The cell division cycle 25 (CDC25) phosphatases include CDC25A, CDC25B and CDC25C. These three molecules are important regulators of several steps in the cell cycle, including the activation of various cyclin-dependent kinases (CDKs). CDC25s seem to have a role in the development of several human malignancies, including acute myeloid leukemia (AML); and CDC25 inhibition is therefore considered as a possible anticancer strategy. Firstly, upregulation of CDC25A can enhance cell proliferation and the expression seems to be controlled through PI3K-Akt-mTOR signaling, a pathway possibly mediating chemoresistance in human AML. Loss of CDC25A is also important for the cell cycle arrest caused by differentiation induction of malignant hematopoietic cells. Secondly, high CDC25B expression is associated with resistance against the antiproliferative effect of PI3K-Akt-mTOR inhibitors in primary human AML cells, and inhibition of this isoform seems to reduce AML cell line proliferation through effects on NFκB and p300. Finally, CDC25C seems important for the phenotype of AML cells at least for a subset of patients. Many of the identified CDC25 inhibitors show cross-reactivity among the three CDC25 isoforms. Thus, by using such cross-reactive inhibitors it may become possible to inhibit several molecular events in the regulation of cell cycle progression and even cytoplasmic signaling, including activation of several CDKs, through the use of a single drug. Such combined strategies will probably be an advantage in human cancer treatment. Reversible protein phosphorylation is an important molecular mechanism for the regulation of protein activity; this is true for many proteins that are essential for the regulation of important cellular events like intracellular signaling, cell cycle progression and proliferation, cellular differentiation and induction of apoptosis . ProteinCyclin-dependent kinases (CDKs) are important regulators of cell cycle progression, and CDK inhibition can be accomplished through inhibition of activating phosphatases . Several5R motif typically of tyrosine phosphatases [CDC25 phosphatases are conserved eukaryotic proteins . The thrphatases . The dipphatases . Accordiphatases ,12, the phatases . As depiphatases . It seemphatases . Recent phatases . The regphatases . The domphatases ,19.CDC25 phosphatases function as cell cycle regulators: they activate CDKs by dephosphorylating two residues within the ATP-binding loop . The CDC0-phase. In middle G1-phase, CDC25A activates CDK4 and CDK6 that are in complex with cyclin D [1/S-transition rate-limiting step [1-phase, where it together with CDC25B facilitates the transition into S-phase by dephosphorylation of CDK2/cyclin A, which in turn activates DNA replicating proteins [2-phase [2 to mitosis transition [2/M-transition but CDC25B appears to take the key role in this process [2-phase and during mitosis, and is localized to the centrosome where it dephosphorylates CDK1/cyclin B [The CDK3/cyclin C complex initiates the re-entry of quiescent cells into the cell cycle from Ging step , thus peing step ,28. CDC2ing step ,29. CDC2proteins . CDC25C proteins . At late[2-phase . Dephospansition . Active ansition , is the ansition . All thr process . CDC25B cyclin B ,36. The cyclin B . CBC25B cyclin B . At the cyclin B . It alsocyclin B .Results from mice lacking CDC25B , CDC25C 1/S-phase transition [CDC25 gene transcription, but through Rb recruitment it can also exhibit an inhibitory effect [Because the CDC25s are essential for regulation of the cell cycle, their expression needs to be highly controlled. Dysregulation in the case of CDC25A, for example, accelerates the Gansition , whereasansition . The phoy effect . At the y effect . CDKs ary effect , in addiy effect ,45, whery effect ,47. PLK1y effect . An over1/S checkpoint, DNA synthesis is inhibited, whereas intra-S phase arrest blocks mitotic entry until the S-phase is completed [2/M checkpoint, damaged cells are arrested in order to allow for cell repair or apoptosis [Cell cycle progression can be arrested at three stages: before entry into S-phase, during S-phase and prior to mitosis. At the Gompleted . Finallypoptosis . CDC25s poptosis ,50. Actipoptosis  resultinpoptosis ,52. On tpoptosis ,54, and poptosis ,55. Alsopoptosis ,56. The poptosis , and thepoptosis . PLK1 alpoptosis .In addition to the checkpoint kinases, several other proteins are involved in CDC25 inhibition, for example protein kinase B (PKB/Akt) and mitogen-activated protein kinases (MAPKs). The latter negatively regulate CDC25 upon DNA damage mediated by heat shock, oxidative stress, irradiation, feed deprivation and chemotherapy ,59. The et al. provide a comprehensive overview of the current CDC25 inhibitor development [5R) by reactive oxygen species (ROS) generated in cultured cells treated with quinone derivatives [Over the past few years, several synthetic and natural molecules with different structural features targeting CDC25 activity have been reported. Reviews by Lavecchia elopment ,69. The elopment ,71,72,73elopment ,75 or oxivatives ,77. This50 values in the mid nM range [2/M phase transition of the cell cycle.NSC95397, an unspecific naphthoquinone isoform shows ICnM range , signifi1 and G2/M phases and induces significant growth inhibition of human breast cancer cell lines [NSC 663284 is a potent, cell-permeable, and unspecific irreversible CDC25 inhibitor, arrests cells in the Gll lines .50 = 250, 250, and 170 nM, respectively) and blocks the growth of human pancreatic tumor Mia PaCa-2 cells xenografted into athymic nude mice [®), inhibits the proliferation of colon cancer cells [The quinone-based compound BN 82685 inhibits all three mammalian CDC25 phosphatases in the high nM range in biochemical assays  with micromolar activity by means of a structure-based high-throughput virtual screening [in vitro Ki values for CDC25A and CDC25B of 70 and 80 nM, respectively, generates intracellular ROS in cells, arrests cells in the G0/G1 and G2/M phase transitions of the cell cycle, and significantly suppresses the growth of human MCF-7 breast, PC-3 prostate, and K562 leukemia cancer cell lines.Recently, Lavecchia creening . MechaniRelatively few studies have investigated the role of CDC25 in human AML. The most important studies are presented in detail in i.e., there are only quantitative but not qualitative differences in their activity with regard to inhibition of CDC25A/B/C [CDC25 inhibitors can inhibit AML cell proliferation induced by integrin adhesion to fibronectin ; this inCDC25A. Upregulation of CDC25A enhances lymphoma cell proliferation [feration . Its expferation . Loss offeration . This isferation ; this FLCDC25B. High expression is associated with resistance against the antiproliferative effect of PI3K-Akt-mTOR inhibitors [2/M phase transition delays mitotic entry and inhibits transcription of G2/M-specific genes, including cyclin B, PLK1, and CDC25B [hibitors , but thehibitors ; whereashibitors . These rhibitors . Finallyd CDC25B . NFκB isd CDC25B , but it CDC25C. The molecule is encoded on chromosome 5 and CDC25C seems important for the phenotype of AML cells with chromosomal deletions involving this chromosome [romosome . Malignaromosome .Cell cycle progression is a multistep process regulated by an interacting molecular network, and the CDC25 family is important at different steps. However, the expression or function of several other regulators can also be altered in human AML; leukemia-associated alterations of other important regulators will be described below.1/S-phase progression [1 phase thus seems to differ between patients according to their AML-associated genetic abnormalities.Cells that have exited mitosis can become quiescent, differentiate or progress to another cell division cycle; initiation of a new cell cycle can be stimulated by the downstream signaling from various growth factor or cytokine receptors and at the same time the transcriptional regulation can be altered to suppress differentiation . Howevergression ,107,108.1 can be caused by various molecular mechanisms and not only by CDC25, and several of these molecules are regarded as potential therapeutic targets in human malignancies. The optimal targeting of G1 progression is therefore likely to differ between patients, and the possible therapeutic strategies include p53 activation and inhibition of various receptor tyrosine kinases, CXCR4 and CDK4/6 [CDK2 and CDK4/6 activation in Gd CDK4/6 ,109,110.The regulation of S-phase progression is summarized in 2 phase that ends up with CDC25 mediated activation of CDK1/cyclin B; the described abnormalities of G2 regulation in primary human AML cells are summarized in the table [1 phase, the molecular context of CDC25 varies between patients also for this phase, but in contrast to the G1 phase where the heterogeneity is mainly due to the direct effects of AML-associated genetic abnormalities, the heterogeneity of G2 regulation is mainly determined by differences in expression levels  or isoform profiles .he table ,139,140.CDC25 is not an important mediator in regulation of the mitotic step that includes the spindle assembly checkpoint members together with the Aurora kinases and PLK1 . HoweverThe mitotic checkpoint. Several regulators of the mitotic checkpoint show decreased levels in primary human AML cells and this weakens the mitotic checkpoint response [response ,142,143.Chromatin modeling. 