Lysosomes, since their discovery, have been primarily known for degrading cellular macromolecules. However, in recent studies, they have begun to emerge as crucial regulators of cell homeostasis. They are at the crossroads of catabolic and anabolic pathways and are intricately involved in cellular trafficking, nutrient signaling, energy metabolism, and immune regulation. Their involvement in such essential cellular functions has renewed clinical interest in targeting the lysosome as a novel way to treat disease, particularly cancer. Acute myeloid leukemia (AML) is an aggressive blood cancer with a low survival probability, particularly in older patients. The genomic landscape of AML has been extensively characterized but few targeted therapies (with the exception of differentiation therapy) can achieve a long-term cure. Therefore, there is an unmet need for less intensive and more tolerable therapeutic interventions. In this review, we will give an overview on the myriad of functions performed by lysosomes and their importance in malignant disease. Furthermore, we will discuss their relevance in hematopoietic cells and different ways to potentially target them in AML. Lysosomes were first discovered by Christian de Duve in 1955 while working on a specific enzyme, glucose-6-phospatase, a target of insulin in hepatic tissue. Glucose-6-phosphatase was found to be encased in what was described as “sac-like particles” [In the 1990s, the discovery of autophagy , generatIn this review, we will focus on the key signaling functions of lysosomes, their potential role in the pathophysiology of malignant disease and consider why lysosomes might represent a good therapeutic target for acute myeloid leukemia (AML).+ ATPase (V-ATPase) on the lysosomal membrane [Lysosomes are acidic organelles with an internal pH of 4.5 to 5.5, maintained by the presence of the vacuolar-type Hmembrane . This pHmembrane .+ ) transporter [The outer lysosomal membrane is rich in transmembrane proteins, predominantly lysosome-associated membrane proteins (LAMPs). Five types of LAMPs have been identified so far: LAMP-1/CD107a, LAMP-2/CD107b, LAMP-3/DC-LAMP, LAMP-4/Macrosialin/CD68 and LAMP-5/BAD-LAMP. LAMP-1 and LAMP-2 account for almost 80% of the lysosomal membrane proteins. Other key lysosomal membrane proteins (LMPs) include, ion channels and various receptors for specific cargo, tethering/fusion or signaling, such as the chloride channel protein 7 (CLC7), Niemann-Pick C1 protein (NPC1), synaptotagmin (SYT7), and the Vacuolar (V)-ATPase proton —enabling a rapid response to metabolic demand. This gene network is regulated by transcription factors belonging to the microphthalmia/transcription factor E (MiT/TFE) family, including TFEB, TFE3, and MITF. They bind to a 10-bp palindromic motif sequence in the promoter sequence of lysosomal genes referred to as the CLEAR element. TFEB has also been shown to co-regulate the expression of several autophagy genes .2 + release from the lumen of the lysosome [Lysosomes are the terminal compartment for the delivery of cargo from clathrin- and caveolin-dependent and -independent endocytosis, autophagosomes, phagosomes, and chaperone-mediated autophagy (CMA) Fig. . Interaclysosome . The prilysosome . Howeverlysosome .Fig. 1Ma2 + /Fe2 + /Zn2 + , H+ /Na+/K+, Cl-, and ATP. Intra-luminal levels of amino acids such as arginine and leucine are also regulated by lysosomal receptors and linked to external signaling [The luminal compartment of lysosomes is the hydrolytic engine of the organelle, containing more than 60 acidic hydrolytic enzymes including proteases, nucleases, lipases, glycosidase phosphatases, and sulfatases. Deficiencies in any of these enzymes can have a major impact on cellular homeostasis. The lysosome is also a reservoir for important ions and metabolites including Caignaling .Cargo digestion is a key function of lysosomal enzymes. Several classes of enzymes will however also influence events outside the lysosome. Cathepsins are lysosomal proteases classed into, serine (A and G), aspartic (D and E), and cysteine proteases . The release of these proteases into the extracellular space has been associated with degradation of the extracellular matrix, cell migration, and invasion of cancer cells . In addiLysol). Relative to the cytoplasm, the lysosome has high concentrations of Ca2 + , which is a key regulator of many lysosomal functions. There are at least three main types of Ca2 + channels in mammalian lysosomes: Transient receptor potential mucolipin sub-family (TRPML)/mucolipin 1-3, (Two-Pore) TPC1-2 and P2X4. These respond to a variety of signals including cell stress, phospholipids, nutrients, and ATP depletion. The best-characterized channel is probably TRPML1/mucolipin 1, which regulates several lysosomal processes including lysosomal reformation, exocytosis, motility, and fusion with other organelles. Calcium efflux from TRPML1 also activates the calcium-dependent phosphatase calcineurin, which is important for TFEB nuclear translocation [Lysosome membranes contain several ion channels that establish concentration gradients and maintain the lysosome membrane potential  GTPase and Ragulator complex localized on the lysosomal surface. This recruits the mechanistic target of rapamycin complex 1 (mTORC1), which is one of two protein kinase complexes incorporating the serine-threonine kinase mTOR. The mTORC1 complex consists of the mTOR kinase itself, Raptor, GβL, and DEPTOR. mTORC1 is then activated by Rheb (Ras homolog enriched in brain) GTPase, on the lysosomal membrane. Simultaneously, SLC38A9 also allows efflux of other amino acids such as leucine into the cytosol. This enables cross talk with other cytosolic amino acid sensors associated with mTORC1 complex (outlined below) , 18. HigAs with other organelles, most intra-organelle communication and signaling is orchestrated at the surface. The lysosomal surface is a platform for the assembly of several signaling hubs that link metabolic functions to environmental signaling. A central link in this signaling is the mTORC1 complex. The primary role of active mTORC1 is to drive anabolic/biosynthetic pathways to fuel growth and proliferation and to inhibit catabolic pathways such as autophagy. Several signaling cues converge at mTORC1 including (i) nutrients (ii) growth factors and (iii) energy status.Amino acids promote the translocation of mTORC1 to the lysosomal surface where it can be activated. This process is mediated by the coordinated actions of several complexes including Ragulator and Rag GTPases . The Ragulator complex consists of 5 subunits; p18, p14, MP1, C7orf59, and HBXIP . The p18 subunit (LAMTOR-1) is anchored to the lysosomal membrane. In response to amino acids, Ragulator acts as a guanine nucleotide exchange factor (GEF) for Rag A/B—promoting their GTP bound state and anchoring both the Rag GTPases and mTORC1 to the membrane where it can be activated by the small GTPase Rheb , 21.The presence of specific amino acid residues also influences other GEFs and GTPase-activating proteins (GAPs), upstream of GTP-RagA and GDP-RagC and mTORC1 signaling. A key integrator of amino acid signaling is the GTPase-activating proteins toward Rags (GATOR) complex . GATOR i3), which stimulates protein kinase B (Akt) phosphorylation and inactivation of TSC2. This inactivation leads to an increase in GTP-loaded Rheb, which fuels activation of mTORC1 [Many external signals, such as growth factors converge at a large protein complex referred to as tuberous sclerosis complex (TSC), which negatively regulates the MTORC1 kinase . TSC is f mTORC1 .In contrast to growth factor signaling, energy depletion activates AMP kinase at the lysosomal surface, which stimulates TSC2 through phosphorylation and inhibits RAPTOR. This leads to inhibition of mTORC1 and promotion of catabolic pathways .2 + -dependent phosphatase, calcineurin. During starvation, Ca2 + is released through the lysosomal calcium channel, TRPML1/mucolipin-1, activating calcineurin, which dephosphorylates TFEB. A zinc finger family DNA-binding protein, ZKSCAN3, has been shown to be a transcriptional repressor of autophagy by repressing the expression of more than 60 essential genes associated with lysosomal biogenesis and autophagy including Microtubule Associated Protein 1 Light Chain 3 Beta (Map1lc3b) and Wipi2. ZKSCAN3 and TFEB have also been found to be oppositely regulated by starvation [mTORC1 also plays a major role in the phosphorylation of MiT/TFE factors. Under nutrient-rich conditions, mTORC1 phosphorylates TFEB on Ser 122 and Ser 211 and sequesters it to the cytoplasm. Inhibition of mTORC1 under a stressful condition, such as starvation, disrupts this association allowing nuclear translocation of TFEB and transcription of CLEAR network genes . Anotherarvation  Autophagy is a process by which cells transport dysfunctional or excessive cellular components into the lysosomes for degradation. Autophagy can eradicate harmful organelles, pathogens or aggregated material and the recycling of macromolecules from the lysosome enables cells to cope with stresses such as nutrient deficiency . The thrThe class III phosphatidylinositol 3-kinase (PI3K-III/ VPS34) complex containing Beclin-1 , hVps34, p150 , and Atg14-like protein (Atg14L) and (ii) the ULK1 kinase complex (ULK1-Atg13-FIP200-ATG101)—this activates the class III PI3K/ Beclin-1 complex enabling autophagosome nucleation. These complexes are regulated by phosphorylation from the following pathways:Macroautophagy is mediated by several protein conjugation reactions, eventually leading to the formation and expansion of a double-membraned autophagosome which carries the cargo to the lysosome to be degraded . The genThe PI3K/AKT/mTOR signaling pathway: Under abundant supply of nutrients and growth factors, cells activate the PI3K/AKT/mTOR signaling pathway and suppress autophagy, by inhibitory phosphorylation. In contrast, when cells are experiencing stress or nutrient starvation, they respond by inactivating the PI3K/AKT/mTOR pathway, which de-represses and activates autophagy.The AMPK signaling pathway: AMPK (AMP-activated Protein Kinase) is a critical energy-sensing protein, which detects changes in the ratio of ATP to AMP or ADP. Phosphorylated AMPK phosphorylates TSC2 thereby inhibiting mTORC1 and activating autophagy. AMPK can also induce autophagy by directly phosphorylating and activating ULK-1/2. [ULK-1/2. CMA is a client-specific, selective autophagic pathway. CMA clients contain a signature motif containing a short stretch of 5 amino acid residues, KFERQ  . This moIn microautophagy, the dynamics of the lysosomal membrane allows it to wrap itself around cytosolic contents and engulf them into the lumen for degradation. Molecular details of microautophagy have not been elucidated