The ability of Ca2+ to bind to proteins to alter their charge and conformation is essential to achieve its signaling role. Cytosolic Ca2+ (cCa2+) concentration is maintained low at ~100 nM so that the impact of elevations in cCa2+ is readily sensed and transduced by cells. However, such elevations in cCa2+ must be transient to prevent detrimental effects. Cells have developed a variety of systems to rapidly clear the excess of cCa2+ including Ca2+ pumps, exchangers and sequestering Ca2+ within intracellular organelles. This Ca2+ signaling toolkit is evolutionarily adapted so that each cell, tissue, and organ can fulfill its biological function optimally. One of the most specialized cells in mammals are the enamel forming cells, the ameloblasts, which also handle large quantities of Ca2+. The end goal of ameloblasts is to synthesize, secrete and mineralize a unique proteinaceous matrix without the benefit of remodeling or repair mechanisms. Ca2+ uptake into ameloblasts is mainly regulated by the store operated Ca2+ entry (SOCE) before it is transported across the polarized ameloblasts to reach the insulated enamel space. Here we review the ameloblasts Ca2+ signaling toolkit and address how the common electronegative non-metal fluoride can alter its function, potentially addressing the biology of dental fluorosis.Most cells use calcium (Ca The chemical properties of Ca2+, its radius (0.99 Å), hydration energy and charge, are such that they facilitate its role as a signaling messenger in cells favored over other cations, such as magnesium (Mg2+), that have a smaller radius and higher hydration energy  concentration is maintained low at ~100 nM compared to ~2 mM outside the cell and therefore, small changes in cCa2+ have important effects  and mitochondria as Ca2+ sinks  entry, b) transit and c) extrusion. For a very long time, the molecular pathways regulating these steps in ameloblasts had remained undefined. Recently, several studies of human mutations and rodent models indicate that the store operated Ca2+ entry (SOCE) pathway is essential for enamel mineralization, being the primary Ca2+ uptake systems in ameloblasts  entrloblasts –13.2+ release activated Ca2+ (CRAC) channels, is an essential and widely expressed Ca2+ influx channel to the extent that SOCE dominates the ability of non-excitable cells to uptake Ca2+ in physiological and pathological conditions  found in the membrane of the ER, and the channel pore formed by the ORAI proteins in the plasma membrane (ORAI1-3) , muscle weakness and anhidrosis  –16. CRAC1-3)  synthesis , may be an important factor in SOCE modulation in ameloblasts. SARAF  is an ER membrane protein that associates with STIM to promote Ca of SOCE . We founloblasts , suggestTrpm7-inactive knock-in mutant mice which showed hypomineralized enamel in the heterozygous mice  linked to an intrinsic kinase domain, enabling it to modulate cellular functions  but have low transport capacity, pumping 1 Ca2+ per ATP consumed  are expressed in enamel cells, appearing to be upregulated during the secretory stage (Plasma membrane Ca2+-ATPases (PMCAs), or Ca process . PMCAs hconsumed , 46. PMCry stage . However2+ exchangers are also important in removing cCa2+ out of the cell and unlike PMCA, the exchangers do not require ATP hydrolysis. Instead, the Na+/Ca2+ exchangers use the chemical energy of the Na+ gradient (the Na+ concentration is much higher outside of the cell than inside the cell) to remove Ca2+ from the cytosol , which is their physiological function, as well as, in a reverse mode (Ca2+ uptake)  .2+ exchangers, the NCX and NCKX. The NCX are Na+/Ca2+ exchangers in the plasma membrane, coded by the SLC8A genes, that remove 1 Ca2+ in exchange for 3 Na+  in humans and mouse models results in enamel defects  , providing the best balance of protection from dental caries while limiting the risk of dental and/or skeletal fluorosis. At this concentration, fluoride has a positive effect on enamel strengthening chemical bonds in the formed enamel crystals, decreasing the risk of caries once the enamel crown has erupted –64. Excese (UPR) –69. Thisse (UPR) . Interesse (UPR) .via diffusion as hydrogen fluoride (HF), as reported in other cells  than rats drinking the same fluoridated water but without receiving a Ca2+ supplement, strongly suggesting that Ca2+ has a protective role in fluorosis toxicity   , 75. Thein vitro showed decreased internal Ca2+ in the ER and SOCE   awards (DE025639 and DE027679) to RL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, the exact mechanisms for this remained unspecified so far. This study shows that