11q23 translocations resulting in a fusion protein with septin cause disturbed chromatin remodeling [modeling . The chrmodeling ,146 therAurora kinases and PLK1. The Aurora kinases [ kinases ,148,149  kinases ,139,150 The t abnormality. This AML-associated abnormality can also disturb this step through downregulation of securin. Furthermore, cyclin B is involved in regulation of mitosis and can be downregulated by the fusion protein encoded by the t fusion gene [ion gene .CDK1 and FLT3 mutations. An alternative pathway for activation of CDK1 is constitutive activation of FLT3-initiated signaling by FLT3-ITD; this signaling can activate both ERK1/2 and CDK1 and these kinases then phosphorylate the transcription factor C/EBPα; this phosphorylation will inhibit its transcriptional activity and thereby contribute to the differentiation block in these AML cells [ML cells ,128,129.ML cells ,151, andML cells . Thus, tThe summaries given above and the data presented in Inhibition of the PI3K-Akt-mTOR pathway is now considered for the treatment of human AML . HoweverOur recent observation that resistance against PI3K/mTOR inhibitors in primary human AML cells was associated with increased mRNA expression of CDC25B also supports the hypothesis that CDC25 inhibition will increase the anticancer effects of PI3K/mTOR inhibition, but at least for AML this combined strategy showed an increased efficiency only for certain patients .To summarize, the PI3K-Akt-mTOR pathway seems to be closely linked to CDC25, especially the CDC25A and CDC25B isoforms in human AML , but ouri.e., to differ between AML cells with no signs of differentiation and cells showing monocytic differentiation [in vitro [in vivo.AML is characterized by accumulation of immature leukemia cells; these cells have a block in their differentiation but for some patients morphological signs of differentiation can be seen . The funntiation . Similarin vitro . Howeveri.e., inv(16), t, t) are defined to have AML even when the number of blasts is <20%. Secondly, the age of the patient seems to reflect biological differences independent of the WHO classification. Thirdly, both the cytogenetic and molecular genetic abnormalities show a much more extensive heterogeneity than reflected in the WHO classification. Finally, a large number of differences at the protein level has also been detected [AML patients are classified according to the recently published WHO classification , and thidetected ,156,157.Targeting of cell cycle regulation is already considered for the treatment of human AML, and especially inhibition of Aurora kinases or PLK1 has been studied in clinical trials as described in detail in a recent review . These cThe 11q23 chromosomal abnormalities involving the H3K4 methyltransferase MLL give rise to an aggressive subtype of AML . A recen1/S and G2/M transitions. Combination of E7070 and CDC25 inhibition would thus represent an extensive inhibition of several steps in the cell cycle together with inhibition of adaptation to the hypoxic bone marrow microenvironment of human AML [The sulfonamide derivative E7070/indisulam ,161,162 uman AML ,164,165.E7070 has been investigated in a recently reported clinical trial . The stuPLK/Aurora kinase inhibitors seem to enhance the antileukemic effect of several cytotoxic drugs used in the clinical treatment of human AML, including cytarabine, daunorubicin and etoposide . Furthertrans-retinoic acid (ATRA) plus valproic acid [One possibility would be to combine CDC25 inhibitors with intensive chemotherapy to increase the antileukemic effect and thereby improve the long-term AML-free survival. However, an alternative strategy could be to combine CDC25 inhibitors with low-toxicity disease-stabilizing strategies in patients that are unfit for the intensive and potentially curative treatment. If this last strategy is considered, CDC25 inhibitors may be combined with all-oic acid , low-dosoic acid , hydroxyoic acid ,170,171.Several side effects have been observed with other pharmacological agents targeting cell cycle regulation . HematolMost CDC25 inhibitors inhibit all three CDC25 isoforms, but the relative strength of inhibition for each of the three isoforms varies between inhibitory agents [CDC25 has its own network of interacting kinases  21,25,2,65,172. As outlined above, CDC25 inhibition can be achieved directly and indirectly through inhibition of its upstream signaling pathways or its interacting molecules. The direct inhibitors have not been investigated in clinical trials  and their pharmacokinetic properties have to be better characterized, but despite this they should be regarded as promising therapeutic agents in human AML because they offer the possibility to target several proteins, including various CDC25 isoforms as well as several kinases, through the administration of single pharmacological agents. Their possible use in AML treatment either as single drugs or in combination with other drugs should therefore be further investigated. The first step will then be a further characterization of the effects at the cellular level and especially in primary human AML cells and this should be followed by studies in AML animal models ,174 as a


During the light response, the Ca2+-free Rcv releases GRK, which in turn phosphorylates visual pigment, ultimately leading to the cessation of the visual transduction cascade. Technological advances over the last decade have contributed significantly to a deeper understanding of Rcv function. These include both biophysical and biochemical approaches that will be discussed in this review article. Furthermore, electrophysiological experiments uncovered additional functions of Rcv, such as regulation of the lifetime of Phosphodiesterase-Transducin complex. Recently, attention has been drawn to different roles in rod and cone photoreceptors.This review article focuses on Rcv binding properties to Ca2+, disc membrane and GRK, and its physiological functions in phototransduction and signal transmission.Recoverin (Rcv) is a low molecular-weight, neuronal calcium sensor (NCS) primarily located in photoreceptor outer segments of the vertebrate retina. Calcium ions (Ca Specialized Ca2+-binding proteins, belonging to the neuronal calcium sensor (NCS) family, detect neuronal Ca2+  spectroscopy  state  state. The Ca2+ induced exposure of the myristoyl group, known as Ca2+ myristoyl switch, allows Rcv binding to the phospholipid membranes only when intracellular Ca2+ concentration ([Ca2+]i) is high  is known as a powerful tool to measure interaction modes of proteins, peptides, and lipids Koch, . The sub2+ and the presence of the myristoyl group  via strain-promoted azide-alkyne cycloaddition. In this way, the membrane translocation of Rcv is visualized in a Ca2+ dependent manner and the membrane with negative spontaneous curvature or higher fluidity shows stronger Rcv binding in the presence of Ca2+.Another useful technique to study the Rcv-membrane interaction is site-specific fluorescent labeling  has been implicated as an internal modulator of Ca2+ sensitivity and therefore may also affect GRK binding  influx across the plasma membrane into the outer segment. This Ca2+ influx is reduced by the closure of the CNG channels during light response, while Ca2+ efflux via Na+/Ca2+ K+ exchanger continues, leading to a decrease in [Ca2+]i in the outer segment. This decline in [Ca2+]i modulates the quenching of the phototransduction cascade, which requires the shutoff of both active intermediates (R* and PDE*) and the resynthesis of cGMP. Under steady background illumination, the [Ca2+]i decline is proportional to the reduction in the circulating current and is crucial for lower photosensitivity and faster response kinetics during this process. What are the targets of Ca2+? The light-induced decrease in [Ca2+]i is postulated to accelerate phosphorylation of R* via Rcv, speed up cGMP synthesis via guanylyl cyclase activating protein (GCAP), and increase cGMP affinity of the CNG channel via calmodulin .Despite the fact that Rcv has been extensively shown to inhibit GRK in a Ca2+ is high, its deletion should result in a shortened lifetime of R* and decrease the gain of signaling pathway. This in turn should lead to lower light sensitivity, without strongly affecting the time course of response decay. However, electrophysiological recordings from Rcv−/− mice showed an accelerated dark-adapted flash response, but only a minor effect on response sensitivity  and the exponential decay constant of the response (τREC) are significantly accelerated in Rcv−/− rods  is slower than R* decay (around 50 ms) and dominates the overall photoresponse recovery in mouse rods .During light adaptation, rod sensitivity is reduced and response recovery is accelerated when background illumination increases, which extends the working range of rods to brighter light levels  may still be involved. Is this mechanism Ca2+ dependent? Background light can still accelerate the response in Rcv−/− rods recorded in low extracellular Ca2+ concentration ([Ca2+]o) which in theory can clamp [Ca2+]i to its maximal light-adapted level, indicating a possible Ca2+-independent process  and the deactivation of PDE*. GTPase-activating proteins (GAPs) accelerate the turn off rate of Transducin. Deletion of GRK in GAPs deficient rods produces little effect on the time course of response, suggesting that the target protein(s) may be involved in the PDE-Transducin pathway  but not saturating light  activity in cones is more than 100 times greater than the range of regulation of rod homolog of GRK (GRK1) activity in rods, suggesting that the phosphorylation mediated by Rcv may contribute more during light adaption in cones  must be at work to regulate the photoresponse, for example via GCAPs , therefore, they presumably work together in a similar way in both cell types. Electroretinography (ERG) on Rcv deficient cones shows not only an accelerated response recovery, but also an around twofold decrease in photosensitivity in darkness, which differs from rods  GRK affinity among Rcvs. Indeed, a recent biochemical work shows different Ca2+ affinity and Ca2+-induced conformational changes among zebrafish Rcv isoforms and bovine Rcv  of Rcv which is independent from GRK in other cellular compartments of photoreceptors.Rcv is expressed in all photoreceptors and also in some subtypes of bipolar cells  and the potential function difference between rod and cone Rcv will need to become a focus of future research.Here, we reviewed the molecular process underlining CaJZ and SN drafted and revised the review article.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.