as thoroughly as the other two pathways. Client specificity has been reported in endosomal microautophagy. Similar to CMA, the protein clients harbor a KFERQ-like motif and are delivered to the lysosomes by HSPA8 with the help of the endosomal sorting complex required for transport (ESCRT) machinery . The ESC2 + -regulated mechanism, which plays a role in various physiological processes such as, plasma membrane repair and immunogenic ATP release [The signaling properties of lysosomes are affected by their position within the cell. Lysosomes can move from the center of the cell to the periphery or vice versa with the help of kinesin motors KIF2A and KIF1Bβ as well as the small GTPase ARL8B . The con release .The elimination of defective lysosomes by autophagy is referred to as ‘lysophagy’. Several β-galactoside-binding proteins  have been implicated in mediating lysophagy. GAL 3, 8 and 9 are recruited by exposure of luminal glycans following lysosomal damage. Galectin 8 can inhibit mTORC1 and Galectin 9 activates Mitogen-activated protein kinase kinase kinase 7 (TAK1 or MAP3K7) that stimulates AMPK . MembersLysosomes are important regulators of lipid homeostasis and play a key role in cholesterol trafficking . Low-denLysosomes can participate at several stages in the generation of an immune response. This includes pathogen sensing, phagocytosis, antigen processing and presentation, and inflammation/secretion.Sentinel cells such as macrophages and dendritic cells (DCs) use toll-like receptors (TLR) to sense pathogens and elicit an immune response. Members of the TLR family, TLR 3 TLR7, TLR 8, and TLR9 have been shown to signal from endolysosomes. In addition, TLR9 responds to mitochondrial DNA, delivered to the lysosome via mitophagy , 52. ForCell transformation and cancer progression require an increase in biomass production and adaptation to nutrient stress, processes that are aided by lysosomes. Accordingly, cancer cells have higher lysosomal activity compared to healthy adjacent tissue . LysosomDirect impairment of lysosomal function can be achieved through several strategies. Most of the therapeutic agents being developed to target lysosomes either raise the luminal pH to inactivate cathepsins thus rendering them unable to mediate invasion, or aid in lysosomal membrane permeabilization and induce LCD. Lysosome targeting agents and the diseases they are being tested against has already been adequately reviewed . In TablHematopoiesis is a complex process that gives rise to cells with strikingly distinct morphology and functions. Such processes require drastic reorganization of cellular homeostasis. Lysosomes play a vital role here as they maintain a balance between anabolic and catabolic pathways. Due to their association with the master regulator of cell growth, mTORC1, and the ability to degrade macromolecules via autophagic pathways, they are in a central position to modulate metabolism, differentiation, proliferation, and cell death. All of these components are essential in the maintenance of functioning hematopoiesis and their dysregulation can be a feature of hematological malignancies.Auer rods (or Auer bodies) are large structures sometimes observed in AML, acute promyelocytic leukemia (APL), and myelodysplastic syndromes Fig. . Auer roIn positive AML cases, the percentage of cells with Auer rods and the number of Auer rods per cell can vary greatly. Auer rods are found in AML M1, M2, M3, M4, M5, and M6, but not in AML M0 or M7 of the French–American–British (FAB) classification, or in blast crisis chronic myeloid leukemia, suggesting an association with a more mature myeloid phenotype. While Auer rods clearly have diagnostic value in determining the neoplastic nature of myeloid cells, they do not seem to have a clear prognostic significance .HSC are responsible for repopulating blood cells and maintaining blood homeostasis. Therefore, in order to protect themselves from stress, they usually remain quiescent. Liang et al., reported that, quiescent HSCs are defined by their low mitochondrial membrane potential and glycolysis. Enlarged lysosomes are found in quiescent HSCs, which are speculated to remove toxic products and contribute to the maintenance of healthy HSCs . LysosomAtg7 in the hematopoietic system of mice results in severe clinical symptoms , leading to their death within 12 weeks. The Atg7−/− LSK (Lin− Sca-1+ Kit+) cells showed accumulation of mitochondria and DNA damage with complete loss of stem cell function [Atg5 knockout mice [Autophagic pathways, in particular, macroautophagy contributes to the maintenance of energy metabolism, which is crucial in HSCs. The transcription factor FOXO3A, drives a gene expression program that induces autophagy to protect HSCs during stress . The spefunction . Similarout mice . FIP200,out mice . A recenout mice .Atg5 in Mixed Lineage Leukemia-Eleven Nineteen Leukemia (MLL-ENL) induced murine HSCs led to a higher proliferation rate and a more aggressive leukemia in vivo due to a shift towards glycolytic metabolism [Atg7 targeting shRNA [Atg5 accelerated the initiation of leukemia but had no effect on therapeutic intervention [Atg5 or Atg7 in bone marrow cells reduced the frequency of leukemia-initiating cells (LICs) and decelerated leukemia progression [Autophagy dysregulation can lead to and drive AML. An in silico analysis has shown that a deleted chromosomal region often found in AML coincides with the location of autophagy genes and that lowered autophagic flux is advantageous to AML . Heteroztabolism . Depleting shRNA . In a MLrvention . Conversgression . In FLT3gression . In contgression . The afoSeveral studies indicate that autophagy plays a key role in myeloid differentiation. Macroautophagy is upregulated during colony-stimulating factor-1 mediated monocyte-macrophage differentiation and is necessary for cell survival as it can direct cells towards differentiation in lieu of apoptosis , 79. Mectrans retinoic acid (ATRA). Autophagy can facilitate degradation of the PML-RARA onco-protein in APL, but it is also important for differentiation of AML cells that do not have a fusion protein [Granulocytic differentiation can be induced in the AML subtype APL by treatment with all- protein , 82. ATR protein . More re protein .The lysosome is clearly a multi-functional organelle and there may be many ways to target it for therapeutic gain in leukemia. Moreover, lysosomes in AML cells are bigger in size which could make AML cells more susceptible towards lysosomal disruption than normal cells . SeveralIn 1994, Craig Rosenfeld demonstrated the antileukemic property of the lysosomotropic compound phenylalanine methyl ester in leukemic specimens derived from patients .pig7), which encodes for the small integral membrane protein of lysosome/late endosome (SIMPLE) was found to induce lysosomal membrane permeabilization in AML cell lines and render them more susceptible to chemotherapies such as VP16 and daunorubicin [A screen of a library of 100 drugs showed that the anti-malarial drug, mefloquine, is effective against AML cell lines as well as leukemia patient samples. A gene set enrichment analysis carried out in yeast identified genes associated with lysosomal functions to be enriched in gene deletion strains that were sensitive to mefloquine, indicating that mefloquine targets lysosomes. Mefloquine disrupts the lysosomal membrane of AML cells allowing the release of cathepsins B and L, which results in cell death . Anotherorubicin .The compound, deoxysappanone B 7,4′-dimethyl ether  was identified from a chemical screen of small molecules with anti-leukemic activity. It is a microtubule inhibitor, which indirectly increases lysosomal V-ATPase activity causing hyper-acidification. This effect leads to lysosomal disruption and the death of AML cells. Interestingly, AML cells were found to be more vulnerable to Deox B 7,4, compared to normal hematopoietic cells . ConversSeveral mTORC1 inhibitors, including rapamycin and its analogous  have been developed. Many second-generation inhibitors have also been developed that target both mTORC1 and mTORC2, or mTORC1 and PI3K. Phase I/II studies with rapalogs as single agents in AML have been disappointing, whereas combination studies with chemotherapy have demonstrated some promise . InteresAn innovative approach to target AML cells involved the development of a biohybrid with a tumor-associated peptide somatostatin and the photosensitizer ruthenium, named RU-SST. The stomatostatin receptor type 2 (SSRT2) is expressed more highly on AML cell lines and leukemic cells of patients with AML compared to HSCs from healthy donors. Therefore, the biohybrid more specifically targets AML cells. Interestingly, RU-SST localizes within the lysosomes, indicating their involvement in the eradication mechanism . Non-theLysosomes can be utilized in a rather unique manner by harnessing them as a biologically derived nano-carrier. Encapsulation of cancer drugs within nano-carriers such as liposomes has already been put into practice. Lysosomes, due to their robustness within the biological environment and low immunogenic reaction can serve as a suitable carrier. Yeast-derived lysosomes, engineered to carry the anthracycline drug, daunorubicin, have exhibited efficacy against the AML cell line, HL60 .This review highlights the variety of functions associated with lysosomes and emphasizes their central role in maintaining cellular homeostasis. It is hardly surprising that deregulation of lysosomal structure and function can play a key role in malignant diseases such as AML. Tumor-specific lysosomal alterations have been identified, opening the possibility of targeting lysosomes as a way to exert maximum effect on cancer cells while sparing normal ones. To date, the cornerstone of chemotherapy treatment in AML is the combination of cytarabine (Ara-C) with an anthracycline and/or allogeneic stem cell transplantation. However, patients above the age of 65 respond poorly to such a taxing treatment regime. On the other hand, targeted therapies, while less strenuous, are usually not effective as a monotherapy . In the One major issue in the field of lysosomal research is that the lysosome has become synonymous with its degradative functions and more attention needs to be drawn towards the lysosome as a signaling organelle. Importantly, targeting the lysosome is independent of the molecular lesions in AML and is not dependent upon the disruption of up-stream receptor-mediated signaling events—for which resistance is inevitable. New targeting or chemo-sensitizing strategies of this type would therefore be extremely valuable. We hope that this review reiterates the importance of studying lysosomes and reinforces the growing interest in exploring its value as a therapeutic target in leukemia.