Ca2+/calmodulin‐dependent protein kinase II (CaMKII)‐specific phosphorylation of ryanodine receptor type 2 at Ser‐2814 is the pivotal mechanism by which SR Ca2+ leak develops downstream of β1‐adrenergic stress by increase of the leak/load relationship. Cardiomyocytes with a Ser‐2814 phosphoresistant mutation (S2814A) were protected from isoproterenol‐induced SR Ca2+ leak and consequently displayed improved postrest potentiation of systolic Ca2+ release under adrenergic stress compared to littermate wild‐type cells.Adrenergic stimulation, while being the central mechanism of cardiac positive inotropy, is a universally acknowledged inductor of undesirable sarcoplasmic reticulum (SR) Ca 2+/calmodulin‐dependent protein kinase II‐specific phosphorylation of ryanodine receptor type 2 at Ser‐2814 is the pivotal mechanism by which sarcoplasmic reticulum Ca2+ leak develops downstream of β1‐adrenergic stress by increase of the leak/load relationship.This study shows that Ca A/AlaalanineCaMKII2+/calmodulin‐dependent protein kinase IICaISOisoproterenolNTnormal Tyrode’s solutionPKAprotein kinase APLBphospholambanRyR2ryanodine receptor type 2S/SerserineSERCA2a2+ ATPase type 2asarcoplasmic reticulum CaSRsarcoplasmic reticulumWTwild‐type2+ leak  from 0.1 to 1.4 mmol·L−1, cardiomyocytes were plated onto superfusion chambers, which had been coated with laminin to allow cell adhesion.Isolation of cardiomyocytes was performed as described previously . Briefly2+ measurements were performed as described previously  as well as the positive lusitropic acceleration of Ca2+‐transient decay  are strong in both genotypes and not different between genotypes , the S2814A mutation had no effect on tetracaine‐sensitive SR Ca2+ leak . As expected, β1‐adrenergic stimulation with ISO strongly increased the SR Ca2+ leak in WT cardiomyocytes . In S2814A mice, however, ISO stress did not lead to a significant increase of basal SR Ca2+ leak  and these mice were, consequently, protected from ISO‐induced SR Ca2+ leak as compared to WT  = 17; n(WT‐NT) = 18, P < 0.05], implying increased ‘leakiness’ of the RyR2 upon this adrenergic stimulation, S2814A cells were completely protected from this worsening of leak/load relationship under β1‐stress , suggesting that the extensively increased SR Ca2+ load during ISO stimulation has already reached its maximum. However, while in S2814A mice postrest potentiation was also somewhat lower under β1‐adrenergic stress compared to basal conditions , it was significantly improved compared to WT cells (P < 0.05)  Fig. . Thus, pIn order to look into phosphorylation of RyR2 at Ser‐2808 and Ser‐2814 under β‐adrenergic stress and to exclude off‐target effects of the S2814A mutation on Ser‐2808 phosphorylation, we performed western blots in WT and S2814A cells with or without ISO incubation. We can report that the S2814A mutation indeed completely prevents RyR2 Ser‐2814 phosphorylation also under adrenergic stress Fig. . In cont2+ leak in cardiomyocytes. Phosphoresistance at this site almost prevented ISO‐induced SR Ca2+ leak and increase of leak/load relationship. Furthermore, as SR Ca2+ leak and leak/load relationship were not different between genotypes under basal conditions, it appears that Ser‐2814 only becomes relevant for SR Ca2+ leak upon (phosphorylation) challenge such as under adrenergic stress.In conclusion, our study defines for the first time phosphorylation of RyR2 at the CaMKII‐specific RyR2 Ser‐2814 phosphorylation site as the pivotal mechanism mediating β1‐adrenergic induced SR CaThe authors declare no conflict of interest.SN conceived and supervised the study. MJB, JN, and MTS designed and performed experiments and analyzed data. MJB, LSM, and SN interpreted data for the work. MJB and JN wrote the manuscript. MTS, LSM, and SN made manuscript revisions.


The UPRmt-mediated transcriptional program requires the participation of various upstream signaling pathways and molecules. The factors regulating the UPRmt in Caenorhabditis elegans (C. elegans) and mammals are both similar and different. Cancer cells, as malignant cells with uncontrolled proliferation, are exposed to various challenges from endogenous and exogenous stresses. Therefore, in cancer cells, the UPRmt is hijacked and exploited for the repair of mitochondria and the promotion of tumor growth, invasion and metastasis. In this review, we systematically introduce the inducers of UPRmt, the biological processes in which UPRmt participates, the mechanisms regulating the UPRmt in C. elegans and mammals, cross-tissue signal transduction of the UPRmt and the roles of the UPRmt in promoting cancer initiation and progression. Disrupting proteostasis in cancer cells by targeting UPRmt constitutes a novel anticancer therapeutic strategy.The mitochondrial unfolded protein response (UPR UPRmt induces a transcriptional program involving numerous genes through retrograde mitochondrial-to-nucleus communication .