Francisella tularensis (Ft). While there have been a number of attempts to develop a vaccine for Ft, few candidates have advanced beyond experiments in inbred mice. We report here that a prime-boost strategy with aerosol delivery of recombinant live attenuated candidate Ft S4ΔaroD offers significant protection  in an outbred animal model, New Zealand White rabbits, against aerosol challenge with 248 cfu (11 LD50) of virulent type A Ft SCHU S4. Surviving rabbits given two doses of the attenuated strains by aerosol did not exhibit substantial post-challenge fevers, changes in erythrocyte sedimentation rate or in complete blood counts. At a higher challenge dose , protection was still good with 66% of S4ΔaroD-vaccinated rabbits surviving while 50% of S4ΔguaBA vaccinated rabbits also survived challenge. Pre-challenge plasma IgG titers against Ft SCHU S4 corresponded with survival time after challenge. Western blot analysis found that plasma antibody shifted from predominantly targeting Ft O-antigen after the prime vaccination to other antigens after the boost. These results demonstrate the superior protection conferred by a live attenuated derivative of virulent F. tularensis, particularly when given in an aerosol prime-boost regimen.Tularemia, also known as rabbit fever, is a severe zoonotic disease in humans caused by the gram-negative bacterium  Francisella tularensis, a gram-negative facultative intracellular bacterium [F. tularensis; subsp. tularensis (a.k.a. type A) is the most virulent, followed by subsp. holarctica (a.k.a. type B) and subsps. mediasiactica and novicida which are highly attenuated or avirulent in humans [F. novicida is a subsp of F. tularensis). Tularemia is readily treatable with antibiotics, with an overall case fatality rate less than 2%. Antibiotic resistance, either natural or engineered, is a concern; in the pre-antibiotic era fatality rates reached as high as 30–60% with the pneumonic and septicemic forms of the disease [Tularemia  is a severe zoonotic infection in humans caused by acterium . Under cn humans   of SCHU S4 protected rabbits against respiratory tularemia better than LVS when given as a single vaccination by scarification, although protection was not complete [aroD or S4ΔguaBA would improve survival of rabbits after aerosol SCHU S4 challenge.With the development of systems for genetically manipulating the irulence –34. Somecomplete . Based oaroD by scarification or aerosol and then boosted 14 days later with S4ΔaroD by the alternate route. One group received both prime and boost vaccinations by aerosol. The groups and doses achieved for each vaccination are shown in aroD induced a fever response regardless of route . Mock-vaccinated controls succumbed rapidly to the infection, within 7 days post-exposure all had become moribund and were euthanized (p < 0.0002) protection was seen with 83% of rabbits surviving in the APAB group and 50% of rabbits surviving in both the APSB and SPAB groups when compared with mock-vaccinated and historical controls   and erythrocyte sedimentation rate (ESR) as a measure of the inflammatory response. Bacteremia levels were generally low or non-existent, even in mock-vaccinated rabbits at all time points tested . In mock 0.0001) . The ave 0.0001) . ESR lev 0.0001) . At no p3/µl) (CBC results showed a decrease in WBC at day 4 in the mock-vaccinated rabbits and in the APAB rabbits . This dr3/µl) . ESR and3/µl) .aroD vaccination against a higher challenge dose as well as evaluate whether a similar improvement in protection could be seen with S4ΔguaBA using an APAB regimen, another vaccine that had shown promising results when given once by scarification. In addition, because the initial prime-boost vaccination study used a lower challenge dose (~10 LD50) we included a third APAB vaccine group given S4ΔaroD to evaluate protection against an even higher challenge dose (~300 LD50). Eighteen rabbits were split into three groups of six; one group received S4ΔguaBA while the other two groups received S4ΔaroD. Vaccination doses are shown in guaBA & S4ΔaroD rabbits (and associated controls) were aerosol challenged with SCHU S4 at a median dose of 1,475 cfu (64 LD50) and the higher challenge dose S4ΔaroD group and associated controls received 6,316 cfu (275 LD50). As expected from prior studies, mock-vaccinated rabbits at both challenge doses succumbed within 4–5 days of infection. In the ‘low’ challenge dose groups, 66% that received S4ΔaroD survived challenge while 50% that received S4ΔguaBA survived challenge. At the higher challenge dose, 66% that received S4ΔaroD survived infection. Similar to what we had observed with the first prime-boost study, rabbits in the vaccine groups at either challenge dose had less fever and weight loss post-challenge compared to the mock-vaccinated rabbits (We sought to repeat these promising results for APAB S4Δ rabbits .aroD were significantly higher than mock-vaccinated rabbits (p < 0.0001). Antibody titers to prime-boost vaccination (regardless of route) were also significantly higher than antibody responses to a scarification prime only (aroD or S4ΔguaBA (p < 0.0001) but were not significantly different from one another (p = 0.7413). We also looked at earlier time points for plasma IgG titers induced by APAB S4ΔaroD. At all timepoints post-vaccination, plasma IgG titers were highly significantly different from pre-vaccination IgG titers (p < 0.0001). We then compared whether plasma IgG titers taken 2 days pre-challenge across all vaccine studies . Rabbitsime only . We nextS4ΔguaBA . Plasma < 0.0001). Post-vaion only , 36) cou= 0.5183). It shou= 0.5183).wbtA) Ft LVS revealed that the plasma IgG response to a single vaccination (prime-only) with either S4ΔaroD or S4ΔguaBA is largely directed at the bacterium’s O-antigen and a small subset of proteins most readily observed in an O-Ag Ft mutant, wbtA  with some modest divergence between the S4ΔaroD- and S4ΔguaBA-induced Ab responses (compare reactivity of the prime-boost S4ΔguaBA and S4ΔaroD plasma with the wbtA strain in the ~35 and ~22 kDa range). Identification of these putative, differentially-recognized bands and a more-accurate quantitative and qualitative analysis of these Ab responses is ongoing. The results of our antibody ELISA and western blot analyses suggest that antibody responses to specific Ft antigens may be of value as a potential correlate of protection. We are continuing to investigate this question.Western blot analysis using lysates of wild type (WT) and O-antigen-deficient  survived aerosol challenge with SCHU S4 . We subsequently demonstrated that the protection elicited by LVS vaccination could be improved by delivering the vaccine by either oral gavage or small-particle aerosols [aroD warrants further evaluation as a promising tularemia vaccine candidate.We have previously shown that a single vaccination by scarification with deletion mutants of SCHU S4 outperformed LVS in the outbred rabbit model of lethal aerosol SCHU S4 challenge. In thos50) compared to prior studies. However, mock-vaccinated rabbits did not survive challenge and the time to death for mock-vaccinated rabbits was not significantly different from prior studies with higher challenge doses  to whole, heat-killed SCHU S4 did not survive long. Survival time was extended at higher titers, although titers >3log10 EC50 did not insure survival. IgA titers in plasma did not correspond with survival at all, but it is possible that we would have seen different results had we looked at IgA in mucosal secretions. Data from the literature support a role for both cellular or antibody immune responses in vaccine-mediated protection against F. tularensis. In some studies, antibody titers have been shown to predict protection and passive immunization has provided at least limited protection in animal studies [F. tularensis is a facultative intracellular bacterium yet can have a significant extracellular phase, it seems likely that antibody and cellular immune responses would have complementary roles in a protective response. The results we present here suggest plasma IgG titer may serve as a correlate of protection against tularemia. That does not mean that antibody is the mechanism by which S4ΔaroD (or S4ΔguaBA) immunized rabbits are protected against SCHU S4.While it was expected that a prime-boost vaccination regimen would improve protection, we did not expect that delivering both doses by aerosol would be better than the combinations of scarification and aerosol . We thought that scarification would provide stronger systemic immunity while aerosol delivery would provide stronger immune responses at the site of entry. Aerosol-primed rabbits, regardless of the route of boost, had less morbidity than the scarification primed-group. Rabbits primed with  studies –44. Othe studies , 44, 45.aroD and S4ΔguaBA-vaccinated rabbits suggested that the prime vaccination generated antibody mostly against O-antigen while the boost vaccination resulted in stronger antibody responses against protein antigens. The identity of these protein antigens and their relationship with rabbit survival is an active area of investigation. Analyses of the immunoproteome to F. tularensis have been reported by others in mice and humans [F. tularensis antigens, not whole, heat-killed bacteria, may prove to be a better correlate. This is an area we are actively exploring, as well as evaluating antibody in mucosal tissues, antibody subtypes, and functional antibody responses.A more refined approach, looking at specific antigens rather than a crude, killed preparation of bacteria should provide a better answer on the role of antibody. Western blots of plasma IgG response from S4Δd humans , howeverAnother striking finding was the overall low or non-existent fever and ESR response after challenge in the surviving prime-boost vaccinated rabbits. In our prior study with single vaccinations, all rabbits challenged with SCHU S4 including survivors developed a fever by no later than day 4 post-challenge . In prioIn summary, we have demonstrated