Whether mitochondrial Ca2+ influx through the mitochondrial Ca2+ uniporter (MCU) controls T cell metabolism and effector function remained, however, enigmatic. Using mice with T cell-specific deletion of MCU, we here show that genetic inactivation of mitochondrial Ca2+ uptake increased cytosolic Ca2+ levels following antigen receptor stimulation and store-operated Ca2+ entry (SOCE). However, ablation of MCU and the elevation of cytosolic Ca2+ did not affect mitochondrial respiration, differentiation and effector function of inflammatory and regulatory T cell subsets in vitro and in animal models of T cell-mediated autoimmunity and viral infection. These data suggest that MCU-mediated mitochondrial Ca2+ uptake is largely dispensable for murine T cell function. Our study has also important technical implications. Previous studies relied mostly on pharmacological inhibition or transient knockdown of mitochondrial Ca2+ uptake, but our results using mice with genetic deletion of MCU did not recapitulate these findings. The discrepancy of our study to previous reports hint at compensatory mechanisms in MCU-deficient mice and/or off-target effects of current MCU inhibitors.T cell activation and differentiation is associated with metabolic reprogramming to cope with the increased bioenergetic demand and to provide metabolic intermediates for the biosynthesis of building blocks. Antigen receptor stimulation not only promotes the metabolic switch of lymphocytes but also triggers the uptake of calcium (Ca Mitochondria play a pivotal role in cellular metabolism by producing large amounts of ATP through oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO). In addition, intermediates of mitochondrial metabolism, such as tricarboxylic acid (TCA) cycle, glutaminolysis and FAO, control the fate and function of immune cells by regulating signaling pathways, the cellular redox balance, apoptosis and epigenetic rewiring through DNA and histone modifications. Not surprisingly, mitochondrial metabolism has been (re-)discovered as an important regulator of lymphocyte function and “immunometabolism” has become a major research focus.2+) from the cytosol was made over half a century ago , the production of reactive oxygen species (ROS) and apoptosis through opening of the mitochondrial permeability transition pore (mPTP)  is tightly regulated and requires specific transporters. Following an intensive search over decades, two groups identified the predicted mitochondrial Ca2+uniporter (MCU) using an in silico approach and demonstrated that the MCU protein forms a highly selective, inwardly rectifying Ca2+ channel. After the molecular identification of MCU, it became clear that mitochondrial Ca2+ uptake is mediated by a larger protein complex that not only contains MCU as the pore-forming subunit but is composed of additional regulatory and structural elements. Essential MCU regulator (EMRE) and its homologue MCUb were shown to be part of the channel pore, whereas the two EF hand domain-containing proteins mitochondrial Ca2+uptake 1 (MICU1), 2 and 3 function as “gatekeepers” of the MCU complex  . Althoug complex .2+ influx into the mitochondrial matrix is driven by a negatively charged membrane potential at the IMM that is established by the ETC. The opening of the MCU channel is initiated by conformational changes of MICU1/2 after binding of free cytosolic Ca2+ to their EF hand domains. Given the relatively low affinity of MICU1/2 for Ca2+ it was originally believed that the extramitochondrial Ca2+ levels required for MCU activation could not be achieved under physiological conditions. However, most Ca2+ signals are not uniformly distributed throughout the cytosol but are locally restricted to the site of Ca2+ influx. These Ca2+ “microdomains” can be generated by Ca2+ release from ER stores and/or extracellular calcium influx pathways, such as store-operated Ca2+entry (SOCE) through Ca2+release-activated Ca2+ (CRAC) channels, and reach the required local Ca2+ concentration to induce MCU channel opening. In activated T cells, mitochondria are in proximity to the ER and move actively towards the immunological synapse at which Ca2+ influx via SOCE is elicited  and Cd4Cre mice (strain 022071) were purchased from the Jackson laboratories and housed at the University of Würzburg. All animals were on a pure C57BL/6J genetic background and maintained under SPF conditions.cufl/fl  (strain T cells were isolated using MojoSort Mouse T cell isolation kits (BioLegend) and cultured in complete RPMI 1640 medium (Gibco) containing 1 g/L glucose. 24-well plates were pre-coated with 12 μg/ml polyclonal anti-hamster IgG, washed with PBS and activated with either 0.25 μg/ml (for Th17) or 0.5 μg/ml (for Th1 and iTreg subsets) of anti-CD3 (clone 145-2C1) together with 1 μg/ml anti-CD28 (37.51). The following culture conditions were used: Th1 cells: 2.5 μg/ml anti-IL-4 (clone 11B11), 10 ng/ml rhIL-2 and 10 ng/ml rmIL-12. iTreg cells: 2.5 μg/ml anti-IL-4, 2.5 μg/ml anti-IFNγ, 10 ng/ml rhIL-2 and 5 ng/ml rhTGF-beta. Th17 cells: 2.5 μg/ml anti-IL-4, 2.5 μg/ml anti-IFNγ, 20 ng/ml rmIL-6 and 0.5 ng/ml rhTGFβ. Tc1 cells: 2.5 μg/ml anti-IL-4, 10 ng/ml rhIL-2 and 10 ng/ml rmIL-12. Tc17 cells: 2.5 μg/ml anti-IL-4, 2.5 μg/ml anti-IFNγ, 20 ng/ml rmIL-6 and 0.5 ng/ml rhTGFβ. All antibodies and cytokines were from Bio X Cell and Peprotech, respectively. To detect cytokine expression, cells were stimulated with 1 μM ionomycin plus 30 nM phorbol myristate acetate  for 5 h in the presence of brefeldin A (BioLegend). In some experiments, T cell were treated for 72 h with 2–10 µM Ruthenium red (Sigma).35–55 peptide (Synpeptide) was emulsified in CFA with 5 mg/ml M tuberculosis H37Ra (BD Difco). 200 mg MOG35–55 was injected subcutaneously at the flanks of the mice, followed by intraperitoneal injection of 250 ng pertussis toxin (Enzo) on day 0 and 2. The EAE score was assigned as described before . LCMV was grown in BHK-21 cells and viral titers (PFU) in the supernatant were determined as described . For chr33-41 and GP66-77 were provided by the NIH Tetramer Core Facility.Cells were blocked with anti-FcγRII/FcγRIII  and debris was excluded using the viability dye eFluor780 (eBioscience). Staining of surface molecules with fluorochrome-conjugated antibodies was performed in PBS containing 0.1% BSA. Intracellular (IC) and transcription factor (TF) staining was carried out with the IC Staining and TF Staining Buffer Kit, respectively (eBioscience). Samples were acquired on a BD FACSCelesta flow cytometer and analyzed with FlowJo Software (Tree Star). The following antibodies were used: anti-mouse CD4 (clones 53–6.7 and GK1.5), anti-mouse IL-17A (C11-18H10.1), anti-mouse IFNγ (XMG1.2), anti-mouse GM-CSF (MP1-22E9), anti-mouse TNFα (MP6-XT22), anti-mouse IL-2 (JES6-5H4), anti-mouse Foxp3 (FJK-16s), anti-mouse T-bet (4B10), anti-mouse RORγt (Q31-378), anti-mouse CD25 (PC61), anti-mouse Ki-67 (B56), anti-CD44 (IM7), anti-CXCR5 (SPRCL5), anti-PD-1 (RMP1-30), anti-CD38 (90), anti-GL.7 (GL7), anti-CD8α (53–6.7), anti-Tim3 (RMT3-23). All antibodies were from BioLegend or eBiosciences. PE-labelled tetramers loaded with the LCMV peptides GPT cells were labelled with CellTrace Violet (Thermo Scientific) according to the manufacturer’s instructions. Cells were blocked with 50% FBS, washed twice with RPMI and stimulated with 1 μg/ml plate-bound anti-CD3 (clone 2C11) and anti-CD28 antibodies  with 50 U/ml IL-2 (Peprotech). CTV dilution was monitored daily by flow cytometry.2+-free Ringer solution. Changes in intracellular Ca2+ concentration were analyzed using a FlexStation3 plate reader (Molecular Devices) at 340 and 380 nm. Cells were stimulated with 1 μM thapsigargin (TG) (Sigma) in Ca2+-free Ringer solution and SOCE was analyzed after re-addition of 2 mM Ca2+. Alternatively, whole splenocytes were loaded with 2 μM Fluo4-AM (Thermo Scientific) and incubated with anti-CD4-APC and anti-CD8a-BV421 antibodies (BioLegend). Measurements of intracellular Ca2+ changes were recorded by flow cytometry. Baseline cytosolic Ca2+ levels acquired in 0 mM Ca2+ Ringer solution, before cells were stimulated with 1 μM thapsigargin and re-addition of 2 mM Ca2+. Mitochondrial uptake of free Ca2+ was monitored in the presence of digitonin-permeabilized T cells . 1 × 106 cells per well were re-suspended in PTP buffer  containing 2 mM succinate, 2 µM thapsigargin and 1 μM CalciumGreen-5N (Thermo Scientific). Fluorescence (503/535 nm) was monitored using a Tecan M200 Pro reader in a 96-well plate with injection of 25 µM Ca2+ pulses.T cells were labelled with 2 μM Fura-2-AM (Thermo Scientific) as described earlier . Cells wCNS specimens were fixed in 4% PFA, embedded in paraffin and stained with Luxol fast blue and Cresyl violet (both Carl Roth) to detect myelin and nuclei, respectively.CT values were normalized to 18S gene expression and analyzed using the 2−ΔCT method. Mcu-for: TTAGCAGAAAAGCAGAGAAGAG, Mcu-rev: TGATGAAGTAGGTGACGGG, Letm1-for: CGGGGTAGTCTGAGGGATCG, Letm1-rev: TGGAGTACAGCAACGAGACAG, Slc8b1-for: CTGGAAGTGTCAACCAGACTG, Slc8b1-rev: AGTCACAGCGATCAGATGTGT, 18S-for: CGGCGACGACCCATTCGAAC, 18S-rev: GAATCGAACCCTGATTCCCCGT.RNA was extracted using the RotiPrep RNA mini kit (Carl Roth), followed by cDNA synthesis with the iScript cDNA synthesis Kit (BioRad). qRT-PCR was performed using iTaq Universal SYBR Green SuperMix (Bio-Rad) and the CFX RT-PCR thermocycler (BioRad). For quantitation, Cells were resuspended in RIPA lysis buffer with complete protease inhibitor cocktail (Thermo Scientific). 