and for the obtention of myocardial tissue, isolated cardiomyocytes and mitochondria. Animal handling was approved by Research Commission on Ethics of the Hospital Vall d'Hebron. All procedures conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.Young adult (5–6 months) and old (>20 months) C57BL/6 mice were used for 3 – (LVEDD)3)+0.6.Echocardiographic measurements were performerd in young and old mice under light anesthesia (isofluorane 0.5–1%) with a Vivid-Q portable ultrasound system using a ILS 12 MHz transducer . Conventional parameters (ejection fraction of the left ventricle (LVEF), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), septum wall thickness (SWT) and posterior wall thickness (PWT)) were measured in M-mode recordings at the level of the papillary muscles. Left ventricular fractional shortening was calculated as ((LVEDD–LVESD)/LVEDD) × 100. Left ventricular mass was calculated as 0.8x(1.04 × (LVEDD+ PWT+SWT)39 Spectra were acquired in a 9.4T vertical magnet interfaced to a Bruker AVANCE 400 spectrometer  tuned to 161.97 MHz; each spectra consisted in the accumulation of 400 scans and lasted 14 min. The areas of Pcr and γ-ATP peaks were measured by deconvolution.Myocardial energetic status was evaluated in Langendorf-perfused intact mouse hearts by NMR spectroscopy. ATP/PCr was measured as described.et al.40 Ventricular tissue was minced in cold 'buffer A', containing (in mmol/l): 290 sucrose, 5 MOPS at pH 7.4, 2 EGTA and 0.2% deffated albumin. Minced tissue was mildly homogenized using a Potter–Elvehjem device. Homogenates were centrifuged at 750 × g for 5 min. Pellets and supernatants were processed independently to isolate different mitochondrial pools. For SSM mitochondria fraction, supernatants were centrifuged at 5000 × g for 5 min. For IFM fraction, the resulting pellets were resuspended in cold 'buffer B', containing (in mmol/l): 100 KCl, 5 MOPS at pH 7.4, 2 EGTA and 0.2% deffated albumin with proteinase K  at 2 mg/g wet weight, and quickly homogenized. Homogenates were centrifuged at 750 × g for 5 min. Supernatants were subjected to a subsequent centrifugation at 5000 × g for 5 min to obtain IFM. Protein was determined by Bradford assay. For western blot analysis, SSM and IFM were additionally centrifugated using percoll buffer to increase the purity of preparations. Protein was determined by Bradford assay.SSM and IFM cardiac mitochondria were isolated by differential centrifugation from mouse hearts as originally described by Palmer 2 consumption was quantified in SSM and IFM from young and old mice hearts incubated in respiration buffer  using Clark-type oxygen electrodes  after the addition of specific substrates for each of the respiratory complexes ). Maximal O2 consumption (state-3) was obtained with 0.25 mmol/l ADP. RCR was calculated as ADP-stimulated O2 consumption (state-3)/non-stimulated O2 consumption (state-2). CS activity was determined by colorimetry and was used to normalize data on mitochondrial respiration.41O2+ uptake was fluorometrically quantified in 150 μg of SSM or IFM incubated in 235 μl of respiration buffer with 0.5 μmol/l calcium green-5N . Mitochondria were exposed to an external Ca2+ pulse of 30 μmol/l in the presence of 1 μmol/l cyclosporine A to avoid membrane permeabilization. Changes in 530 nm fluorescence were recorded every 10 s. Fluorescence decay after Ca2+ addition reflected the kinetics of mitochondrial Ca2+ uniporter and could be effectively inhibited by 10 μmol/l of the specific blocker Ru360.Mitochondrial Ca2PO4, 1.2 MgSO4, 11 glucose and 10 butanedione monoxime, pH 7.4) with 0.03% type II collagenase . Perfused tissue was subjected to differential centrifugation. Calcium tolerant rod-shaped cardiomyocytes were selected by sedimentation in 4% BSA  gradient, and plated on laminin-coated cover slips. Only when the initial yield of rod-shaped cardiac myocytes was >50%, preparations were considered suitable for experiments.For the obtention of cardiomyocytes, hearts from young (<6 months) or old (>20 months) mice were perfused in a Langendorff system  for 15 min with a modified Krebs buffer . Lipofuscin pigment was observed as autofluorescence after 488 nm laser excitation. In all cases, cell fluorescence was analyzed in background-substracted images using commercially available software .Lysosomes were visualized in cardiomyocytes loaded with 50 nmol/l lysotracker green  and excited at 560 nm using an Ar/Kr laser confocal system , set on an Olympus IX70  with × 60 oil immersion objective lens. Mitochondrial pool was