here that priming and boosting by aerosol delivery of an attenuated recombinant derivate of SCHU S4 offers strong protection against a virulent Ft aerosol challenge. The level of protection was nearly complete, even at high challenge doses. We would envision pursuing a similar strategy in humans, similar to respiratory delivery of vaccines for influenza or measles –49. AeroF. tularensis was conducted at biosafety level (BSL)-3 in the University of Pittsburgh Regional Biocontainment Laboratory (RBL). For respiratory protection, all personnel wore powered air purifying respirators (3M GVP-1 PAPR with L-series bumpcap) or used a class III biological safety cabinet. Vesphene II SE  was used to disinfect all liquid wastes and surfaces associated with the agent. All solid wastes, used caging, and animal wastes, were steam-sterilized. Animal carcasses were digested via alkaline hydrolysis . The University of Pittsburgh Regional Biocontainment Laboratory is a Registered Entity with the CDC/USDA for work with F. tularensis. The University of Pittsburgh’s Biohazard Committee approved these studies. Work with recombinant Francisella tularensis strains was approved by the University of Pittsburgh’s Institutional Biosafety Committee.All work with live All animal work performed for this publication adhered to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) and the Animal Welfare Act (AWA). All studies were performed under protocols approved by the University of Pittsburgh’s Institutional Animal Care and Use Committee (Protocols: 1105929 & 14053602). The University of Pittsburgh is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Rabbits were monitored at least once daily prior to infection and at least twice daily after infection. For blood collection, rabbits were anesthetized with 2–5% isoflurane and bled from the saphenous vein. Rabbits that were determined to be moribund  were first anesthetized with isoflurane (2–5%) and then euthanized promptly by barbiturate overdose . Five of the forty-two rabbits (12%) in these studies did succumb prior to administration of euthanasia; this was within the 20% anticipated in the approved protocols.Young female New Zealand White (NZW) rabbits  were housed in the University of Pittsburgh Regional Biocontainment Laboratory (RBL) at ABSL-3 for the duration of the studies. Prior to vaccination, IPTT-300 temperature/ID chips  were implanted subcutaneously. Body weight was recorded once in the morning and body temperature was recorded twice daily. Temperature was read using a DAS-7000 reader (BioMedic Data Systems).F. tularensis SCHU S4 was originally obtained from the Dynport Vaccine Company respectively and were stored as single-passage stocks [F. tularensis SCHU S4 ΔaroD and SCHU S4 ΔguaBA, generated as previously described in the laboratories of Dr. Eileen Barry and Dr. Barbara Mann, were shipped to the University of Pittsburgh for these experiments[F. tularensis was grown first on Cysteine Heart Agar (CHA) for two days prior to growing overnight in Brain Heart Infusion (BHI) broth supplemented with ferric pyrophosphate and L-cysteine[periments, 50–52. -cysteine, 54 usin10 cfu/ml were placed in a drop on the shaved area and a bifurcated needle (Becton Dickinson) was jabbed through the drop of bacteria into the skin 17 times. The drop was allowed to absorb into the skin, after which the xylazine was reversed by i.m. injection of 0.2–1 mg/kg yohimibine. The scarification site was monitored daily for the first seven days after vaccination.Rabbits were vaccinated by scarification or by aerosol exposure. For scarification, rabbits were anesthetized by subcutaneous injection of ketamine (80 mg/kg) and xylazine (8 mg/kg); once anesthesia was confirmed a small area of the dorsal surface was shaved. Approximately 0.1 ml of bacteria at a concentration of 1x10F. tularensis were generated inside a class III biological safety cabinet  located inside the RBL as previously described [Aerosols of escribed . Brieflyescribed . Mock-vaF. tularensis SCHU S4. Duplicate wells were used for each dilution. A positive control was included on every plate using plasma drawn on day 28 from a vaccinated rabbit that survived SCHU S4 challenge. After washing with PBS-Tween, secondary goat anti-rabbit IgG-HRP  was added to the plates and incubated for 1 hour at 37°C, after which the plates were washed again with PBS-Tween and BM Chemiluminescence ELISA Substrate  was added to the plates. Plates were then read on an Lmax plate reader . Median effective concentration (EC50) was determined by four-parameter logistical regression of ELISA data using GraphPad Prism 6.ELISA were performed using standard ELISA procedures. Briefly, dilutions of rabbit sera were incubated for 1 hour at 37°C on 96-well plates coated with heat-killed rabbit whole blood collected in EDTA was pipetted using a glass Pasteur pipet into glass Wintrobe tubes. After one hour, the degree of sedimentation was recorded in mm for each rabbit.rabbit whole blood collected in EDTA was analyzed on a VetScan HM2  to determine the white blood cell counts including granulocytes, macrophages, and lymphocytes as well as platelets.Ft protein (~1x108 cells) were mixed with Laemmli sample buffer and boiled for 10 min prior to resolution through 4–12% gradient SDS-PAGE pre-cast gels . The running buffer was NuPAGE MES SDS buffer from Invitrogen; gels were run at 120 V. Resolved gels were stained with either Coomassie blue (BioRad) or transferred to nitrocellulose membranes. Coomassie-stained gels were scanned into Adobe Photoshop using an HP LaserJet Pro 300 color (MFP-m375nw) scanner. Membranes were cut into sections, each containing one lane of WT and one lane of wbtA. Membrane sections were blocked in parallel for 30 min with PBS, 0.05% Tween 20, 5% non-fat dry milk. Rabbit sera  were applied in parallel for overnight incubation at a dilution of 1:1000 in PBS, 0.05% Tween 20. HRP-conjugated goat anti-rabbit secondary Ab (Southern Biotech 4010–05) was used at a dilution of 1:5000 in PBS, 0.05% Tween 20. Development of the chemiluminescent substrate  from the 5 individual membrane sections was visualized simultaneously using an BioRad ChemiDoc Touch Imaging System.Whole cell samples containing 10 μg of Data was collected and organized using spreadsheets in Microsoft Excel 2016; graphing and statistical analyses were conducted using GraphPad Prism 7. Historical controls , 35, 36 S1 FigGraph shows the % of time points post-challenge that were scored as febrile (body temperature ≥ 40oC) for individual rabbits in the different vaccine groups. Individual rabbits that survived challenge are marked with gray triangles while rabbits that succumbed are marked with a gray X. Means and standard deviations are indicated by black bars for each group.(TIF)Click here for additional data file.S2 FigGraphs show bacteremia levels (in cfu/ml of blood) for individual rabbits (gray triangles) with the mean (black center line) and standard deviation (error bars) on different days for rabbits that were A) mock-vaccinated, B) APAB vaccinated with S4ΔaroD, C) APSB vaccinated with S4ΔaroD, D) SPAB vaccinated with S4ΔaroD.(TIF)Click here for additional data file.S3 FigGraphs show WBC for individual rabbits (gray triangles) with the mean (black center line) and standard deviation (error bars) on different days for rabbits that were A) mock-vaccinated, B) APAB vaccinated with S4ΔaroD, C) APSB vaccinated with S4ΔaroD, D) SPAB vaccinated with S4ΔaroD. Mock-vaccinated includes historical controls, shown as open squares.(TIF)Click here for additional data file.S4 FigGraphs show LYM for individual rabbits (gray triangles) with the mean (black center line) and standard deviation (error bars) on different days for rabbits that were A) mock-vaccinated, B) APAB vaccinated with S4ΔaroD, C) APSB vaccinated with S4ΔaroD, D) SPAB vaccinated with S4ΔaroD. Mock-vaccinated includes historical controls, shown as open squares.(TIF)Click here for additional data file.S5 FigGraphs show GRA for individual rabbits (gray triangles) with the mean (black center line) and standard deviation (error bars) on different days for rabbits that were A) mock-vaccinated, B) APAB vaccinated with S4ΔaroD, C) APSB vaccinated with S4ΔaroD, D) SPAB vaccinated with S4ΔaroD. Mock-vaccinated includes historical controls, shown as open squares.(TIF)Click here for additional data file.S6 FigGraphs show MON for individual rabbits (gray triangles) with the mean (black center line) and standard deviation (error bars) on different days for rabbits that were A) mock-vaccinated, B) APAB vaccinated with S4DaroD, C) APSB vaccinated with S4DaroD, D) SPAB vaccinated with S4DaroD. Mock-vaccinated includes historical controls, shown as open squares.(TIF)Click here for additional data file.S7 FigGraphs show PLT for individual rabbits (gray triangles) with the mean (black center line) and standard deviation (error bars) on different days for rabbits that were A) mock-vaccinated, B) APAB vaccinated with S4DaroD, C) APSB vaccinated with S4DaroD, D) SPAB vaccinated with S4DaroD. Mock-vaccinated includes historical controls, shown as open squares.(TIF)Click here for additional data file.S8 FigFigure shows gross pathology in lungs  and spleen  of rabbits at time of necropsy. Representative examples are shown.  mock vaccinated rabbit that succumbed to challenge;  APAB vaccinated rabbit that succumbed to challenge;  APAB vaccinated rabbit that survived challenge;  S4aroD scarification prime only vaccinated rabbit that survived challenge.(JPG)Click here for additional data file.S9 FigRabbits were vaccinated with a prime and boost of S4ΔaroD or S4ΔguaBA by aerosol. Two groups of rabbits received S4ΔaroD in order to evaluate protection against higher challenge doses. Graphs show averaged daily group results (n = 6 per vaccine group and 4 mock-vaccinated controls); error bars indicate standard deviation. Vertical dotted lines indicate days for prime (day 0) and boost (day 14). A) Average fever response after prime (day 0) and boost (day 14) with S4ΔaroD and S4ΔguaBA compared to mock-vaccinated controls. The horizontal dotted line at 40°C indicates the threshold used for fever. B) Average weight change after vaccination with S4ΔaroD or S4ΔguaBA. A horizontal dotted line is shown at 0% weight change to make it easier to see where weight was lost in the vaccine groups.