Its expression was found to be upregulated in endothelial cells (ECs) under cardiovascular disease conditions. Since the role of MCU in regulating cytosolic Ca2+ homeostasis in ECs exposed to shear stress (SS) is unknown, we studied mitochondrial Ca2+ dynamics  in sheared ECs. To understand cause-and-effect, we ectopically expressed MCU in ECs. A higher percentage of MCU-transduced ECs exhibited mitochondrial Ca2+ transients/oscillations, and at higher frequency, under SS compared to sheared control ECs. Transients/oscillations correlated with mitochondrial reactive oxygen species (mROS) flashes and mitochondrial membrane potential (ΔΨm) flickers, and depended on activation of the mechanosensitive Piezo1 channel and the endothelial nitric oxide synthase (eNOS). A positive feedback loop composed of mitochondrial Ca2+ uptake/mROS flashes/ΔΨm flickers and endoplasmic reticulum Ca2+ release, in association with Piezo1 and eNOS, provided insights into the mechanism by which SS, under conditions of high MCU activity, may shape vascular EC energetics and function.The mitochondrial calcium (Ca The MCU is a heteromeric complex that includes the Ca2+-conducting core protein, also called MCU, and the regulatory proteins MCU dominant-negative β-subunit (MCUb), essential MCU regulator (EMRE), the mitochondrial Ca2+ (mCa2+) uptake (MICU) family (MICU1-3), MCU regulator 1 (MCUR1), and solute carrier 25A23 (SLC25A23)6. Due to the formation of mitochondria-associated membranes (MAMs), a type of quasi-synaptic junctions between the endoplasmic reticulum  and neighboring mitochondria, Ca2+ concentration in the MAM region can reach sufficiently high levels to activate the MCU complex and lead to efficient mCa2+ uptake9. Besides being regulated by the Ca2+ concentration in MAMs and, in essence, by the cytosolic Ca2+ (cCa2+) concentration ([Ca2+]c), the complex activity is also regulated by the levels of reactive oxygen species (ROS) in the mitochondrial matrix (mROS), via a posttranscriptional modification of the MCU protein tail that faces the matrix, and by a continuously increasing number of transcriptional, posttranscriptional, and posttranslational modifications of its subunits10. In return, MCU complex activity regulates the mitochondrial free Ca2+ concentration ([Ca2+]m), and, hence, plays a key role in mitochondrial respiration, ATP production, mitophagy/autophagy, and the mitochondrial pathway of apoptosis13. Furthermore, the mCa2+ fluxes  shape the spatiotemporal profiles of [Ca2+]c and regulate all Ca2+-dependent cell functions, including activation of kinases/phosphatases, gene transcription, and cell survival17.Calcium ion  exposure to arterial-level steady laminar shear stress (SS), an in vitro flow condition that mimics the in vivo hemodynamic environment in straight segments of arteries and arterioles and contributes to the anti-inflammatory and atheroresistant EC phenotype in those regions of the vasculature23. Recent studies showed the EC MCU gene/protein to be upregulated under pathological conditions associated with cardiovascular disease (CVD). Specifically, in apolipoprotein E-knockout (apoE−/−) mice fed a high fat diet and in cultured HUVECs treated with hydrogen peroxide (H2O2), EC MCU expression was upregulated leading to mCa2+ overload and cell apoptosis24. HUVEC treatment with either homocysteine or high glucose also upregulated MCU expression/increased MCU channel activity and led to EC dysfunction and apoptosis26. These findings suggest that altered mCa2+ influx may play a key role in EC dysfunction, the earliest and most critical step in CVD development28.MCU gene/protein expression  in cultured human umbilical vein endothelial cells (HUVECs) was found to be necessary in maintaining the [Ca2+]m temporal profiles in SS-exposed HUVECs and test the hypothesis that the SS-induced mCa2+ response may be altered following EC MCU overexpression (OX). Furthermore, we aimed to delineate the underlying molecular mechanisms responsible for any alterations in the SS-induced EC mCa2+ response due to MCU OX. Using the mitochondria-targeted genetically-encoded Ca2+ indicator (GECI) mito-GCaMP6(m) and mathematical analysis of fluorescence imaging data, we discovered that MCU-transduced ECs when exposed to SS exhibit [Ca2+]m oscillations at a frequency of ~ 10 mHz. There were significantly fewer [Ca2+]m transients in untransduced  ECs exposed to SS for the same time of exposure, and barely any [Ca2+]m transients in either MCU-transduced or untransduced  ECs kept under static conditions. By employing appropriate pharmacological agents and based on published work by others, we proposed a molecular pathway where MCU OX synergizes with SS to cause mitochondrial ROS (mROS) generation, transient ΔΨm depolarization and mitochondrial permeability transition pore (mPTP) opening, followed by mROS release into the MAMs and sensitization of the ER inositol 1,4,5-trisphosphate receptor (IP3R). The released Ca2+ from the ER, via MCU-mediated mCa2+ uptake, generates each subsequent [Ca2+]m oscillation. The plasma membrane mechanosensitive Ca2+ channel Piezo1 and the SS-produced nitric oxide (NO) were involved in maintenance of the above mCa2+ signaling pathway. In summary, our study showed that increased MCU activity disrupts the normal Ca2+ signaling in response to arterial-level SS by causing [Ca2+]m oscillations, which may predispose the vascular endothelium to mitochondrial oxidative stress, inflammation and apoptosis, and contribute to CVD initiation.Based on the above, we designed experiments to monitor the (currently unknown) [Ca30 properly overlapped with mitoTracker Red in HUVECs  showed that all ECs in the monolayers responded with a sharp increase in mito-GCaMP6 fluorescence (an index of [Ca2+]m) upon the onset of flow , they exhibited the same temporal profile of ΔF/F0 as the sheared untransduced ECs, which comprised of a steady ΔF/F0 during the 1 min static followed by a sharp rise upon the onset of SS  were found between (untransduced) Static and each of the other conditions  following analysis of 60 ECs per condition, as well as among all conditions tested  upon 2+]m oscillations during SS , there were many cells that fell in the opposite category. These cells, such as cell 2 in Fig. 2+]m oscillations . As a result, the temporal profile of normalized, averaged over the cell area  mito-GCaMP6 fluorescence intensity (ΔF/F0) identified the [Ca2+]m oscillations in cell 1, but it was unable to identify them in cell 2  and was plotted as a 3D topography surface for each cell  ECs loaded with mtSOX Deep Red were exposed to SS, they showed an increasing trend in fluorescence with a few random transient peaks during SS  that accumulates only in active mitochondria34, and monitored both the mito-GCaMP6 and TMRM fluorescence in the same cells over time under SS. TMRM staining was shown to properly overlap with mito-GCaMP6 staining in static ECs at baseline in the absence of any mechanical or chemical treatment  at a final concentration of 5 µM38. The D-isoform of GsMTx4 at 3 µM was previously shown to inhibit whole cell Piezo1 currents by 70 ± 6%37. A characteristic ΔF/F0 temporal profile of MCU-transduced cells in the presence of GsMTx4 is shown in Fig. 2+]m transients compared to that in MCU OX cells exposed to SS in the absence of GsMTx4  were previously shown to lead to local mROS flashes in the mitochondrial matrix and transient ROS emissions in the MAMs, where they sensitized the IP3R to release ER Ca2+ and increase [Ca2+]m in a cancer cell line33. To examine whether mROS play a role in the initial [Ca2+]m peak and the subsequent transients, MCU-transduced ECs were both preincubated and exposed to SS in the presence of the mitochondrial superoxide (O2-.) scavenger 2--2-oxoethyl triphenylphosphonium  at either 25 nM, 100 nM, or 1 µM, concentrations known to effectively scavenge O2-. in activated ECs40. Characteristic temporal profiles of mito-GCaMP6 ΔF/F0 in MCU-transduced cells exposed to SS in the presence of the lowest (25 nM) and highest (1 µM) MT concentrations tested are shown in Fig. 2+]m transients in a dose-dependent manner compared to that in MCU-transduced cells sheared in the absence of MT  in the mitochondrial matrix, to increase the EC mROS production43. To examine the role of NO in the initial [Ca2+]m peak and subsequent oscillations, ECs were both pretreated and sheared in the presence of the NO synthase (NOS) inhibitor L-NG-nitro arginine methyl ester (L-NAME) at a final concentration of 500 µM, previously shown to inhibit the SS-induced endothelial NOS (eNOS) activation/NO generation44. A characteristic temporal profile of mito-GCaMP6 ΔF/F0 in MCU-transduced cells sheared in the presence of L-NAME is shown in Fig. 2+]m transients compared to that in MCU-transduced cells sheared in the absence of L-NAME  inhibited the percentage of ECs with [Ca2+]m transients, confirming the role of mROS in generating SS-induced [Ca2+]m transients in MCU OX cells and, based on the effect of the highest MT concentration, even in untransduced ECs  from the untransduced  monolayers showed 1–2 transients during the 9 min SS, excluding the initial peak at the onset of SS  in over 80% of the cells following the initial large [Ca2+]m peak that all ECs exhibited at the onset of SS. In comparison, SS alone (without MCU OX) caused only a few [Ca2+]m transients in a small percentage of ECs, whereas no [Ca2+]m transients were observed in static MCU-transduced ECs. Our findings using TMRM and mtSOX Deep Red suggested that SS synergizes with MCU to also generate ΔΨm flickers  and mROS flashes. Our findings using GsMTx4, MT, and L-NAME demonstrated that the SS-induced oscillatory mCa2+ response is mediated by the mechanosensitive Piezo1 channel, mROS, and eNOS activation/NO production, providing additional insights on the intracellular signaling that may lead to EC dysfunction under conditions of increased MCU channel activity.In this study, we unveiled the −/− mice fed a high fat diet for 12 weeks compared to mice fed a general diet (twofold)24, as well as compared to H2O2 -treated HUVECs (2.5-fold)24, homocysteine -treated HUVECs (twofold)25, and high glucose -treated HUVECs (1.5-fold)26, in each case, compared to their respective controls.It is noteworthy that, by employing the adenovirus-mediated MCU overexpression method, we achieved a ~ 2.5-fold MCU protein overexpression in HUVECs Fig. D, which m, mROS, and [Ca2+]c or [Ca2+]m in different cell types under stress: Using fluorescent probes targeted to the MAM area of a cancer cell line together with super resolution microscopy, cellular stress  was shown to cause transient mPTP openings that were associated with, and/or led to, ΔΨm flickers, mROS flashes, and miniature ROS bursts in the MAMs. The latter, by oxidizing the ER-mitochondria interface, sensitized the IP3R to release Ca2+ from the ER, followed by MCU-mediated