40 μg of total protein was fractionated by SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were incubated with antibodies against β-Actin , VDAC , GAPDH  and MCU . For detection, peroxidase-coupled secondary anti-mouse or anti-rabbit antibodies (BioRad) and ECL Substrate (Thermo Scientific) were used.Glycolytic proton efflux rate (PER) and oxygen consumption rate (OCR) were measured using a XFe96 extracellular flux analyzer (Seahorse Bioscience) as described before .t-test. Differences were considered significant for p values < 0.05.All results are means with standard error of the means (SEM). The statistical significance of differences between experimental groups was determined by unpaired Student’s 2+ uptake in primary T cells, we generated mice with T cell-specific deletion of the Mcu gene by crossing Mcufl/fl mice  and mitochondrial Ca/fl mice  to Cd4Culations . The freulations . Naïve agulation . We nextgulation  and protgulation . We nextaddition . Althougnchanged , this da2+ elevations. In most studies, genetic or pharmacological inhibition of MCU attenuated ER store depletion and SOCE (2+-dependent inactivation (CDI) of IP3R and/or CRAC channels (2+ levels after ER store depletion with the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin (TG)  . Althouggin (TG) .2+ buffering and elevated SOCE affects the activation and effector function of T cells, we isolated naïve CD4+ T cells from WT and Mcufl/flCd4Cre mice and differentiated these cells into different T helper (Th) subsets. The expression of MCU was similar in all Th cells tested  and memory (Tc17) phenotype but also differ greatly by their mitochondrial activity   cells to support affinity maturation of germinal center (GC) B cells and anti-viral humoral immunity. We did not detect differences in the activation of CD4+ T cells and Foxp3+ Treg cells in Mcufl/flCd4Cre mice compared to WT controls , loss of effector function  and apoptosis of CD8+ T cells  , which ris (EAE) . We firsremities , inflammremities  and the remities . The inf animals . In linee of MCU . Because T cells . Importativation  and the e of MCU , suggestclone 13 . LCMV incontrols . The clonaltered . Furthernaltered  and GC Bnaltered  were int T cells . Using G animals . T cell  animals  and cyto animals  of MCU-d2+ uptake and elevating cytosolic Ca2+ levels after antigen receptor stimulation, MCU is largely dispensable for T cell-mediated immune responses in vitro and in models of autoimmunity and persistent viral infection.Collectively, these data demonstrate that, despite attenuating mitochondrial Ca2+ uptake is crucial for many cellular functions, including oxidative metabolism, ROS production, signal transduction and the regulation of cell death. In stressed or chronically activated cells, MCU triggers mitochondrial Ca2+ overload that causes the opening of the mPTP and the release of pro-apoptotic factors. Thus, tight control of mitochondrial Ca2+ uptake safeguards cellular survival and alterations in mitochondrial Ca2+ handling have been linked to a variety of diseases. Not surprisingly, MCU gained attention as a target for therapeutic interventions in different pathologies, including cancer, cardiovascular, and inflammatory disorders.Mitochondrial Ca2+ signals play fundamental roles in the immune system. They not only control the differentiation and effector function of lymphocytes at the transcriptional level but also promote their metabolic re-programming  adaptions that compensate for the lack of MCU in vivo (We here show, contrary to our expectation, that ablation of MCU has no obvious effects on T cell metabolism, differentiation and effector function  tissues . Of note strains . The fin in vivo .2+ uptake on cytosolic Ca2+ levels remain complex (2+ influx in absence of MCU was explained by an accelerated CDI of IP3R and/or CRAC channels that causes their premature closing when incoming Ca2+ cannot be taken up by adjacent mitochondria (2+ influx in primary T cells. These observations are in line with recent reports of macrophages and lymphocytes from MCU-deficient mice that showed a similar increase of cytosolic Ca2+ (per se and, thus, mitochondrial Ca2+ buffering are not essential for SOCE in primary T cells. Our seemingly contradictory findings compared to other reports may have a simple explanation: defective Ca2+ uptake from the cytosol into the mitochondrial matrix in MCU-deficient lymphocytes causes an accumulation of cytoplasmic Ca2+, resulting in a net increase of SOCE. Enhanced SOCE in MCU-deficient T cells could have been expected to amplify and augment T cell effector function and metabolism, which was, however, not the case. These observations are reminiscent of our previous findings using ORAI2-deficient mice that show a similar elevation of SOCE without increasing the effector functions of T cells in animal models of infection and autoimmunity (The consequences of mitochondrial Ca complex . In most complex . Other s complex . Impairechondria . In thislic Ca2+ . Furtherlic Ca2+ , indicatimmunity .Mcufl/flCd4Cre mice. It was shown before that deletion of MCU alone is not sufficient to completely abolish mitochondrial Ca2+ uptake in neurons (2+ as precipitates and acidification of the mitochondrial matrix during respiration dissolves these crystals and releases intra-mitochondrially free Ca2+ ions in an MCU-independent fashion (2+ channels, such as ryanodine receptors, TRP channels and SERCA pumps (A possible explanation for our finding that MCU is largely dispensable for murine T cell function is that adaptational changes in the mitochondria may compensate for the “chronic” loss of MCU in  neurons . Further fashion . AlthougCA pumps . Thus, f


Therefore, we will first give a short overview of physiological cardiomyocyte Na+ handling and its deterioration in heart failure. On this basis we discuss the salutary effects of SGLT2i on Na+ homeostasis by influencing NHE1 activity, late INa as well as CaMKII activity. Finally, we highlight the potential relevance of these effects for systolic and diastolic dysfunction as well as arrhythmogenesis.Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are emerging as a new treatment strategy for heart failure with reduced ejection fraction (HFrEF) and—depending on the wistfully awaited results of two clinical trials (DELIVER and EMPEROR-Preserved)—may be the first drug class to improve cardiovascular outcomes in patients suffering from heart failure with preserved ejection fraction (HFpEF). Proposed mechanisms of action of this class of drugs are diverse and include metabolic and hemodynamic effects as well as effects on inflammation, neurohumoral activation, and intracellular ion homeostasis. In this review we focus on the growing body of evidence for SGLT2i-mediated effects on cardiac intracellular Na The opposite is true for NCX “reverse mode,” which is promoted by an increase in [Na+]i and positive membrane potentials. Apart from these transporters, which are accountable for most of the Na+-entry (NCX > Nav > NHE) m), which is elevated in parallel to cytosolic Ca2+ levels, e.g., during β-adrenergic stimulation, activates Krebs cycle dehydrogenases to increase the production of reducing equivalents (NADH and NADPH) in order to meet the energetic demand and also to preserve the anti-oxidative capacity of the cell [2+ uptake is mediated via the mitochondrial Ca2+ uniporter (MCU) [+/Ca2+ exchanger (NCLX) primarily causes Ca2+ extrusion linking [Na]i with [Ca2+]m [i results in a decrease in [Ca2+]m, which in turn hampers Krebs cycle activity o et al. . In briethe cell . While mer (MCU) , activit [Ca2+]m ,38. Consactivity .+ homeostasis for cardiac excitation-contraction coupling as well as cardiac metabolism it comes as no surprise that dysregulation of Na+ handling is centrally involved in the development and progression of heart failure.Given the role of Na+ overload as a consequence of a dysbalance between Na+ in- and efflux. Indeed, bulk cytosolic [Na+]i from failing compared to non-failing myocytes is elevated by ~2 to 6 mM i in human heart failure i and [Ca2+]i, as described above  potentially causing cellular pro-arrhythmic events in form of delayed afterdepolarizations (DAD) [+]i indirectly reduces the availability of reducing equivalents, which not only leads to a deficit in ATP but also contributes to oxidative stress  ,83. Moredevelops .