(TIF)Click here for additional data file.S10 FigFigure shows day 28 post-vaccination plasma IgG response to rabbits that received S4DaroD via either scarification prime only (SP) or the different prime-boost regimens; APSB , SPAB , APAB  or mock-vaccinated rabbits. One-way ANOVA was performed to determine significance; * p < 0.05, *** p < 0.05, Δ 0.0001). Includes re-analyzed samples from Stinson et al (2016) and Reed et al (2014).(TIF)Click here for additional data file.S11 FigFigure shows day 28 plasma IgG titer (y axis) in relation to survival after challenge (x axis). One-way ANOVA found no correlation between IgA titer and survival after challenge .(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Acute myeloid leukemia is a hematopoietic neoplasm of dismal prognosis that results from the accumulation of immature myeloid blasts in the bone marrow and the peripheral blood. It is strongly dependent on epigenetic regulation for disease onset, maintenance and in response to treatment. Epigenetic regulation refers to the multiple chemical modifications of DNA or DNA-associated proteins that alter chromatin structure and DNA accessibility in a heritable manner, without changing DNA sequence. Unlike sequence-specific transcription factors, epigenetic regulators do not necessarily bind DNA at consensus sequences, but still achieve reproducible target binding in a manner that is cell and maturation-type specific. A growing body of evidence indicates that epigenetic regulators rely, amongst other factors, on their interaction with untranslated RNA molecules for guidance to particular targets on DNA. Non (protein)-coding RNAs are the most abundant transcriptional products of the coding genome, and comprise several different classes of molecules with unique lengths, conformations and targets. Amongst these, long non-coding RNAs (lncRNAs) are species of 200 bp to >100 K bp in length, that recognize, and bind unique and largely uncharacterized DNA conformations. Some have been shown to bind epigenetic regulators, and thus constitute attractive candidates to mediate epigenetic target specificity. Herein, we postulate that lncRNAs are central players in the unique epigenetic programming of AML and review recent evidence in support of this view. We discuss the value of lncRNAs as putative diagnostic, prognostic and therapeutic targets in myeloid leukemias and indicate novel directions in this exciting research field. The aim of this mini-review is to discuss emerging epigenetic roles of long non-coding RNAs (lncRNAs) in Acute Myeloid Leukemia (AML). AML has a characteristic dependency on chromatin and transcriptional regulators, which has been discussed in many reviews. To date, most of our understanding of epigenetic regulation and its participation in leukemogenesis is based on the enzymatic activities and protein-protein interactions of histone and DNA modifiers. However, a growing body of evidence suggests that recruitment, stability, and function of epigenetic factors can be mediated by non-protein-coding RNAs. In particular, there are incremental examples of the participation of the distinct class of long non-coding RNAs (lncRNAs) in AML epigenetic regulation, some of which have translational potential. In this context, we summarize common epigenetic alterations in AML that are relevant for understanding lncRNA potential contribution to the disease. We define lncRNAs and highlight their general roles of in normal development and in cancer. Finally, we review the developing body of literature on the participation of lncRNAs in epigenetic regulation of AML, and discuss their putative therapeutic relevance. We expect that the ideas advanced in this mini-review will be sedimented and extended in the coming years, thus positioning lncRNAs at the heart of epigenetic regulation and manipulation in AML.de novo after chemotherapy and independently of mutation, and may drive disease progression and relapse in a manner akin to genetic variegation  (NPM1 is the most commonly targeted gene in AML (Epigenetic regulators recurrently mutated in AML include: Histone acetyl-transferases (HATs)—namely location . Histonerognosis . DNA met  . Mutatio (5-hmC) . Other c (5-hmC) . In the e in AML .MLLT1/ENL or MLLT3/AF9, as part of the RNA polymerase (Pol) II super-elongation complex to modify promoters and proximal gene bodies and activate transcription  to repress transcription at specific targets (NPM1 mutation (FLT3-ITD)   that conLT3-ITD) . LSD1, opression , 36. Liknged AML , but hasnged AML . We havenged AML , but it nged AML  and C/EBnged AML , which anged AML . It alsonged AML , 44 and nged AML , and itsnged AML . Specifinged AML –49, and nged AML , 51. On nged AML , 53, as nged AML , depend In summary, AML depends on multiple epigenetic regulators, in genetic abnormality, disease subtype, and disease stage-dependent manners, with and without mutation of the epigenetic regulators themselves. Unsurprisingly, this is an area of intensive research that holds promise in the development of novel anti-leukemia therapeutic strategies.HOX transcript antisense intergenic RNA (HOTAIR), which is transcribed from the HOXC locus in a tissue-specific manner, was found to mediate transcriptional silencing of the HOXD gene locus through direct interaction with the PRC2. This provided the first evidence that non-coding RNAs could regulate epigenetic gene silencing. Subsequently, various other lncRNAs, e.g., Xist, RepA, Kcnq1ot1, Braveheart, or Malat-1, have been shown to interact with PRC2 in a multi-protein-RNA complex  is transcriptionally silenced in various solid cancers by Histone Deacetylase 3 (HDAC3)-mediated promoter inactivation  is regulated by ARID1A, a component of the SWI/SNF chromatin-remodeling complex , and their expression can be modulated by chromatin-modifying complexes. For example, the lncRNA tivation . Also, t complex , and its complex .cis by altering chromatin structure or DNA methylation pattern of neighboring gene promoters. Approximately 40% of all human protein coding genes are co-expressed with a paired natural antisense transcript (NAT), a lncRNA subtype that is expressed from the opposite strand of the host gene ((AChE) gene, which is silenced by AChE-AS lncRNA in hepatocellular carcinoma through triggering of histone methylation  lncRNA family modifies global DNA methylation patterns by protecting host gene promoters from DNMT1-driven de novo methylation after cell division  is dramatically downregulated in lung cancer. Mechanistically, it can silence the HOXB7 oncogene expression by recruiting EZH2 to the core promoter and repressing gene expression through deposition of H3K27me3 , the lncRNA central to X-chromosome inactivation (Wilms' Tumor 1 (WT1) gene result in repression of Maternally Expressed Gene 3 (MEG3) lncRNA expression, a tumor suppressive lncRNA that inhibits tumor growth in a TP53 dependent and independent manner. Inactivation of MEG3 in turn promotes AML initiation  in C/EBPα mutant AML. C/EBPα is a master transcription factor regulator of myeloid cell differentiation, which is recurrently mutated in AML. The mutant truncated p30-isoform of C/EBPα promotes UCA-1 expression, which in turns promotes leukemia proliferation through recruitment of the non-coding RNA-protein complex hnRNP I to repress the cyclin-dependent kinase inhibitor P27Kip1 is a lncRNA located within the HOXA gene cluster (HOXA7-11 genes are central to AML self-renewal programs, namely in the context of MLL rearrangements and NPM1 mutation. Within the locus, HOTTIP activates expression of HOX genes in a candidate positive feedback loop: it guides the WDR5-MLL methyl-transferase complex to the respective promoters, resulting in deposition of the activating H3K4me3 modification (HOTTIP expression leads to increased WBC and neutrophil counts, to a splenomegaly, and an altered self-renewal—differentiation balance of hematopoietic stem and progenitor cells (Locus regulation and downstream interaction with chromatin modifiers further illustrate the epigenetic role of lncRNAs in AML. For example, lncRNA ack loop . One of s in AML . Mechanific loci . In AML,r P27Kip1. HOXA di cluster . HOXA7-1fication . Accordior cells . In summecCEBPA lncRNA directly binds to the DNMT1 protein and protects the CEBPA gene promoter from DNMT1-mediated de novo methylation after cell division. This mechanism tightly controls the transcriptional activity of the CEBPA host gene with promoter specificity; furthermore, it suggests a template for future targeted cancer therapies using lncRNAs as scaffolds to control demethylation of individual hypermethylated targets. In theory, this strategy can increase efficiency and decrease toxicity of untargeted DNA demethylating agents. In addition to demethylating therapies, HDAC inhibitors have also been developed as potential epigenetic therapeutic agents (The field of lncRNAs in cancer, including AML, is in its infancy. However, a growing body of data provides strong evidence for the role of lncRNAs as key mediators and effectors of epigenetic alterations in AML, which will add to more advanced research in the epigenetic field. Indeed, several epigenetic-modifying drugs have either been approved for AML treatment, or are in clinical trials with initial promising results . This noc agents . Howeverc agents . LncRNAsc agents , and, siIn conclusion, preliminary data indicate strong potential for lncRNAs as biomarkers, prognostic factors, and therapeutic targets or mediators in AML, with the ability to individualize therapies, reduce toxicity and enhance treatment success, all of them urgent roles in this disease.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it 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.