Ca2+ uptake and an increase in [Ca2+]m33. Either MCU knockout (KO) or IP3R KO inhibited the ΔΨm flickering suggesting a positive feedback loop33. Although their study did not detect [Ca2+]m transients, studies in cardiomyocytes activated by a laser flash found correlations among ΔΨm flickering, mitochondrial O2-. production, and [Ca2+]c spark frequency45. The antioxidant N-acetyl-cysteine (NAC) was shown to block both the transient ΔΨm depolarization events45 and the [Ca2+]c sparks45, collectively suggesting that mROS drive the transient mPTP openings and, by modulating the local redox environment, the [Ca2+]c sparks. In cardiomyocytes under pressurized flow, Kim et al. showed that shear-induced [Ca2+]c sparks were mediated by mROS through NADPH oxidase (Nox)2 activity, since they were inhibited by either MT or a Nox2 inhibitory peptide46. Kuznetsov et al. found a synchronicity in mROS flashes, ΔΨm flickers, and [Ca2+]m transients in a photoactivated cancer cell line, and showed that ROS scavenging, by either Trolox or manganese O2−. dismutase (MnSOD) OX, prevents the ΔΨm flickers47. Last, in HeLa cells activated by hyperosmotic stress, Hou et al. showed that both mROS and [Ca2+]m needed to increase above a certain level to generate [Ca2+]m transients48.Recent studies have explored the interrelationship among ΔΨ2+]m transients, which supports the notion that both mROS and [Ca2+]m increases contribute to the oscillatory mCa2+ response. SS alone is known to increase mROS levels in (untransduced) ECs49, and the present study showed that it also increases [Ca2+]m  ECs. Genetic ablation of MCU exhibited lower mROS levels in primary hepatocytes30 tempting to speculate that MCU OX may cause an increase in mROS, and, when SS stimulation is added, the levels of mROS and [Ca2+]m may satisfy the requirements for generation of [Ca2+]m transients.Our study showed that a combination of SS stimulation with increased MCU activity is required to generate [Ca+]m Fig. C. MCU OX2+]m transients confirmed the central role of mROS in the positive feedback loop inside sheared MCU-transduced ECs: Increased [Ca2+]m, in concert with mROS generated by the electron transport chain (ETC), triggers the opening of the low conductance mPTP and the redistribution of Ca2+ and H+ across the IMM causing a brief depolarization that temporarily shuts down mitochondrial bioenergetics and contributes to an mROS flash  favors mPTP closure, and, hence, the mPTP switches between on and off states resulting in a ΔΨm flicker51. The transient mPTP opening/ΔΨm flicker leads to an ROS burst in the MAMs33  channels. Piezo1 is known to mediate the SS-induced Ca2+ influx and the initial [Ca2+]c rise in HUVECs36, and, since the MAMs region is continuous with the cytosol, Piezo1 is expected to, at least in part, be responsible for the initial peak of [Ca2+]m and the subsequent [Ca2+]m transients, in agreement with our findings. Others showed Piezo1 to interact with the sarco/endoplasmic Ca2+ ATPase (SERCA) and the Piezo1-mediated Ca2+ influx to maintain the SR Ca2+ content and the [Ca2+]c sparks in cardiomyocytes52, suggesting that, in our case, the SS-induced Piezo1-mediated Ca2+ entry may help maintain the ER Ca2+ content and, hence, the [Ca2+]m transients in MCU OX ECs  the SS-induced, Piezo1-mediated Ca2+ influx phosphorylates/activates eNOS53, (b) the produced NO, either on its own or via formation of reactive nitrogen species (RNS)/ONOO−, increases mROS production by the ETC43, and (c) mROS are necessary for the SS-induced [Ca2+]m transients in MCU OX ECs (this study), it is expected that NO via mROS will enhance the formation of [Ca2+]m transients  channel, which allows for Ca2+ entry in SS-exposed ECs55 and may be regulated by Piezo136, was shown to get activated via NO-mediated cysteine S-nitrosylation56 and, hence, in the absence of NO, the SS-induced Ca2+ influx  will be compromised, in agreement with our findings.Our study also showed a significant inhibition of both the initial [Ca2+]m oscillations remained confined in the regions of the mitochondrial network where they originated. These observations, however, cannot exclude the possibility that, if SS were to be extended (hr-days), it might activate mitochondrial fission, inflammation, and apoptosis. MCU OX alone does not cause apoptosis, but is known to exacerbate it, when cells are exposed to various oxidant stimuli57. SS alone is known to protect ECs from apoptosis by activating nuclear factor erythroid 2-related factor (Nrf)2 and Krüppel-like factor (KLF)259. A study found the mCa2+ influx to be essential for agonist-induced eNOS activation60. If the mCa2+ influx similarly regulates the SS-induced eNOS activation/NO production, then it is possible that exposure of MCU OX ECs to SS would generate pathological levels of NO/RNS, which could lead to apoptosis.The present study did not examine the long-term effects of SS in MCU OX ECs. However, during the 9 min of EC exposure to SS, we did not observe any global mitochondrial depolarization events (detected by TMRM) or significant mitochondrial fission events (detected by either mitoTracker Red or mito-GCaMP6). Furthermore, the m transients are a part of the loop that consists of ΔΨm flickers/mROS flashes/[Ca2+]m transients, it is reasonable to expect that the [Ca2+]m transients regulate the exact same signaling pathways as the mROS flashes. In agreement to that, a recent study showed the [Ca2+]m oscillations generated by laser stimulation in HeLa cells to be responsible for phosphatase and tensin homolog (PTEN)-induced kinase (PINK)1 accumulation on the outer mitochondrial membrane (OMM), parkin recruitment to the OMM, and mitophagy63.The mROS flash frequency is thought to correlate with the mitochondrial metabolic state, because it was shown to increase in response to increased substrate, glucose or fatty acid, supply in cells2+ dynamics requires that the detection range of the probe is appropriate for the size and time course of the Ca2+ transient(s). The GCaMP Ca2+ sensors were designed to probe neuronal function, because neurons have fast Ca2+ dynamics and relative low peak accumulations (< 1 µM)64. The GCaMP6 sensor employed was chosen due to its rapid kinetics and high Ca2+ sensitivity that contribute to an increased temporal resolution of signals. However, GCaMP6 has a relatively high Ca2+ affinity (Kd of 0.17 µM)66, which means that transient peaks close to the maximum dynamic range of ~ 1 µM will be saturated. [Ca2+]m was previously recorded in cultured pulmonary artery ECs transfected to express the fluorescence resonance energy transfer (FRET)-based low-affinity mCa2+ indicator Cameleon D3cpv (Kd of 0.76 µM) during cell exposure to histamine (100 nM)67. [Ca2+]m was shown to rise from 0.1 µM at baseline to 0.8 µM immediately following histamine addition and within 1 min decreased to 0.2 µM and remained at that level at later time points67. Mitochondria, including EC mitochondria, have a phosphate buffering system that does not allow [Ca2+]m to reach above 2 µM69. Based on all the above, and, since [Ca2+]m signals were not calibrated in this study, it is possible that the relatively large [Ca2+]m signal upon the onset of flow may be saturated and that would lead to the absence of a difference in peak amplitudes between sheared MCU-transduced ECs and sheared controls. There is no such concern regarding the mito-GCaMP6-mediated recording of the [Ca2+]m transients following the initial flow-induced peak.The success of monitoring intracellular or intraorganellar Ca2+]c response was not monitored in MCU-transduced ECs under SS. The mito-GCaMP6 fluorescence is not compatible to double-label with Fluo-4. It is compatible with Fura-2, but Fura-2 requires a different LED light source for excitation. For the double-labeling experiments to add meaningful information on Ca2+ homeostasis in sheared ECs, the cytosolic and mitochondrial sensors should have similar Ca2+ affinities and kinetics to allow for optimal detection of large vs. small and fast vs. slow Ca2+ dynamics in the different compartments. Furthermore, it is possible that, in the case of the shear-induced [Ca2+]m transients in MCU-transduced ECs, the [Ca2+]c sensors would not detect any significant perturbations, because these small [Ca2+]m transients would mostly affect the Ca2+ concentration in the MAMs.Another limitation of this study is that the [Ca2+]c and [Ca2+]m responses in the same cells found [Ca2+]m to oscillate concomitantly with [Ca2+]c: Ishii et al. used pericam and Rhod-2 to measure [Ca2+]c and [Ca2+]m, respectively, in HeLa cells stimulated with histamine and, based on published characteristic signals, the oscillation frequency was in the order of tens of mHz with the mitochondrial oscillations showing a time lag compared to cytosolic ones70. Hajnoczky et al. used Fura-2 and dihydro-Rhod-2 to measure [Ca2+]c and [Ca2+]m, respectively, in hepatocytes stimulated with a low dose of vasopressin and, based on characteristic signals, the oscillation frequency was lower than 10 mHz71. Samanta et al. used Fura-2 and pericam to measure [Ca2+]c and [Ca2+]m, respectively, in a monocytic cell line stimulated with a leukotriene and showed signals with an oscillation frequency of ~ 15 mHz72. In our prior work, we used Fluo-4 to study [Ca2+]c transients/oscillations in  HUVECs exposed to 10 dynes/cm2 for 5 min, and found a [Ca2+]c oscillation frequency of ~ 17 mHz43, which differs from the [Ca2+]m oscillation frequency reported in this study (~ 1 mHz) by an order of magnitude. The mismatch in [Ca2+]c and [Ca2+]m oscillation frequencies is not due to differences between Fluo-4 and GCaMP6, since the time course and magnitude of fluorescence response to agonists were reported to be similar for the two indicators73. The mismatch in oscillation frequencies is probably due to the different methods used to detect transients/oscillations: In our SS-induced [Ca2+]c study43, the transients had relatively large amplitudes (mean ΔF/F0 of ~ 0.5) and a threshold of > 15% of the local ΔF/F0 was chosen arbitrarily to identify [Ca2+]c transients. In contrast, in the present study, because of the relatively small amplitude of [Ca2+]m transients (mean ΔF/F0 of < 0.1), we developed a mathematical approach to identify [Ca2+]m transients by selecting an intensity threshold and a drop (percentage of the maximum ΔF/F0) and, as a result, we identified fewer events and a lower [Ca2+]m oscillation frequency compared to the previously reported [Ca2+]c oscillation frequency in sheared (untransduced) ECs. In sheared MCU OX ECs, our mathematical analysis yielded a [Ca2+]m oscillation frequency of ~ 10 mHz, which is within the range of frequencies calculated from the published [Ca2+]m characteristic signals in agonist-stimulated cells, as listed above.The limited number of studies that recorded both [CamCa2+ signaling in human ECs with and without MCU OX during