estimated in confocal images of intact cells after labeling mitochondria with 1 Ψm was analyzed in isolated cardiomyocytes loaded with 10 μmol/l JC-1  and excited at 488 nm . Green (520 nm) and red (590 nm) emission lights were simultaneously recorded at × 60  and 590/520 nm fluorescence ratio was calculated as an index of ΔΨm and expressed relative-to-maximal mitochondrial membrane depolarization achieved with 200 μmol/l dinitrophenol (DNP).Resting Δ2+ uptake that is dependent on SR Ca2+ transfer without the contribution of sarcolemma/cytosol, cardiomyocytes were loaded with 5 μmol/l rhod-2 using the cold–warm protocol (60 min at 4 °C followed by 30 min at 37 °C) and the sarcolemma of the cells was subsequently permeabilized with 10 μmol/l digitonin  in intracellular-like buffer  under Ca2+ free conditions (2 mmol/l EGTA). SR Ca2+ release was induced with a pulse of caffeine (10 mmol/l) and mitochondrial Ca2+ uptake was monitored as changes in rhod-2 fluorescence throughout time with respect to the initial value  using an Ar/Kr laser confocal system , set on an Olympus IX70  at × 60.To analyze the fraction of mitochondrial Caμmol/l DNP. Results were expressed as the ratio of NADH(P)H/NAD(P)+ and as percentage of the reduced form with respect to total NAD(P)H pool according to the calibrated data.15 To test the effect of increasing contractile activity on NAD(P)H consumption/regeneration, electrical pacing of the cells was increased from 1 Hz to 5 Hz.NAD(P)H regeneration from NAD(P) was determined in intact cardiomyocytes as autofluorescence after excitation at 340 nm (Em:450 nm) with the aid of a xenon lamp  on the stage of an inverted microscope , at × 40. For calibration, maximal reduction state of NAD(P)H was achieved after the addition of 2 mmol/l sodium cyanide, and minimal reduction state was achieved after the addition of 200 μmol/l of the cytosolic ROS-sensitive fluorochrome 2',7'-dichlorodihydrofluorescein diacetate  and 5 μmol/l of the mitochondrial ROS-sensitive fluorochrome MitosSOX red . Cells were electrically stimulated at low (1 Hz) and high frequencies (5 Hz). At the end of the protocol, 250 μmol/l of the mitochondrial respiratory complex 1 inhibitor rotenone was added to induce a peak of ROS production. Fluorescence was recorded using an Ar/Kr laser confocal system  on the stage of an inverted microscope (Olympus IX70). Results were expressed as relative changes with respect to peak ROS induced by rotenone. Image acquisition was set at 1 image/10 s to avoid phototoxicity.ROS production in cytosolic and mitochondrial compartments was analyzed in freshly isolated cardiomyocytes simultaneously labeled with 10 2+ indicador fluo-4  were superfused with a control HEPES buffer  and electrically stimulated at 1 Hz . Fluorescence changes throughout time were recorded using an Ar/Kr laser confocal system , set on an Olympus IX70  at × 60, and expressed as changes throughout time with respect to the initial value . Image acquisition was set at 40 images/s. Data were analyzed with VoxCell Scan software (Visitech).Intact cardiomyocytes loaded with the cytosolic Ca2+ sparks were analyzed in intact fluo-4 loaded cardiomyocytes incubated in control HEPES buffer using a spectral confocal microscope . Spark frequency (sparks × (100 μm−1) × (s−1)), amplitude (ΔF/F0), rate (ΔF/F0 × (s−1)) and morphology  were quantified using Spark Master plugin of ImageJ software .Spontaneous SR Ca2+ load was analyzed in intact cardiomyocytes loaded with the cytosolic Ca2+ indicator fluo-4 . Cells were submitted to a brief field-stimulation at 1 Hz for 30 s to allow Ca2+ transient stabilization followed by a single pulse of 10 mmol/l caffeine. Maximal amplitude of caffeine-induced peak fluorescence was normalized by the initial fluorescence value (F/F0) and considered as an index of total SR Ca2+ load.Total SR Caμg/ml Hoeschst-33342. Mounted samples were observed with a spectral confocal microscope . Degree of RyR-VDAC overlap was quantified with Mander's coefficient analysis .Isolated cardiomyocytes placed on laminin-coated coverslips were fixed , permeabilized ) and incubated at 4 °C overnight with mouse monoclonal RyR antibody  and rabbit polyclonal VDAC antibody  in PBS–BSA 1%. This was followed by 1 h incubation at room temperature with secondary Alexa antimouse-561 and Alexa antirabbit-488. Nuclei were stained with 10 Isolated cardiomyocytes were simultaneously labeled with monoclonal RyR antibody  and rabbit polyclonal VDAC antibody , followed by the corresponding secondary antibodies (Alexa antimouse-561 