Mitochondrial calcium uniporter (mtCU) is a protein complex which consists of the pore-forming subunit (MCU) and several regulatory subunits. MtCU is the main contributor to inward Ca2+ currents through the inner mitochondrial membrane. Extensive investigations of mtCU involvement into normal and pathological molecular pathways started from the moment of discovery of its molecular components. A crucial role of mtCU in the control of these pathways is now recognized in both health and disease. In particular, impairments of mtCU function have been demonstrated for cardiovascular and skeletal muscle-associated pathologies. This review summarizes the current state of knowledge on mtCU structure, regulation, and function in different types of muscle tissues in health and disease.Calcium ions (Ca Mitochondria are widely recognized as one of the key organelles maintaining Ca2+ homeostasis .,18.17,18+ filter ,23,24. Hrmeation . 2+ channel in the mitochondrial inner membrane long before its molecular nature was determined [Xenopus oocytes with the human MCU-EMRE complex targeted to the plasma membrane has been recently established [2+ current with mtCU selective inhibitor Ru360 [MtCU had been identified as a highly selective Catermined ,26,27. Ntermined  and MCU-termined  complexetermined ,31. To oablished . Complexor Ru360 . Althoug2+ kinetics in either a suspension of mitochondria isolated from hepatocytes or the mitochondria in intact hepatocytes and, importantly, accounted for the difference between these two systems [2+ currents with consideration of the mitochondrial heterogeneity: The authors assumed that the total mitochondrial volume might correspond to different numbers of individual mitochondria. This consideration is important because the Ca2+ influx strongly correlates with the level of MCU expression and protein abundance in individual organelles. Despite the availability of the experimental data sufficient to create a computer model of Ca2+ currents via mtCU, many questions about the structure and mutual orientation of the subunits in the complex are still unclear. Next, we will focus on the structural and regulatory features of the human mtCU components.MtCU is better investigated as the assembled complex rather than individual subunits. This circumstance, however, does not interfere with effective simulation of the mtCU activity. Wacquier et al. introduced the computational model based on experimental data, which successfully described Ca systems . In addi2+ or Mg2+) destabilizes the tetramer toward monomer, thus causing inhibition of the mtCU activity in response to Mg2+ and Ca2+ binding [The human MCU monomer consists of 351 amino acids and weighs about 40 kDa. Most of the data on the MCU tetramer structure is obtained by the cryo-EM approach and is particularly related to the structural features of the pore-forming tetramer channel discussed above. In this section, we will consider structural features and the role of extra-membrane parts of the tetramer in the functional activity of the whole complex. The matrix part of the tetramer protrudes 70 Å from the membrane , and has binding .2+ uptake, elevating mitochondrial concentrations of ROS, and enhancing the Ca2+-induced cell death [2+ uptake and mitochondrial transport [Another structural feature of MCU is post-translational modifications which affect the functional activity of the protein. Cys is highly sensitive amino acid incorporated into proteins in the thiol (R-SH) form, which subsequently becomes oxidized or converted into disulfides (R-S-S-R). The post-translational modifications of Cys include disulfide formation, S-glutathionylation, S-nitrosylation, persulfidation, etc . Its proll death . NTD is ransport .2+-modulated electrostatic interaction with MICU1 [2+, MICU1 binds to DIME with two arginines and blocks the intermembrane space entrance of the MCU pore. In the presence of Ca2+, its ions bind to MICU1 and disrupt this interaction, leading to the pore opening [2+ threshold for Ca2+ uptake and cooperativity of the whole mtCU complex [The opposite part of the MCU tetramer faces the intermembrane space and acts as an important site of interaction with the MICU1 regulatory subunit, although whether this interaction occurs inside the membrane or in the intermembrane space is still debatable. As recently shown by Phillips et al., Asp in the MCU’s DIME sequence mediates Cath MICU1 . In the  opening ,37. Impo complex .2+-conductive channel but plays an important role in the regulation of mtCU functional activity by forming heteromers with MCU [2+ currents via the channel [MCUb is a protein of 336 amino acids and has a molecular weight of about 40 kDa (similar to MCU). The sequence identity between MCUb and MCU is 48.77% (according to BLAST). MCUb alone is incapable of forming a Cawith MCU ,40. An i channel .2+ concentrations, MICU1 inhibits the Ca2+ current; at high Ca2+ concentrations, the MCU-binding site in MICU1 is blocked by Ca2+ ions and the pore conductance is restored. MICU1 is able to form a homodimer or heterodimer (by disulfide bonds) with another regulatory subunit—MICU2. Both MICU1 and MICU2 have high affinities for Ca2+ [2+ binding capacity of individual subunits (in the nanomolar range of concentrations) is preserved. As demonstrated by Kamer et al., the MICU1-MICU2 heterodimer selectively binds with liposomes containing cardiolipin; this finding suggests a mechanism for association of the complex with the inner mitochondrial membrane [The regulatory subunit MICU1 consists of 476 amino acids and weighs about 54 kDa. As we have already mentioned, MICU1 binds with MCU through the DIME sequence thus serving as a gatekeeper of mtCU ,41. At lfor Ca2+ . When a membrane .2+-binding proteins. This motif is especially important for overall ion selectivity of the uniporter. Kamer et al. have suggested that specific metal-ligand interactions in EF-hand underlie the ability of MICU1 to differentiate between Ca2+ and Mn2+ [2+ levels, Ca2+ binds to EF hands promoting the channel opening and facilitating the passage of Ca2+ and Mn2+ through the pore. When manganese level is high and Ca2+ is low, Mn2+ binds to the MICU1`s EF-hands without structural changes in the MICU1-MCU interconnection and the pore remains closed. In MICU1 knockout cells (HEK-293T), MCU has no such ion selectivity, which leads to Mn2+ loading in mitochondria. Wettmarshausen et al. came to a similar conclusion: Co-expression of MCU and MICU1 in the yeast S. cerevisiae as a model system protects the cells against the uniporter-dependent Mn2+ overload and Mn2+ toxicity [MICU1 has two structural domains separated by a long alpha helix . Each doand Mn2+ . Accorditoxicity . Drosophila [2+ overload, severe neurological and myopathic defects, similar to that observed in MICU1-deficient patients [In vivo deletion of MICU1 leads to and larval lethality in osophila , while ipatients . MICU1 degradation is Parkin-mediated and occurs in proteasome. Interestingly, it proceeds independently of the E3-ubiquitin ligase activity of Parkin protein but depends on its Ubl-domain . Parkin MICU1.1 is a splicing isoform of MICU1 having an extra micro-exon between exons 5 and 6, which encodes four amino acids and is highly conserved among all vertebrates . MICU1.12+ concentration-dependent manner in two types of dimers: Back-to-back or face-to-face dimers. At low Ca2+ levels, MICU2 forms back-to-back homodimers thus blocking the Ca2+ conduction through the pore, while at high Ca2+ levels both MICU2 and MICU1 form face-to-face dimers which do not interfere with Ca2+ currents [2+ concentrations MICU1 mainly performs the gatekeeping function, while MICU2 modulates its inhibitory effect on mtCU [2+ currents, depletion of MICU2 decreases the levels of both MCU and MICU1 proteins without altering transcription of the corresponding genes [2+ homeostasis persisting in the patients’ cells: The MICU2-deficient cells exhibited mitochondrial Ca2+ overload at resting state and showed a slower bradykinin-stimulated Ca2+ influx than control cells from healthy individuals [MICU2, another gatekeeper, contains 434 amino acids and has a molecular weight of about 50 kDa. Kamer et al.  recentlycurrents . Payne eng genes . MICU2 nng genes . Disruptividuals . 2+ uptake rates. All in all, MICU3 is presently considered as mtCU activator with reduced gatekeeping function [2+ signals in neuronal networks. MICU3 contains 530 amino acids and weighs about 60 kDa. According to UNIProt database, the MICU3 structure also has the EF-hand motif (as in MICU1 and MICU2), and its presence in skeletal muscle has been reported . Howeverfunction . The pre2+ sensor [2+ uptake [2+ pore in open state with β-hairpin in its N-domain [EMRE, also known as SMDT1, is a 107 amino acids protein weighing about 11 kDa. It contains a TMD while its N- and C- terminal domains are exposed to the matrix and intermembrane space, respectively ,55,56. A+ sensor . EMRE is+ sensor . Without+ uptake . It is nN-domain ,56. EMREN-domain . Thus, E2+ uptake; therefore, MCUR1 may be considered as a positive regulator of mtCU complex [2+ import, and increased autophagy in cardiomyocytes and endothelial cells [MCUR1 contains 359 amino acids and weighs about 40 kDa. MCUR1 is a regulatory protein which is often overlooked in the context of mtCU functioning. Orthologs of MCUR1 are found in various organisms, but whether to accept MCUR1 as an independent and separate subunit is still questionable. The published data indicates that MCUR1 deletion significantly impairs Ca complex . The hea complex ,61. Tissal cells .2+ handling, provided by proper mtCU functioning, is essential for the well-being of individual cells and the whole organism. Fine-tuning of mtCU function is carried out at multiple levels, including transcriptional, post-transcriptional, and post-translational levels [2+ signals induce CREB activation through its phosphorylation by various kinases. Damping of Ca2+ oscillations reduces CREB phosphorylation leading to significantly lower MCU expression [2+ oscillations essential for cell differentiation and maturation [The proper mitochondrial Cal levels . Severall levels . Shanmugpression . Recentlturation .2+ was recently reviewed [2+ homeostasis [2+ level in cardiomyocytes during pacing and suppressed the hypertrophic gene expression program. The beta-adrenergic receptor/Akt/FOXO axis was proposed as an upstream regulator of miR-1/MCU pathway [Post-transcriptional regulation of mtCU expression and function by microRNAs (miRs) is not yet fully understood, although the influence of miRs on mitochondrial Careviewed . Remarkareviewed . At the reviewed . In the reviewed . More speostasis . It shoueostasis , and a seostasis . The MCUeostasis , the moseostasis . MiR-1 t pathway . DRP1 pr pathway , indicat2+ pathways meet and regulate each other in mitochondria [2+ uptake and ROS production, and, finally, cell death induced by the Ca2+ overload [At the post-translational level of regulation, mtCU undergoes a variety of modifications including phosphorylation, S-glutathionylation, and methylation. It is now accepted that bioenergetic, oxidative, and Cachondria . For thioverload .2+ uptake via mtCU results in increased generation of ROS as well as calcium overload and initiation of the apoptotic signaling [Phosphorylation of MCU by proline-rich tyrosine kinase 2 (Pyk2) after adrenoceptor stimulation in intact cardiac H9c2 myoblasts has been reported . Adrenocignaling . Similarignaling .2+ uptake, respiration, and ROS production during β-adrenergic stimulation and pacing in cardiomyocytes and isolated cardiac mitochondria from the CaMKII knockout mice. The authors pointed out that the results were inconsistent with any relevant role of CaMKII in the control of mitochondrial Ca2+ uptake [2+ uptake which further activates the CaMKII-mediated mtCU phosphorylation thus forming a positive feedback circuit, and triggers migration of vascular smooth muscle cells (VSMCs) [Although direct phosphorylation of MCU by CaMKII in cardiac muscle was demonstrated, its relevance is still debatable. In 2012, Joiner et al.  publishe+ uptake . It is a+ uptake ,78 stimu (VSMCs) . This ob2+ entry leading to the intensification of mitochondrial respiration and energy production necessary for the proper spindle dynamics [2+ uptake induction and thus contribute to the development of colorectal cancer [Mitosis-specific phosphorylation of MCU by AMPK, necessary for the restoration of ATP levels during cell division, was recently reported . The AMPdynamics . The recl cancer . Zeng etl cancer . 