exposure to SS. Our main findings are that (a) SS leads to ΔΨm flickers/mROS flashes/[Ca2+]m oscillations in ECs with increased MCU activity/mCa2+ uptake and (b) the SS-induced oscillatory mCa2+ signaling in ECs with increased MCU activity is mediated, at least in part, by Piezo1 activation, mROS and NO/RNS. Since EC MCU expression was upregulated under pathological conditions, a better understanding of the EC Ca2+ and redox homeostasis under conditions that mimic the in vivo hemodynamic environment may lead to development of improved prevention and/or treatment strategies for EC dysfunction and CVD.In summary, this is the first study that recorded 2/95% air. Plastic parallel-plate flow chamber slides with high optical quality  were coated with 6% fibronectin solution for 40 min. ECs of passage 3–5 were seeded on the fibronectin-coated chamber slides at a seeding density of 36,000–40,000 cells/cm2. At 24 h, cells reached 60% confluency and were incubated with an adenoviral vector that expresses the mitochondria-targeted GECI mito-GCaMP6m  in EGM at a multiplicity of infection (MOI) of 10 for 6 h, as described previously by our group30. Ad.mito-GCaMP6 was constructed by fusing the mitochondrial matrix-targeting sequence to a DNA plasmid encoding GCaMP6m (GenBank), and then inserting the plasmid into a replication-deficient adenoviral vector under the control of the promoter-enhancer region of the human cytomegalovirus (Vector BioLabs)30. Media were changed every 24 h after the 6 h incubation period. At 48 h post-transduction, ECs had formed confluent monolayers and were either exposed to SS or kept static. Some EC cultures at 60% confluency were also transduced with Ad.MCU at an MOI of 50, as previously31, simultaneously with Ad.mito-GCaMP6, in EGM for 6 h with media changes every 24 h after the transduction period. Ad.MCU was made in house by inserting a human gene coding for MCU (GenBank) into a replication-deficient adenoviral vector under the control of the promoter-enhancer region of the human cytomegalovirus31. MCU OX was verified by Western blotting. EC extracts were prepared using RIPA buffer (Abcam: ab156034) and Halt™ protease and phosphatase inhibitor cocktail, EDTA-free (ThermoFisher Scientific: 78441). Equal amounts of protein (20 μg/lane) were separated on 4–12% Bis–Tris polyacrylamide gels (ThermoFisher Scientific: WG1402BOX), transferred to a PVDF membrane (ThermoFisher Scientific: 1B24001) using iBlot 2 PVDF regular stacks (ThermoFisher Scientific: IB21001), and probed with antibodies specific for MCU , MICU2 , and β-actin . ECs either not transduced with Ad.MCU or transduced with a control Ad.βGal vector at an MOI of 50 (Vector BioLabs:1080), always in the presence of Ad.mito-GCaMP6 in EGM for 6 h, were used as controls.Pooled primary HUVECs (Lonza: C2519AS) were grown in complete EC growth medium  in a tissue culture incubator at 37 °C in an atmosphere of 5% CO2 . During SS exposure, the slides were placed in a stage-top incubator  of an inverted epifluorescence microscope  equipped with a high speed sCMOS camera , an LED8 light source (Leica: 11504256), and an environmental temperature control unit . For the first 1 min, EC monolayers were maintained under static conditions in basal/low-serum media to establish a baseline in mito-GCaMP6 fluorescence (representative of resting [Ca2+]m). At the end of 1 min, flow (corresponding to an SS level of 12 dynes/cm2) started and ECs were exposed to 9 min of SS.EC monolayers in ibidi chamber slides at 48 h post-transduction with Ad.mito-GCaMP6, and with/without Ad.MCU or Ad.βGal, were preincubated with basal/low-serum media consisting of EBM  supplemented with 1% FBS (Lonza: CC-4133) and antibiotics (Lonza: CC-4083) for 4 h for the cells to become quiescent and equilibrate with the perfusion media. The slides with the EC monolayers were attached to an ibidi Pump System (ibidi: 10902) using the yellow-green perfusion set  to achieve an arterial-level SS of 12 dynes/cm2+ channel Piezo1 on the SS-induced mCa2+ response, some EC monolayers, with/without MCU or βGal OX, were treated with the Piezo1 specific inhibitor Grammostola spatulata Mechanotoxin 4 38 at a final concentration of 5 µM in basal/low-serum media for the last 5 min of the 4 h preincubation period and throughout SS exposure (1 min static followed by 9 min SS). To investigate the role of mROS on the mCa2+ response, some EC monolayers were treated with the mitochondrial O2−. scavenger 2--2-oxoethyl triphenylphosphonium  at a final concentration of either 25 nM, 100 nM, or 1 μM for the last 5 min of the 4 h preincubation and throughout SS. These concentrations were previously shown to be effective in scavenging EC mROS40. To test for the contribution of the endothelial NO synthase (eNOS) activation/NO production in the SS-induced mCa2+ response, some EC monolayers were preincubated with the NOS inhibitor L-NG-nitro arginine methyl ester  at a final concentration of 500 µM for the last 30 min of the 4 h preincubation period and throughout SS, as in earlier work by our group44. Control ECs were treated with equal volumes of the respective vehicle, either H2O or DMSO .To delineate the role of the plasma-membrane mechanosensitive Ca. MATLAB (MathWorks) scripts were created to track temporal changes in mito-GCaMP6 fluorescence intensity (in arbitrary units) and were averaged over the surface area of each EC in a microscope field of view. Acquired videos were uploaded to MATLAB as a three-dimensional matrix with the first two dimensions representing the x/y dimensions of the video frame and the third one representing the acquisition time/frame number. ECs were identified and segmented using the roipoly function, which prompted for manual drawing of cell boundaries (from phase contrast images) and saved each cell area as a region of interest (ROI). First, an average fluorescence  intensity was calculated for each ROI in each frame. Then, for quantification of the observed mito-GCaMP6 transients, a separate script nnz was used, which adopted the prior segmentation method for analysis of each ROI in each video frame. The nnz function counted the number of image pixels within each ROI that had an intensity level above a grayscale intensity threshold. Thresholds were selected from a 3D topographical surface of pixels above a threshold vs. a range of threshold values vs. time. As a result of this analysis, each ROI corresponded to a 600-element vector containing the pixels above the threshold at each video frame. To quantify the number of [Ca2+]m transients from this data set, the findpeaks function was used. All relative maxima in the dataset were filtered using the parameter ‘MinPeakProminence’, which only considers relative maxima that are greater than the preceding relative minimum by a specified amount. This prominence value or, else called, drop percentage was determined by multiplying the maximum intensity value of the data set (which corresponds to the fluorescence peak right after the flow onset) by a decimal value in the range of 0.05–0.25. The exact drop value for each ROI was determined by examining the persistence of [Ca2+]m transients over a range of drop percentages. Specifically, a value within the widest range of drop percentages with persistent [Ca2+]m transients was selected as the drop percentage value for the data set from each ROI. This determined the number of [Ca2+]m transients in each individual EC over the 9 min of SS exposure. The initial peak in fluorescence intensity due to the onset of SS was excluded, as it was present in the mCa2+ response of every SS-exposed EC and was not considered a transient. The code developed for the above analysis is supplied in Digital images of relative fluorescence intensity changes were collected using a 63 × magnification at a frequency of 1 Hz and stored at 30 frames/s (fps) as .mpeg files using LAS X Life Science Microscope Software (Leica)0; time-averaged over the 1 min static prior to SS) and the peak amplitude (ΔF = F − F0) at each time point under SS for each of 60 ROIs . Normalized fluorescence intensity (ΔF/F0) temporal profiles were plotted for each EC under static and SS with/without MCU OX and with/without each of the chemical treatments . Average mtSOX Deep Red fluorescence intensity data per ROI were also used to plot normalized fluorescence (ΔF/F0) temporal profiles for each EC under SS. To accommodate for the TMRM photobleaching, data acquisition was limited to a few min, and, hence, TMRM temporal profiles were plotted as not-normalized averaged over an EC (or a subregion within a cell) fluorescence intensity data . The calculated number of [Ca2+]m transients per EC from the threshold/drop analysis described in the previous section was used to determine the percentage of total cells (60 from n = 4 independent experiments) that exhibited [Ca2+]m transients/oscillations in each condition tested. Cells with [Ca2+]m transients were also binned based on their number of transients and expressed as a percentage of total cells. The number of transients per cell averaged over a total of 60 cells in each condition was quantified over binned 60 s time intervals. The cumulative number of [Ca2+]m transients over the 9 min duration of SS was calculated and was used to determine the oscillation frequency (in mHz) per cell, which was then averaged over 60 cells from n = 4 independent experiments in each condition tested.Average mito-GCaMP6 fluorescence intensity for each ROI (F) was used to quantify a baseline  at a final concentration of 2 µM in basal/low-serum media for the last 30 min of the 4 h preincubation period. Changes in ΔΨm were recorded during SS exposure in some EC monolayers by loading the cells with tetramethylrhodamine methyl ester  that accumulates only in active mitochondria34, at a final concentration of 20 nM in basal/low-serum media for the last 30 min of the 4 h preincubation period. To visualize all mitochondria, some EC monolayers were treated with mitoTracker Red  at a final concentration of 100 nM in basal/low-serum media for the last 20 min of the 4 h preincubation period.Some EC monolayers were treated with the mitochondrial O2+]m transients per cell at 60-s time intervals in each treatment were presented as mean ± SE from 60 cells . Data on the number of cells that exhibited [Ca2+]m transients expressed as percentage of total cells and on the oscillation frequency per cell were presented as mean ± SE from n = 4 independent experiments for each condition tested. Statistical differences were determined using a one-way analysis of variance (ANOVA) followed by a Dunnett's multiple comparisons test with a P ≤ 0.05 indicating statistical significance. All statistical analyses were performed using GraphPad Prism 9 on a PC.Data on baseline fluorescence, peak amplitude, and the number of [CaSupplementary Information 1.Supplementary Video 1.Supplementary Video 2.