and Alexa antirabbit-488) conjugated with the PLA oligonucleotide probes  for 1 h at 37 °C. For ligation and amplification reactions, Duolink InSitu detection kit recommendations were followed (Olink Bioscience). Cell images were registered with a spectral confocal microscope . Positive fluorescent cross-reactivity was quantified in background-substracted images using BlobFinder software .g for 5 min, 4 °C), nuclei and other cell debris were discarded in the pellet, and supernatant was centrifuged . The resulting pellet contained crude mitochondrial fraction. Supernatant was ultracentrifuged  to obtain cytosolic fraction in the supernatant and microsomal vesicles in the pellet. Crude mitochondrial fraction previously obtained was purified in a 30% Percoll gradient column. Percoll gradient column was ultracentrifuged  in a swinging rotor, resulting in pure mitochondria (heavy fraction) and MAM (light fraction).42 Equal amounts of protein (70 μg) supplemented with 1% SDS  were subjected to PAGE. Samples were preheated  in SDS reducing buffer, separated on 6–12% acrylamide/bis (Bio-Rad) gels and transferred to Amersham Hybond-ECL nitrocellulose membranes . The membranes were blocked in TBS-T solution  with 5% non-fat milk powder. Proteins were detected with RyR , VDAC1 , GRP75 , GAPDH , Mitofusin-2  and ANT1/2  antibodies in TBS-T with 3% BSA (Sigma). Horseradish peroxidase-conjugated IgGs were used as secondary antibodies: antimouse , antirabbit , and anti-goat . Peroxidase reactions were carried out and visualized using Supersignal West Dura Extended Duration Substrate (Thermo Scientific) and the chemiluminescence imaging system LAS-3000 . Band intensities were quantified with Science Lab-2001 Image Gauge (Fujifilm).Mouse heart homogenates, microsomal, MAMs and mitochondria extracts were obtained by protein fractionation. Briefly, fresh cardiac mouse ventricles were minced and homogenized using a Potter–Elvehjem PTFE pestle-glass tube at 1400 r.p.m. on ice-cold isolation buffer . After an initial centrifugation step  was homogenized in 0.1 mol/l citrate (pH 4.5) and centrifuged at 12 000 × g for 7 min. The supernatants were diluted in citrate assay buffer containing 2-nitrophenyl-β-D-galactopyranoside (2 mg/ml) and 1 mmol/l MgCl2. After overnight incubation at 37 °C, one volume of 1 mol/l potassium carbonate was added at each sample, and absorbance of o-nitrophenol, the product resulting from the enzyme activity, was read at 420 nm (EmM pH10 21.3 ). β-gal was expressed as μmols of o-nitrophenol/g tissue.44 based on the reaction between Ellman's reagent (DTNB) with GSH to form a spectrophotometric detectable product at 412 nm. A standard curve of reduced GSh (0–50 mM) was used to calculate GSH amounts in mice extracts. Briefly, frozen heart extracts from old and young animals were thawed (50 μl) and subsequently assessed in phosphate-EDTA buffer containing GSH reductase (17 U/ml), NADPH (0.016 mg/ml) and DTNB (0.042 mg/ml). To determine GSSG, a duplicate of each sample was previously incubated with 3 mmol/l of 1-methyl-2-vinylpyridinium triflate, a thiol-scavenging reagent, which rapidly scavenges reduced form of GSH without interfering with the enzyme activity. Results were expressed in nmol GSH/mg protein.GSHtot concentration and oxidized GSH fraction (GSSG) were determined in heart extracts from young and old animals using the GSH reductase enzymatic method,47 Changes in the abundance and composition of cysteine-thiol redox status of mitocondria and microsomal proteins were quantified using the GELSILOX methodology.48 Analyses of samples by LC-MS/MS were performed as previously described.49 Functional protein classification was done using the Gene Ontology database. Proteomics results were analyzed as previously described.50Mitochondrial and microsomal fractions were isolated by differential centrifugation (as described above). Peptides and proteins were identified and quantified by differential high-throughput proteomic analysis performed by stable isotopic labeling using a previously described protocol.t-test for independent samples was used. For comparison of groups with more than one factor, a factorial ANOVA analysis was performed followed by post hoc comparisons when necessary. Differences of P<0.05 were considered statistically significant. When samples did not follow a normal distribution, the non-parametric test of median was used. All statistical analyses were performed with SPSS v.15 software .Data are expressed as mean±S.E.M. For comparisons between two groups two-tailed Student's