–66.mt [mall HSPs . Therefo2+ transfer from the endoplasmic reticulum (ER) to mitochondria is the main source of mitochondrial Ca2+, which is mediated by mitochondria-associated membranes (MAMs). Ca2+ regulation in mitochondria is involved in various biological processes, including ATP synthesis and metabolism [2+ buffering, and dysregulation of UPRmt-related proteins lead to neuronal decline during aging [2+ is transported to mitochondria through the MAM, which ultimately leads to increased expression of the mitochondrial protease LONP1 [2+ signaling and UPRmt has not been elucidated. The potential crosstalk between Ca2+ regulation in mitochondria and UPRmt urgently needs to be explored.Calcium, as a key intracellular second messenger, plays a pivotal role in many physiological and pathological processes. Catabolism , 70. Preng aging . Under Ese LONP1 , 73. Recse LONP1 . Howevermt and mitophagy. UPRmt and mitophagy are two major axes that maintain mitochondrial proteostasis [mt facilitates the recovery and survival of cells by inducing the expression of mitochondrial chaperones, proteases, antioxidant genes and protein import and assembly factors. When damaged mitochondria cannot be accurately repaired by a small-scale stress response, such as UPRmt, dysfunctional mitochondria can be removed by mitophagy. Mitophagy targets damaged mitochondria through autophagosome phagocytosis and lysosomal degradation to ensure that they are cleared before they become toxic to cells [mt appears to be activated prior to mitophagy. Inconsistent with this conclusion, a recent study revealed that UPRmt was a downstream pathway of mitophagy [mt and mitophagy were slightly activated in LPS-treated cardiomyocytes to sustain mitochondrial function. Treatment with urolithin A, an inducer of mitophagy, significantly reduces sepsis-mediated heart injury by restoring mitochondrial function without influencing the UPRmt [mt-related genes in LPS-treated mouse hearts [mt by oligomycin effectively alleviated sepsis-induced mitochondrial damage and myocardial dysfunction. This cardioprotective effect was not evident in FUNDC1 CKO mice [mt was suppressed, mitophagy-mediated cardiac protection was partially attenuated [mt and mitophagy are important quality control mechanisms. However, the precise molecular mechanism by which mitophagy modulates UPRmt has not been clarified. Whether other classic mitophagy signaling pathways and regulatory proteins mediate UPRmt also needs to be further verified by in vivo and in vitro experiments.Mitochondria are the first-line defense of cells under various stresses. Dysfunctional mitochondria can be repaired by a complex set of adaptive responses, such as mitochondrial biogenesis, mitochondrial fission/fusion, UPReostasis . As discto cells –78. The to cells –78. Theritophagy , 80. Botmt. In mammalian cells, ISR assists in the regulation of UPRmt, and its molecular mechanisms depend on the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), thereby suppressing the ability of eIF2 to transmit methionylated initiator transfer RNA (Met-tRNAi) to ribosomes [S. cerevisiae. GCN-2-dependent phosphorylation of eIF2α is necessary for the development and extension of lifespan in C. elegans. Depletion of GCN-2 could significantly upregulate the expression of mitochondrial chaperones to activate UPRmt, suggesting that GCN-2-dependent translation attenuation is a parallel signaling pathway to maintain mitochondrial proteostasis during mitochondrial stress. In mammalian cells, four kinases have been identified to phosphorylate eIF2α, including GCN2, PKR-like endoplasmic reticulum kinase (PERK), protein kinase R (PKR), and heme-regulated inhibitor (HRI), in response to different forms of cellular stress [mt. The accumulation of unfolded proteins in the mitochondrial matrix of mammalian cells leads to CHOP-dependent transcriptional upregulation of mitochondrial chaperones and proteases but not ER stress proteins [mt process [mt-related genes during mitochondrial stress. Interestingly, overexpression of ATF5, but not ATF4, in worms lacking ATFS-1 restored the ability to induce the expression of HSP60 [mt through the specific activation of CHOP, ATF4 and ATF5.The ISR is also an evolutionarily conserved cellular signaling pathway that facilitates