MYC/MYC rearrangements, but also epigenetic defects and novel concepts in oncogenic and immune pathways. Concerning pDC-AML, they now appear to exhibit an original mutation landscape, especially with RUNX1 mutations, which is of interest for diagnostic criteria and for therapeutic purposes. We highlight here these two different profiles, which contribute to differential diagnosis between BPDCN and pDC-AML. This point is particularly important for the study of different therapeutic strategies between BPDCN and AML.Differential diagnosis between Blastic pDC Neoplasm (BPDCN) and Acute Myeloid Leukemia with pDC expansion (pDC-AML) is particularly challenging, and genomic features can help in diagnosis. This review aims at clarifying recent data on genomics features because the past five years have generated a large amount of original data regarding pDC neoplasms. The genetic landscape of BPDCN is now well-defined, with important updates concerning MYB/MYC rearrangements as well as recurrent deletions involving ETV6, IKZF1, RB1, and TP53 loci. Epigenetic and splicing pathways are also particularly mutated, while original processes are dysregulated, such as NF-kB, TCF4, BCL2, and IFN pathways; neutrophil-specific receptors; and cholinergic signaling. In contrast, cytogenetic abnormalities are limited in pDC-AML and are quite similar to other AML. Interestingly, RUNX1 is the most frequently mutated gene (70% of cases). These typical genomic features are of potential interest for diagnosis, and also from a prognostic or therapeutic perspective.Plasmacytoid Dendritic Cells (pDC) are type I interferon (IFN)-producing cells that play a key role in immune responses. Two major types of neoplastic counterparts for pDC are now discriminated: Blastic pDC Neoplasm (BPDCN) and Mature pDC Proliferation (MPDCP), associated with myeloid neoplasm. Two types of MPDCP are now better described: Chronic MyeloMonocytic Leukemia with pDC expansion (pDC-CMML) and Acute Myeloid Leukemia with pDC expansion (pDC-AML). Differential diagnosis between pDC-AML and BPDCN is particularly challenging, and genomic features can help for diagnosis. Here, we systematically review the cytogenetic, molecular, and transcriptional characteristics of BPDCN and pDC-AML. BPDCN are characterized by frequent complex karyotypes with recurrent  World Health Organization (WHO) Classification of Tumours of Heamatopoietic and Lymphoid Tissues: Blastic pDC Neoplasm (BPDCN) and Mature pDC Proliferation (MPDCP), associated with myeloid neoplasm [+ CD56+ neoplasm, with frequent skin involvement and leukemic dissemination, mainly affecting elderly men [WHO Classification of Tumours of Heamatopoietic and Lymphoid Tissues [MYC/MYC rearrangements, epigenetic defects, oncogenic and immune pathways), while pDC-AML now appear to exhibit an original mutation landscape, which is of interest for diagnostic criteria. Consequently, genetic data are now sufficiently described to allow comparison, and we highlight here the key defects, which is particularly important for diagnosis, prognosis, and future therapeutic strategies.Plasmacytoid Dendritic Cells (pDC), the natural type I interferon producing cells, are key players in innate and adaptive immune responses. Two major types of neoplastic counterparts for pDC are now discriminated by the neoplasm ,2. The ferly men ,4,5,6,7. Tissues ,2. Two tCytogenetic abnormalities are detected in 57 to 75% of BPDCN patients. Most of the karyotypes contain a wide spectrum of cytogenetic abnormalities, leading to a complex karyotype (CK) (≥3 aberrations) in more than 50% of cases ,9. AbnorCDKN2A/CDKN2B), 12p13.2-p13.1 , 13q11-q21 , 5q31 (NR3C1), or 7p12.2 (IKZF1) [IKZF1, required for BPDCN differentiation [RB1, potentially associated with transformation in the case of biallelic inactivation [ETV6, whose deletion would correspond to an early pathogenic event [NR3C1, involved in the glucocorticoid metabolism [MYC and MYB are also originally rearranged at high frequency in this neoplasm. Remarkably, the significant association of these alterations  constitute the special and unique pattern frequently detected in BPDCN  ,9,12. Detivation , ETV6, wtabolism . The train BPDCN .IFNGR1, TNFAIP3), 9p21.3 (cluster of IFNA genes), and 12p13.2-p13.1  [Interestingly, these recurrent deletions also involve genes related to the original function of pDC, i.e., immune response, especially losses of 6q23 (NFRSF1A) . Thus, tTP53 [RB1, involved in the regulation of the G1/S cell cycle transition, is also frequently deleted [RB1 is also reported in chronic lymphocytic leukemia and Acute Lymphoblastic Leukemia (ALL) [CDKN1B/CDKN2B/CDKN2A genes have a role in the G1/S transition, and their deletions are reported in some studies [Similarly to other hematological malignancies, deletions inactivating Tumor Suppressor Genes (TSG), such as TP53 ,17,18,19TP53 , may als deleted ,17,19,21 deleted , RB1 is ia (ALL) . Additio studies ,17,19,24 studies  and lymp studies . This alHINT1 was first proposed to be a key gene [HINT1 encodes a homodimeric purine phosphoramidase, suggesting a transcriptional modulatory role. Moreover, HINT1 deficiency would impair ATM function and thus DNA repair [NR3C1 was found to be recurrently deleted, leading to haploinsufficiency and decreased glucocorticoid receptor activity [EZH2, with dysregulation of the HOXA locus and plasmacytoid dendritic differentiation.Deletion in 5q are particularly recurrent in BPDCN, constituting a specific defect compared to other hematological malignancies ,26. In tA repair . The gluactivity . The delETV6 (TEL) is frequently mutated or deleted [ETV6::RUNX1 in B-cell Acute Lymphoblastic Leukemia (B-ALL) [ETV6::PDGFRB, t ETV6::JAK2 or t  ETV6::PDGFRA in hypereosinophilic syndromes, and other translocations in rare cases of Acute Myeloid Leukemia (AML) [ETV6 remain rare in AML [Similarly to other hematological neoplasms, transcription factor are particularly impacted in BPDCN.  deleted ,16,17,24 (B-ALL) , tia (AML) . ETV6 ine in AML , but gerIKZF1/2/3) is also frequently deleted [ZEB2 may also be altered in BPDCN [The IKAROS family  rearrangements had previously been described in rare cases of CD4+ CD56+ neoplams identified as BPDCN (KMT2A::ENL and KMT2A::MLLT1) [While translocations involving transcription factors have been widely described in ALL and AML, recurrent rearrangements in BPDCN were rare before 2017. Indeed, ::MLLT1) ,35, but ::MLLT1) .MYC rearrangements (8q24) have been largely described in approximately 30% of BPDCN [Initially, the translocation t was sporadically reported by several studies, using conventional karyotyping. Since 2018, of BPDCN ,37,38,39of BPDCN .MYC abnormalities bring BPDCN closer to high-grade B lymphoma [MYC rearranged cases of BPDCN, Sakamoto et al. confirmed the high prevalence of the t—detected in 56% of cases involving the RUNX2 locus at 6p21. Interestingly, Kubota et al. showed that the t juxtaposes the promoter of MYC to the pDCs-specific RUNX2 super-enhancer, leading to overexpression of MYC. In this recurrent t, both MYC and RUNX2 are dysregulated, and cooperate together to promote survival and proliferation of the BPDCN cells. Remarkably, RUNX2 is physiologically involved in differentiation and migration of pDCs and plays a dominant role in controlling transcription networks in BPDCN [These lymphoma ,13,37,41in BPDCN .MYC have been sporadically reported but not clearly identified . It remains to be determined if MYC rearrangement could constitute a primary or secondary genetic event in BPDCN. In this way, the t cannot be considered as a specific genetic abnormality of BPDCN because it has been reported in follicular lymphoma [MYC rearrangement, and this prognostic impact still needs to be confirmed by further independent studies [Other partners of lymphoma . Lastly, studies .MYB rearrangement. Of note, the previous largest report of pediatric BPDCN cases exhibited several cases with 1q and/or 6q abnormalities, or translocation t [MYB rearrangement.In 2017, other recurrent rearrangements were described in nine of fourteen patients, including five children . Remarkaq21;q23) . These oMYB and various partner genes  [MYB transcriptional activity. Indeed, functional analysis of MYB fusions revealed the activation of MYB target genes as a result of induced