+ handling, as set out below -based Ca2+ indicator (mitoCam), increased i or [Ca2+]i in isolated myocytes. Unfortunately, NHE1 function in cardiomyocytes was not assessed in this study. However, inhibition of NHE1 activity by dapaglifozin was demonstrated in human umbilical vein endothelial cells (HUVEC) [+]i by SGTL2i treatment neither in unpaced healthy human nor animal  cardiomyocytes i they even suggest that there is also a detectable decrease in [Na+]i upon treatment with empagliflozin [+]i in healthy cardiomyocytes is debatable, especially when considering that NHE1 activity at normal intracellular pH is low and contributes only little to cellular Na+ loading i and activate NCX forward mode resulting in a depolarizing Na+ influx (Iti) that triggers delayed afterdepolarizations (DAD) i in healthy murine ventricular myocytes after 24 h of treatment. Interestingly, while the effects on CaMKII activity as well as Ca2+ transient amplitude were only observable after incubation with empagliflozin for 24 h, we could detect a decrease in subsarcolemmal [Na+] as assessed by application of the Nernst equation to the reversal potential of NCX currents, which were measured by whole cell patch clamp, as early as 30 min after start of treatment. Consistently, Pabel et al. reported no change in systolic Ca2+ transient amplitude, Ca2+ transient decay kinetics, or diastolic Ca2+ concentration in human failing cardiomyocytes upon acute empagliflozin exposure (15 min) i was not influenced by dapagliflozin i seems unlikely, but might warrant further investigation.Consequently, although conceivable, a relevant contribution of SGLT1 and SMIT1 to SGLT2i-mediated decrease in i, it is conceivable that restoration of Na+ homeostasis contributes to these observations  significantly reduced markers of oxidative stress (Hnd IL-6) . Furthernd IL-6) . Of notend IL-6) . In anot2+ load) . Althougrvations . However+ homeostasis, might be a key to improving cardiovascular outcomes i as well as oxidative stress and inhibit CaMKII, also possess anti-arrhythmic effects  and triggers (early (EADs) and delayed afterdepolarizations (DADs)) coincide . Typicalate INa) ,110,138.overload ,140. Impoverload ,81,141.  effects . Apart feostasis ,142. In eostasis ,144,145.Interestingly, in an explorative analysis of the DECLARE-TIMI 58 trial the incidence of atrial fibrillation (AF)/atrial flutter (AFL) in the dapagliflozin group was reduced by ~19% in this high-risk collective of type II diabetic patients independent of a history of heart failure, cardiovascular disease, or pre-existing AF . In addiThis notion is further supported by a pre-clinical study. In a rat model of streptozotocin-induced type II diabetes 8 weeks of empagliflozin treatment reduced AF inducibility, reduced atrial interstitial fibrosis, and improved mitochondrial function . These aNa activator veratridine in isolated healthy murine cardiomyocytes i ) and [Ca2+]i. However, in regard to these effects, it is still unclear what is chicken and what is egg. Does inhibition of CaMKII normalize ion homeostasis and decrease ROS levels or is it the other way around? To address this issue further research, e.g., using specific knock-out models, is required and will also aid to understand which of the observed beneficial effects of SGLT2i in vivo indeed rely on this common pathway.Due to the great scientific interest in the identification of the cellular targets mediating the cardioprotective effects of SGLT2i a plethora of potential mechanisms of action was proposed in the recent years. As it is unlikely that one substance interacts with an array of pathway specific targets, it is tempting to speculate that SGLT2i positively influence maybe only a couple of targets that are far upstream in the pathological processes of cardiac disease in general and heart failure in particular. CaMKII overactivation, Na


Despite the increased CaV1.1, the mobile intramembrane charge was reduced by ~20% in Triadin-null fibers. The initial peak of calcium release flux by pulse depolarization was minimally altered in the null fibers . The “hump” phase that followed, attributable to calcium detaching from calsequestrin, was 25% lower, a smaller change than expected from the reduced calsequestrin content and calcium saturation. The exponential decay rate of calcium transients was 25% higher, consistent with the higher SERCA1 content. Recovery of calcium flux after a depleting depolarization was faster in triadin-null myofibers, consistent with the increased uptake rate and lower SR calsequestrin content. In sum, the triadin knockout determines an increased RyR1 channel openness, which depletes the SR, a substantial loss of calsequestrin and gains in other couplon proteins. Powerful functional compensations ensue: activation of SOCE that increases [Ca2+]cyto; increased SERCA1 activity, which limits the decrease in [Ca2+]SR and a restoration of SR calcium storage of unknown substrate. Together, they effectively limit the functional loss in skeletal muscles.Triadin, a protein of the sarcoplasmic reticulum (SR) of striated muscles, anchors the calcium-storing protein calsequestrin to calcium release RyR channels at the junction with t-tubules, and modulates these channels by conformational effects. Triadin ablation induces structural SR changes and alters the expression of other proteins. Here we quantify alterations of calcium signaling in single skeletal myofibers of constitutive triadin-null mice. We find higher resting cytosolic and lower SR-luminal [Ca The crucial device in this process is the couplon, a physical continuum of proteins that includes the dihydropyridine receptor , the ryanodine receptor 1 (RyR1), FKBP12, junctophilin 1 (JPh1), stac3, junctin (Jct), triadin (Tr) and calsequestrin (Casq1), among other components SR falls below a certain level  in both cytosol and SR lumen, evaluated Ca2+ release flux kinetically and calculated quantity of releasable Ca2+. Finally, we measured the expression density of Casq1 as well as structural parameters of SR terminal cisternae in living cells, in images of fluorescently tagged Casq1. The separation from wild-type values found in the calcium movements of triadin-null muscle could be assigned only in part to a reduction in SR Ca2+ content associated with the loss of Casq1. Other robust compensatory mechanisms are therefore exposed or implied by the present observations.To interpret the results mechanistically, in terms of alterations of the main proteins and processes required for EC coupling, we quantified intramembrane charge movement   measurements, or a variant, CFP-Casq1, to evaluate the pattern of Casq1 expression inside the lumen of SR. Plasmids were prepared as described by Sztretye et al., (2011b) , [Ca2+]cyto, was monitored by SEER (Shifted Excitation and Emission Ratioing) of Indo-1 fluorescence [2+]cyto was calculated  , [Ca2+]SR, was determined as described in Sztretye et al. (2011b) cyto(t) was calculated byAfter 20 minutes of equilibration of pipette internal solution inside cells, fluorescence of Fluo-4 excited with 488 nm light was acquired in the range 500–545 nm by a confocal microscope . Fmax and Fmin are the fluorescence intensities at zero and saturating [Ca2+]. The kinetic values for Fluo-4 were determined elsewhere (t) by a simplified version of the “removal” method described previously  simultaneously for several evoked transients. This procedure is hampered by lack of knowledge of multiple cellular processes involved in the evolution of [Ca2+]cyto, including the many molecules that bind Ca2+ and transport processes that move it. However, in the presence of a high concentration of a Ca2+ buffer, BAPTA in the present case, the incidence of the endogenous buffers becomes negligible total and [BAPTA]total, were set to 0.5 of the concentration in the pipette for records that started after 30 min in the whole cell configuration. The kinetic constants of BAPTA: Ca2+ reaction were kon = 1000 μM*s-1 and koff = 200 s-1 cyto, has been shown to increase in Tr-null adult myofibers and myotubes [2+ transients induced by chemical RyR1 activation [2+]SR was quantified directly, using SEER of the TC-targeted ratiometric biosensor D4cpV-Casq1 . [Ca2+]cyto  was 20% higher in Tr-null fibers  within the plasma membrane upon membrane depolarization was evaluated during the pulse (Qon) and after its end (Qoff). The evaluation is illustrated in V, between -60 and +30 mV, in 10 mV increments. The “charge movement” current IQ is presented in panels 3A, B. Qon(V), the time integral of the current during the pulse to voltage V, is plotted by thin lines for all WT fibers studied in panel 3C and all Tr-null fibers in 3D. The dependence of Q, on or off, on V, was fitted with a “Boltzmann” function ; that in p = 0.06). Panel E, shows the off portion of intramembrane currents, in pulses to +10, +30 and +50 mV. The decay of these currents could be fitted with exponential functions of time (in red trace). The distribution of the exponential time constants is plotted in F. The decay in Tr-null was faster but the difference was not significant. The average τ was 3.2 ± 0.3, in Tr-null , vs. 3.8 ± 0.36 ms in WT ; p = 0.25. p was calculated by two-level hierarchical analysis.The displacement of charge , in turn calculated from the fluorescence imaged by confocal line-scanning of single muscle fibers, patched and loaded with Fluo-4. A is the “linescan” image of fluorescence, F, upon application of a supramaximal depolarizing pulse (to +30 mV for 100 ms). The average over x, F(t), is overlaid in white. In 4B are Ca2+ release flux (2+ released (R(t), black trace). 