cells and tissues to cope with various stresses, such as ER stress, heme deficiency, amino acid starvation, viral infection and hypoxia –83. A laibosomes . The ancr stress , 85. GCNr stress . HRI actr stress , 85, 87.r stress , 89. In r stress , 89. Ther stress , 82. Theproteins . CHOP biproteins , 91. Morproteins , 93. In  process . Silenciof HSP60 . All themt promotes tumor progression. GCN2-eIF2α-ATF4 axis has been shown to enhance tumor cell proliferation by maintaining metabolic homeostasis [mt and ISR may provide unexpected answers.The imbalance of the ISR pathway is related to a variety of diseases ranging from neurodegenerative diseases to tumors, reflecting the importance of cell stress adaptation to maintain health –83. The eostasis . Additioeostasis . Howevereostasis . For examt in cancer cells maintains proteostasis and regulates metabolic reactions. Furthermore, mtDNA mutations mediate cancer metastasis by activating the UPRmt [mt can be utilized by metastatic cancers [The initiation and development of cancer is a multistep process that involves the acquisition of diverse functions, such as resistance to cell death, prevention of growth inhibition, and activation of proliferation signals . Moreovehe UPRmt . Notably cancers –101.mt is indispensable for the development and progression of cancer [mt promoted by mitohormesis in cancer cells plays a critical role in stimulating the invasion and metastasis of cancer cells [mt-related genes is significantly associated with poor overall survival and metastasis-free survival in breast cancer patients [mt components in breast cancer suggests that UPRmt activation is involved in the progression of breast cancer [mt components are associated with an increased risk of head and neck cancer [mt mediates the secretion of the mitokine GDF15, which in turn promotes the invasion of thyroid cancer cells [Numerous studies have shown that activation of the UPRf cancer , 82. Theer cells . For exapatients . Evidenct cancer . In addik cancer . Strikiner cells .mt facilitates cancer progression by inhibiting cancer cell death and promoting cancer growth. In the following section, we introduce the role of the signaling molecules and several proteins in the UPRmt transcriptional program in cancer development. A further understanding of the signaling molecules in the UPRmt and the role of UPRmt-related proteins in cancer development is expected to provide a new therapeutic strategy for cancer.In principle, the UPRmt, including components of the CHOP/ATF4/ATF5, ERα and HSF1-SSBP1 axis, play an important role in tumorigenesis. These molecules promote tumorigenesis through various mechanisms in many tumors.Many signaling molecules in the UPRATF5 gene in glioma is significantly reduced compared with that in normal tissues. Moreover, this reduction in the methylation level is accompanied by a reduction in glioma differentiation [Previous studies have shown that ATF5 expression is significantly upregulated in a variety of cancers, such as epithelial ovarian cancer, glioblastoma, pancreatic cancer and chronic myeloid leukemia Figs. , 4A 106106–109. ntiation . In addintiation . Mechanintiation , 111. Acntiation . In a prntiation . Due to mt, to tumors range far beyond its function in the UPRmt. As a member of the ER family, ERα has been indicated to play a critical role in a variety of tumors  and mtHSP70 (HSPA9) are mitochondria-localized cytoprotective proteins and are the main molecular chaperones induced by UPRproteins . Moreoveproteins –136. In proteins , 66, 132proteins .HSP60 orchestrates various cell survival programs in cancer. For example, HSP60 binds and stabilizes survivin, protecting it from degradation and thus enabling it to play an inhibitory role in apoptosis , 139. ThKnockdown of mtHSP70 induces the death of melanoma cells via a mechanism related to the MEK/ERK signaling pathway and the mitochondrial permeability regulators cyclophilin D and ANT . In addiMET in the treatment of ovarian cancer. SHetA2, a small-molecule drug, interferes with the mtHSP70-p53 complex, thereby relieving the inhibitory effect of mtHSP70 on p53. PRIMA-1MET binds and modifies mutant p53 to restore the proper protein conformation of p53 and reactivate the wild-type function of mutant p53. Therefore, p53 can exhibit tumor-suppressive effects [Taken together, these observations indicate that the mitochondrial chaperones HSP60 and mtHSP70 rely on their protein-folding ability to maintain the stability of master signaling pathways in cancer cells, thus facilitating invasion and migration. HSP60 and mtHSP70 contribute to the survival