MYB activation [MYB is a nuclear-localized transcriptional activator in hematopoietic cells that interacts with the C/EBP complex to stimulate the transcriptional activity of MYC, BCL2, c-KIT, c-ERBB2, and other targets  and splicing  [TET2 are found in 40 to 60% of cases [NPM1 were initially described in BPDCN [FLT3, KIT, KRAS, and NRAS mutations [In addition to these cytogenetic defects, the mutation landscape of BPDCN has been particularly studied. However, given the rarity of BPDCN, only small cohorts have been studied so far (less than 30 cases), and usually by targeted high-throughput sequencing. Nevertheless, 22 Whole Exome Sequencing (WES) have been performed ,16,50. O, U2AF1) ,57,58,59, U2AF1) . In mult, U2AF1) , and theof cases ,59. Inteutations ,63,64. Tutations ,51. The utations ,65 or AMutations ,67,68,69IKZF1, ETV6, RB1, ATM, and TP53 can also be mutated in some cases (5–10%), also resulting in an invalidation [ETV6 argue for a primordial early event, possibly overexpressing the BPDCN oncogene TCL1A [IKZF1 loss-of-function, either by deletion or mutation, would lead to the increased cell interactions in BPDCN. BPDCN also exhibit KMT2D and SYNE1 mutations or losses, previously reported in follicular lymphoma [Associated with these “myeloid-like”, key deleted transcription factors or tumor suppressor genes lidation ,18,70,71ne TCL1A ,15,72. Ilymphoma ,70. OverEuropean Leukemia Network recommendation [ETV6, TP53, or NRAS/KRAS [NR3C1 [MYC [A major challenge to determine the mutation landscape in BPDCN is to establish a molecular prognostic stratification, as in AML with the endation . NeverthRAS/KRAS , as wellRAS/KRAS ,9, deletS [NR3C1 , abnormaS [NR3C1 ,75, and 3C1 [MYC ,76. At tTET2, IDH1, IDH2) [ASXL1, EZH2) [ARID1A, SUZ12) [As the mutation profile suggests, epigenetic alterations are common in BPDCN, from DNA methylation  ,56,57,771, EZH2) ,50,51,77, SUZ12) ,21,50. I, SUZ12) ,79.LXR targets [Considering their lineage on phenotypic criteria, an original transcriptional program was expected in BPDCN compared to other acute leukemia and was confirmed by various independent studies  15,80,8,880,81. Three different studies focused on the immune response against BPDCN and the tumor microenvironment. Indeed, Summerer et al. recently showed that a group of BPDCN up-regulated the neutrophil-specific receptors CD177 (HNA2A) and CD11b (ITGAM), indicating the presence of an activated subset of neutrophils, with a potential impact on overall survival to be confirmed . On the To sum up, the dysregulated pathways in BPDCN are now analyzed with a microenvironment scope, and dysregulated pathways needs to be correlated to these potential immune defects.Classification of Tumours of Heamatopoietic and Lymphoid Tissues [Classification of Tumours of Heamatopoietic and Lymphoid Tissues) [BPDCN should not be confused with other pDC proliferation, the latter constituting a heterogeneous entity. MPDCP associated with a myeloid neoplasm corresponds to an emerging entity, referred to as a differential diagnosis from BPDCN in the 4th edition of the WHO  Tissues . ContrarTissues) . GeneticNRAS, KRAS, NF1, CBL, PTPN11, PTPN13), the epigenetic regulators , the splicing factors (SRSF2), and intracellular signaling pathway  mutations found in both monocytes and pDCs [RUNX1 mutations, otherwise found in 30–40% of CMML [Nodules of pDC have been commonly described in the bone marrows of CMML ,94,95,96and pDCs . Importa of CMML ,99. In tConversely, pDC-AML have been less described, and only in independent studies, with concordant descriptions ,105,106.KMT2A and inv(16)(p13q22) with CBFB::MYH11 fusion [Classification of Tumours of Heamatopoietic and Lymphoid Tissues [At first, similarly to pDC-CMML, chromosomal abnormalities in pDC-AML seem to be similar to those found in other AML, with a clonal relationship between pDC and blast cells. This clonal relationship has occasionally been reported by karyotype or FISH: notably with isolated del(5q), monosomy 7, trisomy 8, trisomy 13, trisomy 20, or classical recurrent rearrangements such as 11q23 rearrangement involving 1 fusion ,106,107.1 fusion . Importa Tissues , and not Tissues ,102,103.CBFB::MYH11 transcript [FLT3-Internal Tandem Duplication (FLT3-ITD) [CBFB::MYH11 transcript defining a distinct entity, FLT3-ITD are sub-clonal mutations found in many subtypes of AML, and even in BPDCN [Since 2020, the only molecular abnormalities described were the presence of the anscript ,104,107 LT3-ITD) ,104,105.in BPDCN ,62. ThusRUNX1 mutations in 2021 as the most common mutations in pDC-AML, detected in more than 70% of cases, whereas it was detected in 5–15% of AML and absent in BPDCN [RUNX1 defined by the 4th edition of the WHO Classification of Tumours of Heamatopoietic and Lymphoid Tissues [European Leukemia Network stratification [RUNX1 mutations have been largely studied in hematological malignancies with both germline and somatic origins. Indeed, germline mutations are reported in familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML), while somatic mutations are observed in various types of hematological malignancies, including AML. Most of the mutations were clustered in the runt homology domain (RHD) and the c-terminal transactivation domain (TAD)  [RUNX1 are strongly associated with these M0-AML [RUNX1 mutations are an independent adverse prognostic factor in AML [RUNX1 mutation have been withdrawn from the 5th edition drops, considering its molecular heterogeneity [RUNX1 mutations are associated with trisomy 8, but also with trisomy 13 and monosomy 7/loss 7q, similarly to pDC-AML [RUNX1, especially in splicing and epigenetic factors , with sub-clonal mutations in signaling pathways  [RUNX1 mutations are almost mutually exclusive from NPM1 and CEBPA mutations, concordant with their definition of distinct entities [More importantly, two independent studies identified somatic in BPDCN ,108,109.fication . RUNX1 min (TAD) . Found iication) . Interesr in AML ,111,112.3, NRAS) ,111. In entities ,111,112.RUNX1 mutations are detected in all pDC-AML with minimal differentiation (pDC-M0-AML) but not in AML with a monocytic contingent [RUNX1 mutation would constitute a well-defined and more homogeneous entity. However, even if the two studies contain 70% of cases with RUNX1 mutations, the American cohort contains mostly secondary monocytic pDC-AML, and a minority of undifferentiated AML, whereas the French cohort contains mainly M0-AML. Thus, two subsets of pDC-AML can be suggested, pDC-M0-AML and secondary monocytic pDC-AML [RUNX1 and FLT3-ITD mutations in blast cells and in pDCs [Of note, ntingent . In this pDC-AML ,103. Rec in pDCs .RUNX1 mutations, classical early stage mutations in splicing genes  and epigenetic modifiers  are frequent [In addition to frequent ,103. Morfrequent ,103.MYB may be a key gene in BPDCN, RUNX1 appears to be involved in pDC-AML. Xiao et al. showed that leukemic blasts upregulated IFN-driven pDC transcriptional programs, particularly IRF7, MX1, and IFI35, directing toward pDC differentiation and expansion in the case of pDC-AML, but also in AML with the RUNX1 mutation, even in the absence of pDC [RUNX1::RUNX1T1 transcript and upregulated in AML with normal karyotype [Mechanistically, RUNX1 forms with CBFβ the core binding factor (CBF) , a key he of pDC . Moreovee of pDC . Finallye of pDC ,117. In e of pDC ,118. Exparyotype . Of notearyotype ,120.RUNX1 is the most frequently mutated gene in pDC-AML (70% of cases) [RUNX1 mutations are extremely rare in BPDCN [MYC, MYB, ETV6, RB1 in BPDCN, RUNX1 in pDC-AML) but above all from a prognostic or therapeutic perspective. In the era of targeted therapies, recent studies provide a rationale for the use of some of them. In BPDCN, various targeted therapies show promising results: hypomethylating agents have been suggested for epigenetic defects, Bromodomain and Extra-Terminal motif (BET) protein inhibitors for TCF4 expression and MYC rearrangements, bortezomib for activation of the NF-kB pathway, and venetoclax for the hyperexpression of BCL2 [pDC-AML constitutes a challenging differential diagnosis for BPDCN, and genomic feature can help in diagnosis . Indeed,f cases) ,103, whiin BPDCN . There ain BPDCN ,124. Thein BPDCN ,126,127,in BPDCN ,130,131.in BPDCN ,51. Cons of BCL2 ,132,133. of BCL2 .