2+ release  gradient that drives the flux from SR to cytosol SR in the Tr-null, as evidenced by the rate of restoration of peak flux  and of duration sufficient to elicit evacuation of a large fraction of the total SR Ca2+ available for release by depolarization. Thus, they constitute a measure, albeit approximate, of Ca2+ content available to be released. The time courses of quantity of Ca2+ released by a pair separated by 800 ms are illustrated in RT1 and RT2, are clearly separable in the traces. RT2 was generally lower in Tr-null fibers at the intervals evaluated, which is consistent with a lower SR calcium content at all times. By contrast, as a fraction of RT1—the total quantity of Ca2+ released by the first pulse—the release of Ca2+ by the second pulse was greater in the Tr-null. This is illustrated in panels 7B, showing the normalized traces R(t)/RT1. The excess release in the Tr-null as a fraction of the total releasable load is maximal at intervals near 1 s, as shown in panel 7C. Because the quantity released by the 2nd pulse measures most of the releasable content of the SR at the time, it follows that content recovery after a pulse is faster in the Tr-null muscle.The total quantity 2+ release observed in Tr-null muscles suggest a faster recovery of free [Ca2+]SR. An unchanged flux of uptake by SERCA1 accumulating Ca2+ in a lumen with less Casq1 would result in a faster recovery of both free and total SR Ca2+ levels. An increased activity of the Ca2+ pump, expected from the increased content of SERCA1 in microsomes of Tr-null mice  records, superimposed after normalizing to the value at first peak, with single exponentials fitted to the decay phase. On average, the decay was faster in Tr-null, as represented in panel B .The changes in various aspects of Call mice .2+ buffers and binding sites are subsumed into one, “BAPTA”, as the internal solution has sufficient BAPTA that other ligands can be neglected for short-term analysis. The other is a storage compartment, named the SR but comprising all Ca2+ removing organelles . Within this simple framework, R(t), the Ca2+ released by the voltage pulse, differs from Rnet(t), the net released Ca2+, because the SERCA pump removes Ca2+ from the cytosol at all times, including that when release is proceeding. The formal calculation of Rnet is done as described in Methods. Applied to the records in In the simplified form used here, the model that describes the fluxes is closed to the exterior (it neglects flux across the plasma or TT membrane) and only has two compartments. One is the cytosol, where Ca2+]cyto is seen to decay monotonically. Rnet also decays, mirroring recovery of SR content. Within the simple theory used here, this decay is largely determined by SERCA transport. The rate of decay is faster in the Tr-null. As documented with panel E and summarized in N = 9; WT: 3.8 ± 0.19 s, N = 8; p < 0.001) corresponds to a 26% increase in uptake rate in null muscle, in rough agreement with the change in SERCA1 content of Tr-null total fractions, et al. (2012) cyto was found to be significantly higher in the Tr-null. The increase, 17%, is in rough agreement with the 28% increase found by Shen et al. (2007) cyto found in these muscles was substantially lower than that found in Tr-null primary myotubes   in mouses .2+ permeability of the plasma membrane is a known consequence of a decay in SR free Ca2+ concentration SR in Tr-null than WT muscles; this difference is consistent with the lower Ca2+ releasable by depolarization found in the present study and with the difference in Ca2+ releasable by caffeine or depolarization by high potassium, found in myotubes [2+]SR by 1/3 of the resting value was found to activate SOCE in skeletal muscle [2+]cyto in Tr-null muscle.myotubes , 25, 51.l muscle ; therefo2+]SR should be justified in the context of triadin ablation. The following are plausible reasons for the lower [Ca2+]SR: i) the absence of triadin could remove a direct inhibitory effect on RyR1, for which there is evidence [2+]cyto observed in Tr-null muscle.To complete the interpretation, the observed difference in cyto results from changes at the plasma membrane consisting mostly in activation of SOCE in response to a reduction in [Ca2+]SR due to loss via RyRs. In turn, the increased propensity to open of the RyRs may be a release from either a basal inhibitory effect of triadin or an inhibitory effect of Casq1, mediated by triadin.The tentative conclusion in this section is that the increase in [CaRP were changed significantly in the Tr-null. This was unexpected: Ca2+ flux is proportional to the free [Ca2+] gradient between SR lumen and cytosol [2+] gradient amounts to an increase in peak permeability of the SR releasing membrane.Neither the initial peak of release flux nor its time integral  cytosol , a gradiRT max  was 265 μM or 19% lower than in WT. The difference was fully accounted for by the component RH (quantity released in the hump stage), which was lower by 227 μM, or 27% of the WT value. The Ca2+ released in this phase detaches from Casq1  SR. In the hypothesis of proportionality between releasable SR Ca2+ content and free [Ca2+]SR, this change alone should reduce the hump by 30%; however, the Ca2+ buffering power of the SR is known to decrease with free concentration of the ion SR is lowered (as would be expected for ligands of high Ca2+ affinity).The discrepancy between Ca the ion , 66; the storage , and mig2+ release with the other functional and structural changes observed. The presence of four CaV1.1 units per RyR1 channel leaves room for a possible functional reserve; in this view, channels would open even if acted upon by an incomplete set, with fewer than 4 functional, charge-carrying CaV units. A recent theory of physical interactions between RyR1 and CaV1.1 SR and functional voltage sensors, calls into question the assumptions used in the literature to calculate the fraction of releasable Ca2+ contributed by Casq1. We previously estimated it at 75%, equal to the reduction in releasable Ca2+ in Casq1-null mice relative to wild-type SR measured directly SR should be faster if Ca2+ were pumped into the SR at the same rate and had to satisfy a diminished buffer capacity in the SR lumen. That the exponential rate of decay of [Ca2+]cyto was somewhat increased in null muscles points at enhanced SR Ca2+ uptake rate as another contributor to the increased rate of Ca2+ release recovery, and is in agreement with the increased expression of SERCA1 .2+ release, or what was left of it. This change reflects disruption of the Casq1 repolymerization process required to regain the storage capacity needed to contribute the hump of Ca2+ release [We also observed a slowing of recovery of the hump component of Ca release . The dis release .2+]cyto provides a way to calculate the effective Ca2+ buffering power of the cytosol. Indeed, the time constants of decay of both [Ca2+]cyto and Rnet are lower in Tr-null muscles, reflecting faster Ca2+ uptake; however, the time constants, of hundreds of ms for the cytosolic concentration change and a few seconds for Rnet (which reflects replenishment of SR content) differ by nearly an order of magnitude. In the framework of the analysis, the different rates reflect different buffering in the two compartments.The analysis of decay of cyto/dt at the start of the off is ~ Δ[Ca2+]cyto / τcyto (where the numerator is the increase in [Ca2+]cyto from the resting level) and dRnet/dt is Given that RT = 1655 μM, Δ[Ca2+]cyto = 0.5 μM, τcyto = 0.53 s and Bcyto = 460 . The evaluation of Bcyto is therefore consistent with the experimental conditions, which make BAPTA, by far, the dominant buffer.The calculated buffering power of the dominant cytosolic buffer, BAPTA, at the concentration (assumed) of 2.5 mM, with a conventional Bcyto = 486. It can be concluded that the buffering power did not change substantially in the null, a result expected for a feature that here is largely determined by the extrinsic buffer in the pipette.The corresponding calculation in the Tr-null yields 2+ homeostasis and dynamics. The main structural difference, a lower volume of TC, can be ascribed to the reduction in Casq1 expression. The functional effects can be attributed to the loss of conformational, largely inhibitory effects of triadin on RyR1 readiness to open, plus the partial loss of Casq1 and changes in other SR proteins. The altered conformational effects promote both resting and stimulated Ca2+ permeability of the SR membrane. The new steady state features a lower [Ca2+]SR and its usual consequence, higher [Ca2+]cyto, probably due to activation of SOCE. The loss of Casq1 results in lower storage capability and Ca2+ buffering power. Compounded with the reduced [Ca2+]SR, these changes lower the quantity of releasable