2+ level in the mitochondrial matrix, culminating in the ROS production and tumor progression [The large body of data on the mtCU regulation, obtained from cancer cells and models, are highly relevant for the search of new therapies along with the general understanding of mtCU regulation and functioning. For instance, a recently described Akt kinase-mediated phosphorylation of MICU1 at the N-terminal region in cancer is the first demonstration of a phosphorylation event for the MICU1 subunit . More spgression . A more 2+ and reduced the mitochondrial Ca2+ uptake, UCP2 was shown to bind exclusively to the methylated form and to restore the mitochondrial Ca2+ uptake in the cells with pronounced PRMT1 activity [2+ uptake through the inner mitochondrial membrane were contradictory [In addition to phosphorylation, the MICU1 gatekeeper function is also modulated post-translationally through its methylation by arginine methyl transferase 1 (PRMT1) at Arg455 . Interesactivity . Althougadictory ,85, moreadictory ,87. 2+ leakage into mitochondria through the constitutively active MCU-EMRE subcomplex and the increased rates of Ca2+ uptake [2+ overload followed by mPTP opening and neuronal death in murine model [2+ and the increased Ca2+ retention capacity [Drosophila model caused shortened lifespan, progressive locomotor defects, increased sensitivity to chemical and environmental stress, and muscular and neuronal degeneration [Proper mitochondrial functioning requires the tightly controlled turnover of mitochondrial proteins. In particular, the mtCU assembly dynamics is maintained by m-AAA metalloproteases (ATPases associated with diverse cellular activities) . Mitocho+ uptake . On the ne model . Interescapacity . Considecapacity . At the neration .2+ uptake through mtCU is precisely regulated at multiple levels. Several health disorders associated with disturbances in mtCU functioning and involving different types of muscle tissues are discussed in upcoming sections. Summing up, these data indicate that mitochondrial Ca2+ release/uptake cycles in the cytoplasm and buffering organelles of cardiomyocytes [2+ from the cytoplasm after myocardial contraction is mostly provided by activities of the Na+/Ca2+ exchanger (NCX) in sarcolemma and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), whereas in non-muscle cells the Ca2+ buffering and termination of cytosolic Ca2+ signals mostly depend on the mitochondrial Ca2+ uptake [2+ uptake in cardiomyocytes constitutes less than 1% and the mtCU current is surprisingly low as compared to other tissues [2+ handling in cardiomyocytes were described during the last decade [Main function of the heart is to ensure blood supply to all organs and tissues. To provide it, myocardium undergoes constant rhythmic cycles of excitation followed by contraction (excitation–contraction coupling) based on the permanent Camyocytes . Interes+ uptake . Althoug tissues ,94, it i tissues . Preciset decade .After determination of the mtCU molecular identity, its role in the cardiac muscle has been widely investigated by using genetic manipulations. The whole-body MCU KO mice displayed unaffected cardiac function both under basal and stress conditions . On the 2+ entry through the L-type Ca2+-channels in cardiomyocytes induces Ca2+ delivery from the sarcoplasmic reticulum (SR) ryanodine receptors (RyR2) leading to formation of Ca2+ microdomains nearby the SR-mitochondria junction and further Ca2+ signal transduction into mitochondria via mtCU [2+ microdomains in cardiomyocytes is not yet fully understood. Indeed, mtCU was initially reported to be homogeneously distributed in the mitochondrial inner membrane, as observed in intact ventricular cardiomyocytes of adult rabbit [2+ transport [2+ efflux via NCLX from the mitochondrial Ca2+ uptake via mtCU [2+ extrusion at the most Ca2+-exposed area serves for optimization of the Ca2+ signaling efficiency and minimization of the energy costs [Cavia mtCU . It shout rabbit . A more ransport . Along tvia mtCU . The lacgy costs . The con2+ concentration in adult ventricular cardiomyocytes during excitation–contraction coupling is also debatable [2+ dynamics in cardiac mitochondria [2+ concentration in cardiac mitochondria oscillates in a beat-to-beat manner, whereas the second model assumes gradual mitochondrial Ca2+ uptake. The authors emphasize that the mitochondrial Ca2+ dynamics, which may depend on the animal species and pacing frequency, needs further investigation [The kinetics of mitochondrial Caebatable . Cao et chondria . Accorditigation . 2+ microdomains and SR maturation [Myocardium hypertrophy can be defined as an increase in the myocardial mass. It is well known, that in mammals the majority of cardiomyocytes lose the ability to proliferate shortly after the birth, and the subsequent heart growth proceeds mostly by an increase in the cardiomyocyte cell size. This is true under both normal physiological  and pathological  conditions . Physiolturation . Howeverturation . Increasturation . The incturation . Therefo2+ homeostasis, reliably compensated in the case of congenital defects in corresponding genes.As expected, the mtCU inhibition under the overload-induced hypertrophy improved the cardiac function and mitigated the pathological changes . On the Arrhythmias represent one of the primary causes of sudden cardiac death and a severe complication after acute ischemia and myocardial infarction ,105. 2+ handling [2+ uptake via mtCU modulates the spontaneous electrical activity of ventricular-like cardiomyocytes derived from mouse embryonic stem cells, together with RyR2 and IP3Rs at sarcoplasmic reticulum, while the plasmalemmal Ca-L-channels and NCX ensure the cell automaticity [2+ accumulation and spontaneous premature ventricular contractions [2+-uptake after the release of Ca2+ from SR and eliminated the arrhythmogenic Ca2+ waves induced by catecholaminergic stimulation in the CPVT murine cardiomyocytes [Several heart diseases, including nonischemic heart failure and myocardial ischemia, are associated with increased arrhythmia risks and triggered activity. The ischemia-induced abnormalities in the ventricular automaticity are supposed to cause arrhythmias and attributed to the disturbed intracellular and, in particular, mitochondrial Cahandling . Mitochomaticity . The beamaticity , as wellmaticity , were suractions . Consistractions . In contractions . Indeed,myocytes . These c2+ handling by mtCU is likely to provide a versatile mechanism for adjusting the electrical activity of pacemaker cells, the attempts to modulate it with pharmacological agents may lead to adverse consequences and should be studied in more detail. Therefore, although the regulation of mitochondrial CaUcp2−/− mice have more pronounced susceptibility to the Ca2+ overload-induced arrhythmias and increased PRMT1 protein levels, which may be responsible for the decreased mitochondrial Ca2+-uptake in the Ucp2−/− hearts [Disturbances in functioning of the mtCU regulators are also associated with the development of arrhythmias. For instance, − hearts . It is i− hearts . Althoug− hearts ,114, the2+ mishandling in cardiomyocytes during ischemia/reperfusion injury is well documented [2+ overload-induced ROS generation and mPTP opening.Ischemic heart disease remains a major factor of death and disability worldwide , and thecumented . The carMcu deletion in the adult heart does not compromise the myocardial energy production and contractile function due to a compensatory increase in the fatty acid oxidation both under basal conditions and during the acute adrenergic challenge [2+ overload and acute ischemia-reperfusion injury [2+ overload, mPTP opening and cardiomyocyte death activation after hypoxia-reoxygenation [2+ associated with the increased phosphorylation of cyclophilin D at Ser42 were observed in the hearts of conventional (whole-body) MCU KO mice, while the receptor interacting protein (RIP3) kinase-activated cell death pathway had no significant contribution to ischemia/reperfusion injury [The role of mtCU in the cardiomyocyte cell death was assessed on different genetically modified models. In particular, an inducible tissue-specific hallenge . Moreoven injury ,96. In lgenation . On the genation . These cgenation . Indeed,n injury . 2+ overload with consequent ATP depletion and morphological defects [Conductance of mtCU channel is tightly regulated by auxiliary subunits; therefore, the resistance of cardiomyocytes to ischemia may be affected by alterations in either subunits expression or their association with MCU to form the whole mtCU complex. Indeed, the MICU1 protein content in mitochondria was significantly reduced following ischemia/reperfusion, despite the unchanged total MICU1 content, due to the inhibited expression of translocase of outer membrane 70 (Tom70) leading to the impaired MICU1 import into mitochondria . Further defects . 2+-dependent regulation of oxidative metabolism is widely accepted as a key factor in its pathogenesis [2+ concentrations resulting from decreased SERCA expression or activity, along with Ca2+ leakage via RyR2, may lead to mitochondrial Ca2+ overload [2+ entry in cardiomyocytes could be compensated by changes in cytoplasmic Ca2+ homeostasis manifested as unchanged oxygen consumption by the isolated cardiac mitochondria [Heart failure is reported to be the prevalent reason for hospital admissions in some developed countries, and the imbalanced Caogenesis ,123. In overload . Initialoverload . At the overload . The lacchondria . Intereschondria .2+ influx into the mitochondrial intermembrane space during fission [Heart failure progression is accompanied by an increase in the myocardial MCU protein content reaching the highest point during the compensatory hypertrophic phase. The MCU upregulation is accompanied by autophagy blockade, as indicated by an increase in the p62 (SQSTM1) protein content resulting from its reduced degradation . Interes fission . Althoug fission , furtherMcu−/− mice, which show a mild phenotype, the Micu2−/− mice have increased left atrium diameter resulting from the retarded cardiomyocyte relaxation and consequent diastolic dysfunction [2+ handling under pathological conditions. For instance, miR-1 is being extensively studied as a potential therapeutic target, given its contribution to cardiac hypertrophy [In contrast to function . These dfunction , emphasiertrophy  and remoertrophy  and its ertrophy , whereasertrophy . Neverthertrophy , contradertrophy ,31,77 do2+ may also be associated with the disturbed Ca2+ efflux from mitochondria. The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis, as its inducible deletion in mice leads to sudden death caused by myocardial dysfunction and heart failure [The imbalance in mitochondrial Ca failure . The cor2+ concentration in the sarcoplasm is essential for skeletal muscle contraction [The major role of skeletal muscle is the conversion of chemical energy into mechanical movements necessary for the realization of many vital functions ; an incrtraction . 