This mechanism strongly amplified the pericyte [Ca2+]i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slowed the ischemia-evoked pericyte [Ca2+]i rise, capillary constriction, and pericyte death; reduced neutrophil stalling; and improved cerebrovascular reperfusion. Genetic analysis implicated altered TMEM16A expression in poor patient recovery from ischemic stroke. Thus, pericyte TMEM16A is a crucial regulator of cerebral capillary function and a potential therapeutic target for stroke and possibly other disorders of impaired microvascular flow, such as Alzheimer’s disease and vascular dementia.Pericyte-mediated capillary constriction decreases cerebral blood flow in stroke after an occluded artery is unblocked. The determinants of pericyte tone are poorly understood. We show that a small rise in cytoplasmic Ca  Cerebral blood flow (CBF) is regulated both at the arteriole and at the capillary level , 2. Capi2+ concentration ([Ca2+]i) rising in pericytes after a fall of ATP level ([ATP]i) inhibits ion pumping; however, release of vasoconstrictors, such as endothelin-1 (ET-1) and thromboxane, in ischemic stroke may also contribute  channels or by Gq protein–coupled receptors (GqPCRs) triggering Ca2+ release from intracellular stores. However, Ca2+-activated Cl– channels  and pericytes have a high intracellular Cl– concentration . This depolarization is expected to activate Cav channels and amplify the increase in [Ca2+]i and contraction that occur , 90% of the TMEM16A expression was in pericytes . With an intracellular solution containing 0.25 μM free [Ca2+]i, the steady-state current-voltage relationship was outwardly rectifying (n = 14) near the value of ECl (–25 mV). When [Ca2+]i was raised to 1.3 μM, the membrane current on either side of ECl was increased, and its rectification was reduced, as observed for cloned TMEM16A channels -N′-(2-methoxybenzylidene)acetohydrazide] reduced the current  to a level only slightly larger than when using Ca2+-free intracellular solution, for which TMEM16A channels should be closed , implying that TMEM16A has considerable scope to alter the membrane potential of the cell.To assess the importance of TMEM16A, pericytes were whole-cell clamped in rat cortical slices, with a Cs-based internal solution that enables recording of Clctifying , with a channels , 43. Wite closed . Near th2 analog), which act on GqPCRs  and approximately 20%  by ET-1 (10 nM) and U46619 (200 nM), respectively . ET-1 increased [Ca2+]i in pericytes on (at least) the first-, second-, and third-order branch capillaries from penetrating arterioles (PAs), where first order refers to the first branch off the PA, and second order refers to a branch off the first order, etc.  evoke depolarization and, thus, activate Cav channels, causing an influx of extracellular Ca2+ ([Ca2+]o) and myofilament contraction. To distinguish between these possibilities, we applied ET-1 in the absence of [Ca2+]o. At 0 [Ca2+]o, ET-1 neither significantly raised [Ca2+]i  and processes , presumably via [Ca2+]o entry (see below). This [Ca2+]i rise evoked a significant capillary constriction at the soma of the pericytes   or 20 μm  along the capillary from the pericyte soma  and also after 15 minutes in 0 [Ca2+]o and ET-1 , consistent with measurements in vivo  , where myte soma , where tyte soma . Capillayte soma  in the a2+]i rise and contraction in 0 [Ca2+]o was the result of preventing Ca2+ entry via Cav channels, or alternatively a result of depleting internal stores of Ca2+, we examined the effect of the L-type Cav blocker nimodipine on the ET-1 response in normal [Ca2+]o solution. Nimodipine (3 μM) inhibited [Ca2+]i rise by 86% . The [Ca2+]i rise seen in nimodipine was similar in first-, second-, and third-order pericytes . However, MONNA and Ani9 greatly reduced the capillary constriction evoked by ET-1 (10 nM) amino]-5-nitro-benzoic acid (MONNA)  and Ani9 (10 nM)  and U466 (10 nM) . Furtherh orders . The 94%2+]i rise and pericyte contraction is unlikely to result from Ani9 indirectly affecting pericyte tone by acting on nearby endothelial cells, astrocytes, or neurons, because TMEM16A is nearly exclusively expressed in cortical pericytes and SMCs . With 0 free [Ca2+]i, the resting potential and input resistance were –42.0 ± 4.5 mV and 1.1 ± 0.5 GΩ, respectively, in 8 cells. With 0.25 μM [Ca2+]i, an inward current of –12.8 ± 2.3 pA/pF (n = 10)  was present at –40 mV, increased from a value of +0.3 ± 1.2 pA/pF (n = 8) in 0 [Ca2+]i (mean cell capacitance: 10.3 ± 1.0 pF [n = 8]). In the presence of 2 μM Ani9, this current was reduced to –5.3 ± 0.7 pA/pF (n = 5) , implying a TMEM16A-mediated Cl– conductance of approximately 5 nS. This Ca2+-activated current depolarized the resting potential to –18.3 ± 4.1 mV (n = 10)  .qPCR activation, altering the transmembrane Cl– gradient should alter the amplification of constriction produced by TMEM16A. This gradient was altered in cortical slices using 3 different strategies: (a) exposing slices to bumetanide (40 μM) to inhibit the Cl‑ importer NKCC1 and, thus, reduce the intracellular Cl– concentration ([Cl–]i) and the depolarizing influence of TMEM16A; (b) reducing the extracellular Cl– concentration ([Cl–]o) to increase TMEM16A-mediated depolarization; or (c) a combination of these treatments. In slices not exposed to a GqPCR agonist, alterations in the Cl– gradient did not affect capillary diameter (–]i gradient), the resulting capillary constriction was attenuated approximately 4-fold  crossed with NG2-CreERT2 mice (to KO TMEM16A in NG2-expressing pericytes), we assessed the effect of ET-1 on the pericyte-mediated capillary constriction. This showed that KO of TMEM16A reduced the ET-1–evoked constriction of capillaries by pericytes  to 92.5 mM, which is expected to activate Cav channels without the need for TMEM16A activation, elicited a capillary constriction , in the absence or presence of Ani9 (2 μM). Ani9 strongly delayed pericyte-mediated capillary constriction  that associated with (a) TMEM16A gene expression  activity . The genTMEM16A expression, 1.01; 95% CI, 0.99–1.03; P = 0.25), increased genetically proxied TMEM16A expression associated with worse functional outcome  at 60–190 days following ischemic stroke . The association of genetically proxied TMEM16A activity with outcome after ischemic stroke, but not with risk of ischemic stroke, makes effects on systemic blood pressure an unlikely mediator, as this affects ischemic stroke risk more than recovery  of NG2-dsRed mice or NG2-CreERT2-GCaMP5G mice   was performed in mice to mimic the ischemia/reperfusion injury that occurs during severe ischemic stroke . Using sely) see .2+]i rise after 70–90 minutes of reperfusion (Ani9 (10 μM) reduced the CCAO-evoked pericyte i much more than occurs as a result of the store-released Ca2+ alone ; (b) in vivo procedures ; (c) genetic analyses; and (d) statistics are reported in the Detailed descriptions of (a) ex vivo procedures  provided the NG2-Cre mouse, and Walter Marcotti  provided the floxed TMEM16A mouse, which was previously generated in-house.Human cortical tissue was obtained from patients undergoing brain resection for glioblastoma or thymus cancer, as detailed in the The experimenters were blinded to conditions during the execution of the experiments and/or during analysis. The methods of analysis were established during study design, and prior to execution of the experiments, to remove possible operator bias.P values of less than 0.05 were considered significant, and all tests were 2 tailed.Statistical tests used are detailed in the The use of human cortical tissue samples was undertaken with ethical approval from the National Health Service  (REC no. 15/NW/0568 and IRAS project ID 180727), and written consent was obtained from all participants donating tissue, as detailed in the NK, ZI, DA, and PT designed the research. NK, ZI, CLP, TP, PS, DG, DA, and PT performed experiments and/or analyzed data. JRR generated the floxed TMEM16A mice. HS collected and provided human cortical tissue samples. DA and PT obtained funding and supervised the research. NK, ZI, DG, DA, and PT wrote the paper. NK and ZI are co-first authors; authorship order reflects the fact that NK had a uniquely important role in driving key developments in the work.