Ca2+. The diminished Ca2+ bound to Casq1 results in a smaller “hump” component in the flux of Ca2+ release upon pulse depolarization. Unexpectedly, the peak component of Ca2+ release is largely unchanged in Tr-null muscle. This conservation implies an increase in peak Ca2+ permeability of the SR junctional membrane, due to a greater Popen of individual RyR channels or a greater number of activatable channels. The decrease in buffer capacity of the SR, which results from the lower Casq1 content, is reflected in a faster recovery of peak flux after a depleting pulse. The faster Ca2+ reuptake into the SR, which results in a greater rate constant of decay of the cytosolic Ca2+ transient, is consistent with the increase in SERCA content and contributes to the faster recovery of Ca2+ release.The ablation of triadin causes major changes in the expression of SR proteins. These result in structural alterations and functional effects on both Ca2+ release is much less than that predicted by the combined losses in Casq1 endowment, [Ca2+]SR and functional voltage sensors; the discrepancy highlights the operation of compensatory mechanisms, the nature of which is yet to be established.The deficit in CaS1 FigA, representative immunofluorescence images in (a) WT and (b) Tr-null FDB cross-sections. Fiber type was determined by isoform-specific myosin heavy chain immunostaining. Red: type I; green: type IIa; blue: type IIx. The fiber type composition was calculated by manually identifying and counting single fibers across the cross-section of each FDB muscle. B, Box plots compare the distribution of fiber types in Tr-null . Symbols represent averages of measurement in one cross-section per animal.(TIF)Click here for additional data file.S2 FigA, images F of CFP-Casq1, locating terminal cisternae, and Di-8-Anepps, marking the t-tubular system, in a WT myofiber. Shown are central images from a z-stack, after correction for optical spread.  corresponding images of a Tr-null myofiber.  merged images.  y-averaged profiles of selected areas in insets, which reveal the relative location of the triad components . B, averaged Casq1 profiles in triads of WT and Tr-null myofibers. The distance labeled “Triad width” evaluates the joint span of the t tubule plus the two TC. C, representation of individual measurements of triad width and two levels of averaging. Individual measures from 40 images of 14 WT myofibers and 135 images of 28 Tr-null myofibers from 3 and 5 mice respectively, are represented as vertical histograms. The horizontal segments at bottom of each histogram provide the scale for numbers of replicas at each ordinate value. Averages of image values for individual fibers are represented by symbols; different symbols identify 3 WT and 5 Tr-null individual mice. Box plots represent distribution of individual measurements (Tr-null: 0.45 ± 0.02. WT: 0.56 ± 0.025 μm). p = 0.007 was calculated using a three-level hierarchical analysis.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


Hypo) triggers intracellular Ca2+ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca2+‐dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca2+ oscillations by promoting extracellular Ca2+ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4‐mediated Ca2+ entry, in turn, promoted the concerted interplay between inositol‐1,4,5‐trisphosphate‐ and nicotinic acid adenine dinucleotide phosphate‐induced endogenous Ca2+ release and store‐operated Ca2+ entry (SOCE). hAFS‐CMHypo‐induced intracellular Ca2+ oscillations resulted in the nuclear translocation of the Ca2+‐sensitive transcription factor p65 NF‐κB. Finally, inhibition of either intracellular Ca2+ oscillations or NF‐κB activity prevented hAFS‐CMHypo‐induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS‐CMHypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic‐related myocardial injury.Second trimester foetal human amniotic fluid‐derived stem cells (hAFS) have been shown to possess remarkable cardioprotective paracrine potential in different preclinical models of myocardial injury and drug‐induced cardiotoxicity. The hAFS secretome, namely the total soluble factors released by cells in their conditioned medium (hAFS‐CM), can also strongly sustain in vivo angiogenesis in a murine model of acute myocardial infarction (MI) and stimulates human endothelial colony‐forming cells (ECFCs), the only truly recognized endothelial progenitor, to form capillary‐like structures in vitro. Preliminary work demonstrated that the hypoxic hAFS secretome (hAFS‐CM Therefore, in the present investigation, we have characterized for the first time the mechanisms whereby hypoxic hAFS secretome induces pro‐angiogenic intracellular Ca2+ oscillations and one of the downstream Ca2+‐dependent decoders, that is NF‐κB, in circulating ECFCs.Intracellular Ca22.1Blood samples (40 mL) collected in EDTA (ethylenediaminetetraacetic acid)‐containing tubes were obtained from healthy human volunteers aged from 22 to 28 years. The Institution Review Board at “Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Foundation” in Pavia approved all the protocols. Informed written consent was obtained according to the Declaration of Helsinki of 1975 as revised in 2008. ECFCs were isolated from circulating mononuclear cells, as described in 2.2+ hAFS were then cultured in Minimal Essential Medium (MEM)‐alpha with 15% FBS (Gibco—Thermo Fisher Scientific), 18% Chang B and 2% Chang C Medium (Irvine Scientific) with 1% L‐glutamine and 1% penicillin/streptomycin (Gibco—Thermo Fisher Scientific), at 75% confluency before being used to isolate their secretome.Human amniotic fluid‐derived stem cells were isolated from leftover samples of second trimester amniotic fluid obtained by prenatal screening from the Prenatal Diagnosis and Perinatal Medicine Unit, IRCCS San Martino Hospital, and the Fetal and Perinatal Medical and Surgery Unit and Human Genetics Laboratory at IRCCS Istituto Gaslini hospital . Informed written consent was obtained from all donors, according to local ethical committee authorization (protocol PR 428REG2015) and in compliance with Helsinki Declaration guidelines. After obtaining adherent amniotic fluid mesenchymal cells, hAFS were isolated by immunomagnetic sorting for c‐KIT expression , as previously defined2 and 5% CO2 at 37°C) or stimulatory hypoxic  conditions.In order to trigger the hAFS paracrine potential and to enrich their secretome with trophic soluble factors, cells were primed in vitro for 24 hours in serum‐free medium  under normoxic (20% O2.3Normo) or hypoxic preconditioning (hAFS‐CMHypo), was collected as previously described.g for 90’ and then at 3000 ×g for additional 30’. hAFS‐CM protein concentration was assessed by BiCinchoninic Acid (BCA) assay (Gibco—Thermo Fisher Scientific). hAFS‐CM was used for in vitro experiments as 80 mg/mL solution to be added to the cell culture medium as from previous studies.The total cell secretome, as represented by the cell‐conditioned medium (hAFS‐CM) from either hAFS in control normoxic condition , Ca2+ was substituted with 2 mmol/L NaCl, and 0.5 mmol/L EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The osmolality of PSS as measured with an osmometer (Wescor 5500) was 338 mmol/kg.Physiological salt solution (PSS) had the following composition (in mmol/L): 150 NaCl, 6 KCl, 1.5 CaCl2.3.22+ recording set‐up have been described in REFEndothelial colony‐forming cells were loaded with 4 µmol/L fura‐2 acetoxymethyl ester  in PSS for 1 hour at room temperature. The details of the Ca2.3.3Hypo and the specific blockers of intracellular Ca2+ signalling, 6 × 104 ECFCs were plated onto 13 mm coverslips in 24‐well plates. ECFCs were fixed in 4% formaldehyde in PBS for 15 minutes at room temperature, permeabilized for 7 minutes in PBS with 0.1% Triton X‐100 and blocked for 30 minutes in 2% gelatin. Then, primary (incubated for 1 hour at 37°C) and secondary (incubated for 1 hour at room temperature) antibodies were applied in PBS with 2% gelatin. The primary anti‐p65 (NF‐κB subunit) antibody specific for immunocytochemistry  was used at 1:50 dilution, whereas the AlexaFluor 488 secondary antibody from Invitrogen  was used at 1:200. After washing (3 times for 5 minutes each), nuclei were stained with 40,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) for 15 minutes at room temperature. Fluorescence images were acquired using a Leica epifluorescence microscope equipped with S Fluor X40/1.3 objective using MetaMorph software.Twenty‐four hours before treatment with hAFS‐CM2.3.4Hypo for 24 hours. Capillary network formation was assessed starting from 4 up to 24 hours later. The angiogenic response was measured by evaluating both dimensional and topological parameters. The length of endothelial tube‐like structures (tubules or TLS), number of polygon structures established by TLS, referred to as meshes and indicative of endothelial cell migration, and number of master junctions were measured from acquired bright‐field pictures by using the Angiogenesis Analyzer plugin of ImageJ .2+ signalling, the same protocol was