2+ handling is crucial for the adaptation of energy production to the immediate energy demands of the muscle, and also provides a link between excitation and transcription, the mtCU function in skeletal muscle is widely investigated [Mcu depletion on body weight, muscle weightm and fiber size regardless of the age of gene loss induction [2+ level. Whereas the basal mitochondrial Ca2+ concentrations were substantially reduced in the whole-body KO and adult muscle-specific Mcu depletion models [2+ uptake in skeletal muscle in response to different stimuli [Mcu depletion may also depend on the experimental approach. For instance, impaired performance of the animals in the intense muscle exercise tests was demonstrated for the whole-body [Mcu deletion [Since mitochondrial Castigated . Large bstigated ; these fstigated ,132 and stigated . Howevernduction . Some din models ,133, in n models . Neverth stimuli . A metab stimuli . Consist stimuli ,133. Intdeletion ; after adeletion .Mcu deletion in skeletal muscle tissue not only affects muscle metabolism, but also triggers systemic metabolic adaptations [Mcu deletions. At the same time, reduced blood levels of glucose were accompanied by increased glucose uptake in muscles. Taken together, these observations indicate systemic metabolic adaptations allowing the liver to counteract excessive muscle glucose uptake and maintain euglycemia [ptations . Indeed,glycemia . 2+ directs the conversion of nutrient energy into ATP by activating the Ca2+-dependent dehydrogenases of the tricarboxylic acid cycle, regulation of mitochondrial Ca2+ uptake is a crucial point for the proper muscular contractions [2+ uptake at much lower cytosolic Ca2+ concentrations than conventional MICU1-MICU2 does. At low Ca2+ concentrations, MICU1.1 is also capable of gatekeeping. The effect is explained by the increased Ca2+ affinity of MICU1.1, which is an order of magnitude higher than that of MICU1, as estimated by isothermal titration calorimetry [2+ binding affinity by MICU1.1 extra exon remains obscure. Therefore, in this case alternative splicing works as a means of adaptation of mitochondrial Ca2+ uptake to energetic demands of a particular tissue.Skeletal muscle tissue is one of the top energy consumers in the body and therefore has special needs of rapid and tightly controlled energy production. Since mitochondrial Caractions . An alteorimetry . The exa2+ uptake at low cytosolic Ca2+ concentrations and the damping of cytosolic Ca2+ signals due to increased mitochondrial Ca2+ buffering [2+ efflux via NLCX, counteracting the increased mitochondrial Ca2+ uptake [2+ uptake is also substantially impaired in the cells derived from these patients [MtCU dysfunction associated with MICU1 intronic insertions causing frameshifts and leading to significant downregulation of MICU1 mRNA levels has been identified. Clinical phenotype of these patients comprising proximal muscle weakness, learning difficulties and progressive extrapyramidal movement disorder . In accouffering . Moreoveuffering  in the Muffering ) result + uptake . As a re+ uptake . In line+ uptake . Mitochopatients . Surprispatients ,137; thipatients . Anotherpatients , previoupatients . The autpatients . Overall2+ uptake in skeletal muscles following muscle injury has also been demonstrated [2+ activates mitochondrial ROS production which, in turn, facilitates the myocyte membrane repair by local activation of RhoA and triggering F-actin accumulation at the site of injury. Both mtCU inhibition and ROS quenching compromise the plasma membrane repair. Interestingly, this mechanism is common for myoblasts, mature skeletal myofibers, and nonmuscle cells [The beneficial role of mtCU-dependent mitochondrial Canstrated . Specifile cells . These f2+ overload, myocyte cell death, and skeletal muscle atrophy [Drp1 inhibition downregulates miR1, leading to increased MCU protein levels. Simultaneously, MCU inhibition normalizes Ca2+ handling and ameliorates myofiber survival [2+ balance, achieved by proper regulation of mtCU activity, for normal skeletal muscle functioning and prevention of muscular atrophy.Since mtCU has been characterized as a trophic factor in skeletal muscle, therapeutic approaches leading to increased mtCU expression or activity should be considered for the treatment of various atrophic muscle diseases, for example, age-related sarcopenia. Leg press exercise training and neuromuscular electrical stimulation of the anterior thigh quadriceps muscles beneficially affected the structure and function of the muscle tissue in 70-year-old sedentary volunteers . In deta atrophy . This efsurvival . AltogetSPG7 in Drosophila lead to multiple degenerative changes, including skeletal muscle degeneration accompanied by accumulation of morphologically and functionally abnormal mitochondria [MtCU-associated function disturbances may be associated not only with structural and functional impairments of the complex itself, but also with its abnormal regulation. In particular, null mutations in the m-AAA protease chondria . AlthougThe role of mtCU in smooth muscle cells has been mainly investigated in terms of vascular smooth muscle physiology and dysfunction, since VSMCs are crucial players in the pathophysiology of hypertension, which contributes to the development of heart failure, myocardial infarction, stroke and other severe disorders . For ins2+ uptake via mtCU is essential for mitochondrial mobility and the isolated aortic VSMCs migration during neointima formation, with CaMKII as an upstream regulator of this process [VSMC plasticity (the capability of the contractile-to-synthetic shift in phenotype) is pivotal for the vascular wall repair during the phase of cell migration and proliferation following injury. By contrast, if the vascular function is impaired chronically, as occurs in atherosclerosis and neointima formation, VSMC plasticity contributes to the pathology progression . Mitocho process . The CaM process  suggesti2+ in combination with increased content of cytosolic Ca2+ which contribute to PASMC proliferation and migration. PASMC Ca2+ disturbances are attributed to MCU downregulation and coordinated reciprocal upregulation of MICU1 resulting in the impaired mtCU function. Along the same line, inhibition of mtCU in normal PASMCs reproduced the pulmonary artery hypertension phenotype [2+ handling was also identified for maternally-inherited hypertension associated with a mutation in mitochondrial DNA [2+ in combination with the highest cytoplasmic Ca2+ in lymphoblastoid cell lines derived from hypertensive patients carrying the mitochondrial DNA mutation in comparison with both hypertensive patients without the mutation and normotensive carriers of the mutation. The decreased MCU expression was characteristic of the cells from hypertensive patients both with mutation and without it [The excessive artery smooth muscle cells proliferation, migration, and apoptosis resistance are also typical of pulmonary arterial hypertension . In conthenotype . Disturbrial DNA . In partthout it . UnfortuMicu2-depleted mice, led to a progressive increase in the aortic diameter and eventually to aneurysms in some animals, although the initial aortic diameter in Micu2−/− mice was only slightly larger than in normal animals. Interestingly, this effect was specific for angiotensin II and was not reproduced by the treatment of Micu2−/− animals with norepinephrine. Transcriptional profiling of aortic VSMCs revealed dysregulation including proinflammatory signature, increased extracellular matrix remodeling, and cell proliferation [The hypertension, induced by chronic infusion of angiotensin II in the feration . Therefo2+ mishandling in smooth muscle tissue is typical for a number of diseases. For instance, hyper-responsiveness of the airway smooth muscle in asthma results from the elevated cytoplasmic Ca2+ very probably associated with dysregulated mitochondrial Ca2+ buffering [2+ buffering of cytosolic Ca2+ elevations in the histamine-stimulated airway smooth muscle cells [2+ buffering in shaping of cytoplasmic Ca2+ responses. Interestingly, the observed differential responses of perinuclear and peripheral mitochondrial populations indicate the tight control of local Ca2+ concentrations and adaptation to specific energy demands in different cellular compartments [The data concerning mtCU function in the smooth muscle from non-vascular locations are extremely scarce and indirect, although the mitochondrial Cauffering . Inhibitle cells , suggestartments . Expressartments . The oppartments . Overall2+ influx to mitochondria, and its contribution to pathophysiology has been demonstrated for a number of cardiovascular and skeletal muscle diseases. Therefore, modulation of mtCU expression and activity represents a promising molecular target. Ruthenium red and its derivatives are usually used for inhibition of mtCU in the experiments. However, ruthenium red, along with the inhibition of mtCU, has nonspecific activity against some other ion channels [2+ overload and increases cardiac contractility without affecting heart rate [2+-triggered arrhythmias [MtCU is the major route for the Cachannels , which pchannels . Two celchannels  and ruthchannels . DS16570art rate . Ru265 iart rate . Anti-caart rate . MtCU acart rate  and cardart rate  propertihythmias . Even th2+ uptake via mtCU is a crucial process for the life of a cell. The controllable influx of Ca2+ ensures proper energy production and its adaptation to the immediate needs in the tissues with high energy demands, such as cardiac and skeletal muscle tissues. In addition to the energy production, mitochondrial Ca2+ regulates a variety of myocyte functions including trophism, cell division, and cell death. MtCU expression and activity are tightly regulated by the number of intracellular signaling pathways, with some peculiarities in different types of muscle tissues. Disruptions in mitochondrial Ca2+ handling significantly increase the risks of cell and tissue dysfunction and ultimately result in the development of different pathologies. Cardiovascular diseases, currently known as one of the leading causes of death, and some skeletal muscle pathologies are shown to be associated with mtCU dysfunction. Therefore, a better understanding of fundamental mechanisms of mtCU function and dysfunction in muscle tissues may be useful for the future development of new modulators of mtCU activity and perspective therapy approaches.The mitochondrial Ca