We review the recent findings on how risk factors cause Ca2+ dyshomeostasis and how aberrant Ca2+ handling triggers dopaminergic neurodegeneration in the SNc in PD, thus putting forward the possibility that manipulation of specific Ca2+ handling proteins and subcellular Ca2+ homeostasis may lead to new promising strategies for PD treatment.Parkinson’s disease (PD), a common neurodegenerative disease characterized by motor dysfunction, results from the death of dopaminergic neurons in the substantia nigra pars compacta (SNc). Although the precise causes of PD are still unknown, several risk factors for PD have been determined, including aging, genetic mutations, environmental factors, and gender. Currently, the molecular mechanisms underlying risk factor-related neurodegeneration in PD remain elusive. Endoplasmic reticulum stress, excessive reactive oxygen species production, and impaired autophagy have been implicated in neuronal death in the SNc in PD. Considering that these pathological processes are tightly associated with intracellular Ca Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder in humans . It is cGBA, LRRK2, PRKN, PINK1, and SNCA , mitochondria, and lysosomes has been proposed to be the primary cause of neuronal death in PD patients . Dysfuncis of PD . Mitochois of PD . Maintenis of PD . Furtheris of PD . Howeveris of PD . Ca2+, aganelles . Recentlogenesis . Therefo2+ dyshomeostasis is induced by PD risk factors, which eventually leads to neurodegeneration and death of SNc DA neurons in PD.We review recent findings and discuss how intracellular Ca2+ is fundamental for neuronal development and survival by controlling diverse cellular processes including gene expression, synaptogenesis, synaptic transmission, energy production, membrane excitability, and neuronal plasticity . Neuronal VGCCs include L-type (Cav1.2-1.4), P/Q type (Cav2.1), N type (Cav2.2), R type (Cav2.3), and T type (Cav3.1-3.3) VGCCs. Cav1.2 and Cav1.3 are the main VGCC types in neurons. (2) Receptor-operated Ca2+ channels (ROCs). The main ROCs in neurons are glutamate and purinergic receptors. They are further divided into ionotropic and metabotropic receptors. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-sensitive receptors (AMPARs) and N-methyl-d-aspartate sensitive receptors (NMDARs) are the principal types of ionotropic glutamate receptors. Ionotropic purinergic receptors (P2XRs) mediate Ca2+ influx and membrane depolarization, while metabotropic purinergic receptors (P2YRs) activate the Gq/11 signaling pathway upon activation by ATP binding. (3) Store-operated Ca2+ entry channels (SOCs) activated by depletion of ER Ca2+ stores. SOCs communicate with the ER through interactions between STIM  and the ORAI subunit of SOC  volt of SOC .2+ influx, Ca2+ release from subcellular organelles contributes to Ca2+ signaling. ER and mitochondria are major intracellular Ca2+ stores in neurons, while lysosomes are also involved in the control of cellular Ca2+ homeostasis (2+ release from the ER is mediated by the ryanodine receptor (RyR), which is activated by Ca2+ influx, and inositol 1,4,5-trisphosphate receptor (IP3R), which is activated by IP3 from Gq/11 signaling pathways. Both RyR and IP3R are sensitive to cytosolic Ca2+ and oxidation states (+-Ca2+ exchanger (mNCX) and a H+–Ca2+ exchanger , which belongs to the mucolipin subgroup of the TRP ion channel family  and the Na+-Ca2+ exchanger (NCX) by using the energy stored in ATP and the chemical energy of the Na+ gradient, respectively. Uptake of Ca2+ into the ER is mediated by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in an ATP-dependent manner. The mechanism of Ca2+ uptake by the lysosomes is not completely understood. +/Ca2+-exchanger involved in lysosomal Ca2+ refilling in non-placental mammals. SERCA, PMCA, and ion exchangers are sensitive to the cellular oxidative status (2+ from the cytoplasm through voltage-dependent anion channels (VDAC) and mitochondrial Ca2+ uniporter complex (MCU), which is crucially involved in the sequestration of Ca2+ signaling . CaBPs are classified into Ca2+ sensors and Ca2+ buffer proteins  low Ca2+ buffering capacity. SNc DA neurons lack expression of the Ca2+ buffer protein calbindin  SNc DA neurons are constitutively active in the absence of external input, generating slow, broad spikes that lead to a significant increase in resting cytosolic Ca2+ concentration. Although the mechanism underlying the pacemaking activity of SNc DA neurons is not completely clear, Cav1.3 channels have been shown to support pacemaking , SNc DA neurons are extremely susceptible to the increase in cytosolic Ca2+ induced by PD risk factors, such as aging, and genetic and environmental factors. Sustained Ca2+ elevation leads to neuronal damage through both direct and indirect pathways.Cadiseases . Conside2+ can directly activate a variety of Ca2+-dependent enzymes, including phospholipases, proteases, endonucleases, and nitric oxide synthase (NOS). These activated enzymes contribute to neurodegeneration by damaging various cell structures, such as the membrane, cytoskeleton, and DNA, as well as through NO-induced oxidative stress  and glucose-related protein 75 (GRP75). DJ-1 interacts with VDAC, IP3R, and GRP75. GRP75 links IP3R to VDAC. Together, IP3R, VDAC, GRP75, and DJ-1 form a functional tetramer complex (IP3R/GRP75/DJ-1/VDAC complex) at MERCS  and likely ROS transfer from the mitochondria to the ER. MERCSs are regulated by multiple proteins in the ER and mitochondria membrane, which contribute to dynamic physical tethering between the organelles, and regulate the integrity, stability, size, and length of the MERCS . In MERCS, the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane (OMM) is bridged with the ER membrane IPat MERCS , which mhe MERCS . Ca2+ dyin MERCS , which pnd VDACs , and Ca2diseases . Therefoth in PD .3Rs are highly clustered in the ER membrane  mechanism  cycle and oxidative phosphorylation (+ pump chain (respiratory complex I–IV). Respiratory complexes pump H+ out of the mitochondrial matrix and thus establish an H+ gradient and a mitochondrial membrane potential across the mitochondrial inner membrane. Mitochondrial membrane potential promotes ATP synthesis by complex V  and leads to the release of cytochrome c, triggering apoptosis  contribute to cellular Ca2+ signaling generation by transporting Ca2+ from the ER lumen to the cytoplasm; ER resident Ca2+ binding proteins determine the ER Ca2+ buffering capacity; Ca2+ pumps (SERCA) contribute to sequestration of elevated cytosolic Ca2+ by pumping Ca2+ into the ER lumen. Both Ca2+ release channels and pumps are sensitive to cytosolic and ER luminal Ca2+ and ROS. Luminal Ca2+-binding proteins, such as calreticulin, calnexin, protein disulfide isomerases (PDI), and glucose-regulated protein 94 kDa (GRP94), regulate ER function not only by directly buffering luminal Ca2+, but also by regulating Ca2+ release channels, Ca2+ pumps, or ER proteins involved in protein synthesis and folding  by activation of Ca2+/calmodulin (CaM)-dependent phosphatase calcineurin. TFEB is a lysosomal master gene regulator that enters the nucleus and promotes expression of several genes associated with the regulation of the autophagic pathway , damaged organelles and microorganisms . Lysosom pathway . TRPML1   may contribute to aging  that catalyzes the conversion of the sphingolipid glucosylceramide to ceramide and glucose. GBA mutations, such as E326K, are now considered as the single largest risk factor for idiopathic PD. GBA mutations cause decreased expression and activity of GCase, resulting in accumulation of glucosylceramide, which enhances α-synuclein aggregation by stabilizing soluble α-synuclein oligomers  α-synuclein oligomers form Ca2+-permeable pores in the cell membrane that increase Ca2+ influx; (2) aggregated or misfolded α-synuclein interacts with and impairs mitochondrial complex I, causing mitochondrial dysfunction and oxidative stress in cells , a protein containing a GTPase and a kinase domain. LRRK2 mutations are typically associated with late-onset familial PD  gene encodes for the E3 ubiquitin ligase parkin, and mutations in parkin constitute the most common cause of early onset PD  has been associated with increased risk of PD, it is difficult to determine whether an environmental factor is the definitive cause of PD. In other words, each environmental factor may contribute to increased risk, but may not be sufficient to cause PD. It is widely accepted that PD results from complex interactions between two or more environmental factors, possibly independent of gene mutations . CuriousPesticides are used in the agricultural industry to control pests and destroy weeds. Exposure to pesticides has been linked to increased risk of PD. Organochlorines and organophosphates are well-documented pesticides. One of the organochloride pesticides, dieldrin, preferentially targets SNc DA neurons and causes neurotoxic damage through the induction of oxidative stress and mitochondrial dysfunction . ApplicaThere is a link between exposure to various metals and increased risk of PD. Occupational exposure to high-dose manganese during welding is associated with a form of parkinsonism, known as manganism, with symptoms such as tremors, gait abnormalities, and facial muscle spasms. Promotion of aggregation and cell-to-cell transmission of α-synuclein by manganese has recently been suggested to cause neurotoxicity in SNc DA neurons . AccumulExposure to organic solvents may also play an etiologic role in PD development. Organic solvents are commonly used in metal degreasing, dry cleaning, and as detergents. Trichloroethylene (TCE) is an industrial solvent, and exposure to TCE has been implicated as an environmental risk factor for PD. Although the exact mechanism underlying the effect of TCE on PD risk is not completely clear, TCE-associated mitochondrial dysfunction and deficits in complex I contribute to PD pathogenesis .2+ signals. Whether the increased DA release is due to a direct action of estrogen or an indirect effect through an estrogen-induced increase in cytosolic Ca2+ requires further investigation. Recently, estrogenic control of mitochondrial function and biogenesis has drawn more attention. It has been extensively reported that estrogen enhances electron transport chain activity, maintains the mitochondrial membrane potential, increases ATP production, inhibits excessive ROS production, and prevents mitochondrial Ca2+ overload (2+ and cellular homeostasis. Furthermore, estrogen has been reported to support stable, helically folded α-synuclein tetramer that resists aggregation (Epidemiological studies have shown that PD is 1.5–2 times more frequent in men than in women . The preoverload , which hregation , consistregation . TherefoThere are also other risk factors for PD, such as some medications which block the action of dopamine, and head trauma. In this review, the possible effect of head trauma on PD development is briefly discussed.Growing evidence shows a clear link between head trauma and an increased risk of PD. Head injuries are common and can be caused by unexpected incidents, such as traffic accidents and falls, or some specific sport activities. The risk of PD due to head injuries is higher with more severe or recurrent injuries and loss of consciousness . Recent 2+ is the most important signaling molecule that participates in all cellular processes. Ca2+ transfer between subcellular organelles mediates coordinated and concerted interactions involved in cellular homeostasis. Under physiological conditions, cellular Ca2+ homeostasis is tightly controlled by Ca2+-handling