repeated by priming ECFC with hAFS‐CMHypo in the presence of RN‐1734 (20 μmol/L), a selective blocker of transient receptor potential vanilloid 4 (TRPV4),Early passage (P2‐P3) ECFCs were cultured in basal medium EBM‐2 supplemented with 2% FBS in Cultrex (Trevigen)‐coated 96‐well plates, in the absence or in the presence of hAFS‐CM2.3.5Fura‐2/AM was obtained from Molecular Probes (Molecular Probes Europe BV). YM‐58483/BTP‐2 ‐1H‐pyrazol‐1‐yl]‐1,2,3‐thiadiazole‐5‐carboxanilide) was purchased from Tocris Bioscience. Glycyl‐l‐phenylalanine 2‐naphthylamide (GPN) was obtained from Santa Cruz Biotechnology. All the chemicals were of analytical grade and obtained from Sigma Chemical Co.2.3.6P < .05) was evaluated by Student's t test or one‐way ANOVA followed by the post hoc Dunnett's test as appropriate. Data relative to Ca2+ signals are presented as mean ± SE, while the number of cells analysed is indicated in the corresponding bar histograms.All the data have been collected from ECFCs deriving from at least three distinct donors. Pooled data are given as mean ± SE and statistical significance (33.1Hypo)  , thereby confirming the findings recently reported in.2+ signal was significantly (P < .05) larger when hAFS‐CMHypo was administered . The endothelial Ca2+ response to extracellular stimuli impinges on two Ca2+ sources: the extracellular milieu and the endogenous Ca2+ stores located within ER cisternae and EL vesicles.Hypo induced intracellular Ca2+ oscillations in the presence  reduced the percentage of oscillating cells  Figure , but not) Figure , immediad Figure . The free Figure , but note Figure , of extrn Figure . Therefoo Figure , but sigs Figure  and the e Figure . Furtherh Figure . These d3.22+ response to chemical stimulation in circulating ECFCs and vascular endothelial cells.2), to generate InsP3 and DAG. InsP3 induces ER Ca2+ release by priming InsP3Rs to be activated by cytosolic Ca2+,Hypo‐induced intracellular Ca2+ oscillations  or RN‐1734 , which, respectively, inhibit DAG lipaseHypo‐induced intracellular Ca2+ oscillations in circulating ECFCs  , which selectively blocks Sarco‐Endoplasmic Reticulum Ca2+‐ATPase (SERCA) activity, in the presence of extracellular Ca2+ to deplete the ER Ca2+ store and full activate SOCE.2+]i. When hAFS‐CMHypo was delivered upon 20‐minutes exposure to CPA, it induced a sustained elevation in [Ca2+]i ,Hypo‐induced intracellular Ca2+ oscillations has been reported in Figure Phospholipase C (PLC) plays a crucial role in the onset of the Cas Figure . The sams Figure , a selecs Figure . Similar) Figure , a less i Figure  that was4 Figure . Interes3.32+ signals in a pro‐angiogenic output in ECFCs.2+]i.Hypo , a membrane‐permeable Ca2+ buffer,P < .05) inhibited hAFS‐CMHypo‐induced nuclear translocation of p65 NF‐κB , which prevents the onset of the intracellular Ca2+ oscillations, and of thymoquinone   Figure , a selec4Hypo stimulated circulating human ECFCs to form capillary‐like networks through an oscillatory increase in [Ca2+]i.2+ spikes were not dissected. Understanding how the hAFS‐CMHypo elicits intracellular Ca2+ oscillations in ECFCs will provide the biological bases required to decipher the components of the Ca2+ toolkit that could be specifically targeted to induce vascular regrowth in infarcted hearts.Paracrine therapy through stem cells‐secreted mediators is emerging as an alternative, promising strategy to treat AMI by instructing resident cells, for example cardiac stromal cells, cardiomyocytes and endothelial cells, to optimize endogenous mechanism of cardiac repair.4.12+ oscillations in circulating ECFCs, thereby confirming previous results from our group.2+ overload during ischaemia/reperfusion injury.2+ oscillations induced by hAFS‐CMHypo in circulating ECFCs strongly resembled those induced by VEGF.Hypo does not contain VEGF, whereas it is enriched with multiple cytokines and chemokines, including interleukin 8 (IL‐8), angiogenin, Extracellular matrix metalloproteinase inducer and monocyte chemoattractant protein‐1 (MCP‐1).2+]i can be evoked by IL‐8 in mouse lymphokine‐activated killer (LAK) cells2+ signals do not adopt an oscillatory pattern.Human amniotic fluid‐derived stem cells ‐conditioned medium obtained from human amniotic fluid stem cells maintained under hypoxic over control normoxic conditions induced intracellular Ca2+]i, as well as in circulating ECFCs,2+ entry and/or endogenous Ca2+ release.2+ prevented the onset of hAFS‐CMHypo‐induced intracellular Ca2+ oscillations, which promptly resumed upon restitution of external Ca2+. This finding further supports the notion that VEGF is not involved in the spiking Ca2+ signal. Indeed, VEGF is still able to trigger 1‐4 intracellular Ca2+ spikes in the absence of extracellular Ca2+ entry in circulating ECFCs.2+ entry through two distinct DAG‐sensitive pathways, that is TRPC1 and TRPC3, induced intracellular Ca2+ oscillations, respectively, in primary myelofibrosis‐derived ECFCs2+ store.2+ entry through TRPV4 in circulating ECFCs2+ entry triggers hAFS‐CMHypo‐induced intracellular Ca2+ oscillations in circulating ECFCs. First, the pharmacological blockade of TRPV4 with two structurally distinct inhibitors, RN‐1734 and ruthenium red, suppressed the onset of the oscillatory signal. RN‐1734 is a selective TRPV4 antagonist,2+ response to hAFS‐CMHypo. Interestingly, the intracellular Ca2+ signals evoked by MCP‐1, which is quite abundant in hAFS‐CM,2+ transients evoked by hAFS‐CMHypo. Therefore, while SOCE is required to maintain the intracellular Ca2+ oscillations by reloading the ER with Ca2+ in a SERCA‐dependent manner,2+ response.Extracellular stimuli cause an increase in endothelial i, which was caused by TRPV4 activation. Therefore, we propose that TRPV4‐mediated extracellular Ca2+ entry provides the source of Ca2+ that is required to induce the Ca2+‐dependent recruitment of InsP3Rs by hAFS‐CMHypo. The subsequent InsP3‐induced depletion of the ER Ca2+ store can, in turn, engage STIM1, a sensor of ER Ca2+ concentration, to bind to and gate Orai1 and TRPC1 to mediate SOCE.2+ release through TPCs may cooperate with InsP3Rs to trigger endothelial Ca2+ waves.2+ spikes in circulating ECFCs.2+ release with either GPN or NED‐19, which, respectively, disrupt the EL Ca2+ store and inhibit TPC1, also impairs hAFS‐CMHypo‐induced intracellular Ca2+ oscillations. Therefore, NAADP‐induced Ca2+ release is likely to contribute with TRPV4 to ignite periodical InsP3‐induced ER Ca2+ release under these circumstances. Interestingly, IL‐8, which is also enriched in hAFS‐CM,InsP4.32+ oscillations support in vitro tubulogenesis and neovessel formation in vivo.2+]i with BAPTA‐AM impaired hAFS‐CMHypo‐induced ECFC tubulogenesis in Matrigel scaffolds. The Ca2+‐sensitive transcription factor, NF‐κB, may translate intracellular Ca2+ signals into a pro‐angiogenic input in circulating ECFCs. For instance, NF‐κB decodes VEGF‐induced intracellular Ca2+ oscillations in ECFCs2+ entry to induce proliferation and tube formation upon optical excitation of the photosensitive conjugated polymer, P3HT.Hypo promotes the nuclear translocation of NF‐κB to counteract doxorubicin‐induced cardiotoxicity in murine neonatal ventricular myocytes.2+ oscillations with either RN‐1734  or BAPTA‐AM (to buffer intracellular Ca2+ levels). Of note, repetitive increases in [Ca2+]i represent the most suitable Ca2+ waveform to recruit NF‐κB.Hypo‐induced intracellular Ca2+ oscillations, that is ~9 oscillations/hour or 2.5 mHz, is within the same range as that required to efficiently activate NF‐κB, that is 0.56‐10 mHz.2+ oscillations with RN‐1734 and the downstream recruitment of NF‐κB with thymoquinone potently inhibited hAFS‐CMHypo‐induced ECFC tube formation. This finding is strongly supported by the evidence that multiple pro‐angiogenic genes, for example intercellular adhesion molecule 1, selectin E and various matrix metalloproteinases, are expressed upon the Ca2+‐dependent activation of NF‐κB in circulating ECFCs.2+‐dependent engagement of NF‐κB is likely to play a crucial role in hAFS‐CMHypo‐induced revascularization in murine models of AMIHypo2+ in ECFCs,Endothelial Ca5Hypo induces pro‐angiogenic Ca2+ oscillations in circulating ECFCs, which represent the most suitable cellular substrate to achieve therapeutic angiogenesis in ischaemic disorders. hAFS‐CMHypo promotes TRPV4‐mediated extracellular Ca2+ entry, which thereby results in InsP3‐dependent periodical ER Ca2+ release accompanied by SOCE activation ; Investigation (lead); Methodology ; Writing‐review & editing (supporting). Pawan Faris: Formal analysis (supporting). Carolina Balbi: Investigation . Ambra Costa: Investigation . Sharon Negri: Formal analysis . Vittorio Rosti: Conceptualization ; Investigation ; Validation ; Writing‐review & editing . Sveva Bollini: Conceptualization ; Funding acquisition ; Investigation ; Supervision ; Validation ; Writing‐review & editing . Francesco Moccia: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead); Validation (lead); Writing‐original draft (lead).Supplementary InformationClick here for additional data file.