proteins. However, in pathological conditions, prolonged impairment of cellular functions may directly or indirectly cause dysregulation of Ca2+ handling, resulting in cellular Ca2+ dyshomeostasis. SNc DA neurons are highly susceptible to an additional increase in cellular Ca2+, based on their features in Ca2+ handling. Sustained Ca2+ overload in SNc DA neurons may convert Ca2+ from a physiological regulatory factor to a pathological trigger. Indeed, growing evidence suggests that long-term excessive cytosolic Ca2+ contributes to ER, mitochondria, and lysosomal dysfunction in PD pathogenesis. Furthermore, recent studies support the hypothesis that Ca2+ dyshomeostasis is central in perpetuating the vicious cycle between the ER, mitochondria, and lysosomes, because increasing Ca2+ buffering capacity by overexpression of Ca2+-binding proteins or manipulation of subcellular Ca2+ handling can reduce oxidative stress, ameliorate autophagy, and decrease protein aggregation (2+ overload and Ca2+ dyshomeostasis in subcellular organelles by application or expression of cell-specific and organelle-specific Ca2+-handling molecules may represent a promising strategy for PD treatment.Parkinson’s disease is a chronic and progressive neurodegenerative disorder initiated by a number of risk factors, suggesting that risk factor-induced cellular dyshomeostasis accumulates over time. When cellular repair and compensatory systems cannot overcome sustained pathological injuries within cells, cell damage occurs. The ER, mitochondria, and lysosomes are crucial for the maintenance of cellular homeostasis, they interact with each other, and change their functions in response to signal stimulation. Caregation . TherefoJX and MK wrote the manuscript. EM participated in discussion and collection of the manuscript. All authors approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ROS-dependent dissociation of FKBP12.6 mediates pulmonary artery vasoconstriction and pulmonary hypertension. 2+ concentration ([Ca2+]i), which is generated and regulated by various ion channels. Several studies by us and others have shown that ryanodine receptor 2 (RyR2), a Ca2+-releasing channel in the sarcoplasmic reticulum (SR), is an essential ion channel for the control of [Ca2+]i in pulmonary artery SMCs (PASMCs), thereby mediating the sustained vasoconstriction seen in PH. FK506-binding protein 12.6 (FKBP12.6) strongly associates with RyR2 to stabilize its functional activity. FKBP12.6 can be dissociated from RyR2 by a hypoxic stimulus to increase channel function and Ca2+ release, leading to pulmonary vasoconstriction and PH. More specifically, dissociation of the RyR2–FKBP12.6 complex is a consequence of increased mitochondrial ROS generation mediated by the Rieske iron-sulfur protein (RISP) at the mitochondrial complex III after hypoxia. Overall, RyR2/FKBP12.6 dissociation and the corresponding signaling pathway may be an important factor in the development of PH. Novel drugs and biologics targeting RyR2, FKBP12.6, and related molecules may become unique effective therapeutics for PH.Pulmonary hypertension (PH) is a devastating disease characterized by a progressive increase in pulmonary arterial pressure leading to right ventricular failure and death. A major cellular response in this disease is the contraction of smooth muscle cells (SMCs) of the pulmonary vasculature. Cell contraction is determined by the increase in intracellular Ca Pulmonary hypertension (PH) is a group of life-threatening lung diseases characterized by an increase in pulmonary arterial pressure ≥25 mmHg at rest or ≥30 mmHg during or after exercise . AccordiRegardless of differences in underlying pathogenic mechanisms, the pathophysiological features of the most common forms of PH include damage, i.e., plexiform lesions and vessel obliteration, and hyperproliferation of endothelial cells and smooth muscle cells (SMCs), vascular remodeling, and inflammation . Inflamm2+ concentration ([Ca2+]i). In SMCs, [Ca2+]i is precisely regulated by several ion channels in the cell membrane, including voltage-dependent Ca2+ channels (VDCCs), transient receptor potential canonical channels (TRPCs), and store-operated Ca2+ channels (SOCCs). In addition, Ca2+-permeable channels in the sarcoplasmic reticulum (SR), including ryanodine receptors (RyRs), and inositol triphosphate receptors (IP3Rs) contribute to the regulation of [Ca2+]i , which in turn stimulates myosin light-chain kinase. This enzyme phosphorylates the myosin light chain, favoring actin to move across myosin and triggering contraction of SMCs -dependent pathways that lead to vascular smooth muscle (VSM) proliferation and remodeling . This cellular response is triggered by an increase in intracellular Ca [Ca2+]i . Further cytosol . The inc of SMCs . In addigh TRPCs , activatmodeling . Increasmodeling . The sigferation . The Ca2S in VSM ; Fig. 1.2, PGI2) PGI2 analogs, non-prostanoid agonists of the PGI2 receptor, and Ca2+ channel blockers. These drugs target three major signaling pathways involved in abnormal pulmonary artery proliferation and contraction  inhibitors, inhaled nitric oxide, guanylate cyclase stimulators, prostacyclin  through the proliferation of SMCs and endothelial cells . Moreove, and PH . Hypoxia, and PH . RISP-me, and PH . In this2+ into the cytosol and trigger cellular functions such as contraction and proliferation (2+ to the cytosolic side of the channel. The opening of RyRs is sensitive to [Ca2+]i from 1 to 10 μM while millimolar [Ca2+]i promotes their closed state (3 and nicotinic acid adenine dinucleotide phosphate (NAADP), are important messengers involved in intracellular Ca2+ release in VSMCs , all of which are found in VSM. These channels are responsible for the release of Caferation . RyRs ared state . RyRs ared state . cADPR, in VSMCs . Synthesding VSM . CD38 cading VSM  and endoding VSM . The mecchannels . In addichannels . Severalchannels . For insns >5 mM .2+ increases (2+ influx through plasma membrane Ca2+ channels or by Ca2+ release from neighboring IP3Rs or other RyRs. This mechanism is known as Ca2+-induced Ca2+ release (CICR), which plays an essential role controlling vascular tone and excitation–contraction coupling (2+ sparks are associated with vasodilation. Ca2+ sparks trigger K+ efflux through Ca2+-activated K+ channels with high conductance (BK). K+ efflux leads to spontaneous transient outward currents (STOCs) that result in cell membrane hyperpolarization and disrupt Ca2+ entry through L-type VDCC  were used  has been known for years . HPV aids of HPV . Phase In of VSM . Acute hn of VSM . It is an of VSM . All of 2 sensing and contraction in PASMCs. Nonetheless, controversy exists about the source of ROS, their target mechanisms, and whether they increase or decrease in response to hypoxia  is increased, triggering RyRs-mediated Ca2+ release and the inhibition of KV channels . ROCK phosphorylates the myosin-binding subunit of MLC phosphatase (MLCP), MYPT-1, and the MLCP inhibitor protein CP-17. Both phosphorylations inhibit the activity of MLCP and enhance contraction of SMCs  for 5 d prevented hypoxia-evoked increased vascular resistance , is associated with regulation of resting membrane potential and vascular tone because of its voltage independence  of thiol groups present in the channel  in PASMCs, each appears to have distinct functional roles with the RyR2 subtype being the major player in hypoxic responses . We havent of PH .2+]i in freshly isolated PASMCs, which was blocked in cells from RyR2 knockout mice. These in vitro assays demonstrate that acute hypoxia increases Ca2+ release through RyR2 in the pulmonary artery . Moreover, 5 min of hypoxia exposure caused a strong increase in i in PASMCs. Moreover, increased mitochondrial [ROS] ([ROS]m) was observed in isolated mitochondria after PASMCs were exposed to caffeine or norepinephrine. Using mit-2mutAEQ, the mitochondria-targeted double mutant aequorin Ca2+ sensor, Yang and colleagues also found that norepinephrine, caffeine, and hypoxia can increase [Ca2+]m and this increase was blocked by the mitochondrial Ca2+ uniporter (MCU) inhibitor Ru360 (10 μM) in the pulmonary artery  enhanced the formation of ROS in mitochondria and the complex III, which were isolated from PASMCs, showing that CIRG occurs in these cells , which facilitates the dissociation of FKBP12.6 from the channel and leakage of Ca2+  and atrial fibrillation AF; . Multipl of Ca2+ . Therefo of Ca2+ .2+-induced myocardial damage  cardiomyopathy (K201, also called JTV519, was developed to provide a stronger protective effect against Cal damage . This drl damage  and AF (l damage . Althougchannels  and blocreceptor , this drreceptor . In thishythmias . In addiocardium . S107, thythmias . As wellmyopathy . Finallymyopathy .2+ sparks. Moreover, intraperitoneal administration of this drug attenuated the progression of right ventricular failure and prolonged the survival of 23% of rats with PH induced by MCT (2+ sparks in hypertrophied right ventricular cardiomyocytes (2+ leak. S107 also abolished PA remodeling by hampering chronic hypoxia-induced muscularization and SMC proliferation and eliminated right ventricular hypertrophy (Regarding PH and pulmonary vessels as a target, no treatments based on the interaction of RyR2/FKBP12.6 or other proteins associated with this channel have been developed or tested in humans. However, because right ventricular failure is a major cause of death in patients with PH, stabilization of RyR2 in the heart is postulated as a treatment option to increase survival. A study conducted by Huang and colleagues showed that administration of the RyR2 stabilizer dantrolene to decompensated right ventricular cardiomyocytes reduced the frequency of Cad by MCT . Furthermyocytes . In the myocytes . We demoertrophy . In this2+]i. Several channels are involved in Ca2+ processing in SMCs. This review article highlights the importance of RyR2 in the control of Ca2+ homeostasis and ROS generation mediated by hypoxia, as well as its role in the development of PH. We and other investigators have well documented that hypoxia is highly specifically involved in the generation of ROS and increases in [Ca2+]i in PASMCs, serving as of the main cause of PA vasoconstriction, remodeling and PH. Accordingly, a complex signaling pathway involving the hypoxia-induced mitochondrial ROS generation and then RyR2-dependent Ca2+ release, termed RICR process, is well described in this article.Exacerbated PASMC contraction and remodeling are common markers of PH. These processes appear to be mediated to a large extent by an enhanced increase in [Ca2+ from the SR. The released Ca2+ causes PA vasoconstriction, PA remodeling, and eventually PH. Moreover, RyR2-mediated Ca2+ release also causes RISP-dependent mitochondrial ROS production, which further enhances hypoxia-induced ROS generation and cellular responses. The signaling that links the SR to mitochondria through a CIRG process represents an important pathway in hypoxia-induced PH. It is conceivable that pharmacological and genetic stabilization/inhibition of the RyR2/FKB12.6 complex and RyR2 per se in SMCs could be the novel and effective therapeutic options in the treatment of PH.Nevertheless, acute and chronic hypoxia both can increase RISP-dependent mitochondrial ROS generation in PASMCs. The increased mitochondrial ROS can enter the cytosol and activate PKCε. The activated PKCε stimulates NOX to trigger further ROS generation. This ROS-induced ROS, together with the hypoxia-induced direct mitochondrial ROS production, synergistically dissociate FKBP12.6 from RyR2, which increases channel activity, and then induces the release of Ca