of cancer cells by inhibiting proapoptotic proteins and activating antiapoptotic proteins Fig. . Thus, H effects . In summmt-induced mitochondrial matrix proteases ClpP and LONP1 maintain mitochondrial homeostasis by removing harmful proteins. As major mitochondrial proteases, ClpP and LONP1 play important roles in tumors [The UPRn tumors , 169. Mun tumors , 170–174ClpP interacts with multiple respiratory chain proteins and metabolic enzymes in mitochondria that are essential for metabolic regulation in cancer cells . For exaLONP1 has been found to be involved in tumor metabolic reprogramming, which is related to remodeling the components of the ETC and antagonizing cellular senescence . AKT-medCancer cells exploit the functions of ClpP and LONP1 in mitochondrial homeostasis and energy metabolism to accelerate their own invasion and metastasis. Several studies have proven that high levels of ClpP and LONP1 in colorectal cancer, prostate cancer, breast cancer and melanoma are notably correlated with poor prognosis in cancer patients Fig. G, H 173, 179. Acmt is related to many physiological and pathological processes as well as human diseases. The UPRmt plays an important role in aging, the immune response, cancer, heart disease and neurodegenerative diseases [C. elegans, the transcriptional program induced by UPRmt involves numerous genes, which are enriched in mitochondrial chaperones, OXPHOS complex assembly factors and components, and glycolytic genes [mt also boosts the expansion of the mitochondrial network, which is active during normal development, thus satisfying the physiological requirements of individual cells in C. elegans [mt genes, thereby promoting extension of the lifespan and enhancement of immune responses [mt in various human diseases have been gradually revealed. For instance, in decompensated cirrhosis, aging liver cells show the characteristics of mitochondrial dysfunction and an impaired UPRmt. ClpP, an effector in the UPRmt, promotes the elimination of ROS and, thus, delays the senescence of liver cells [mt effector protein SIRT7, and SIRT7 expression is significantly downregulated in senescent HSCs, demonstrating that UPRmt-mediated dysregulation of cell metabolism is one reason for the senescence of HSCs [mt is activated to promote metabolic adaptation [mt activity inhibits the death of cardiomyocytes induced by chronic pressure overload demonstrates the cardioprotective function of the UPRmt [mt signaling promotes neurogenesis in the brains of mice with amyotrophic lateral sclerosis (ALS) [mt is involved in the differentiation of myoblasts [mt plays a critical role in a variety of physiological processes, most of which are accompanied by slight perturbations in mitochondrial homeostasis, thus leading to UPRmt activation. In turn, activated UPRmt maintains mitochondrial function. Specifically, moderate mitochondrial stress contributes to activation of the UPRmt and stabilization of mitochondrial function [mt also acts as a promoter of the maintenance and propagation of deleterious mtDNA. The OXPHOS defect caused by the mutant mtDNA induces the UPRmt. Conversely, to promote mitochondrial recovery, UPRmt activation leads to intracellular accumulation of harmful mtDNA, which ultimately results in cellular dysfunction. This harmful mtDNA hijacks the UPRmt to facilitate its own dissemination [Accumulating studies have indicated that the UPRdiseases , 189–194 elegans . CBP-1 iesponses . The roler cells . The wea of HSCs . In contaptation . Evidenche UPRmt . Activathe UPRmt . Activatis (ALS) . Additioyoblasts , 56. In function . Howevermination , 200.mt promotes the recovery of mitochondria from damage, maintains proteostasis, remodels the ETC, and eliminates accumulated ROS in response to various intracellular and extracellular stresses. Mitocytosis, a migrasome-mediated mitochondrial quality control mechanism, maintains mitochondrial membrane potential and mitochondrial respiration by removing damaged mitochondria from migrating cells [mt and mitocytosis. In a sense, the UPRmt in C. elegans is very similar to the UPRmt in mammals. In both, the efficiency of mitochondrial protein transport determines the activation state. In addition, both of these UPRmts involve intricate cell signaling pathways and dynamic epigenetic regulation. However, the UPRmt in mammals is more complex than the UPRmt in C. elegans because it involves more factors and regulatory branches. Although the UPRmt in C. elegans is well understood, the UPRmt in mammals needs more exploration. What are the other potential branches of the mammalian UPRmt? What functions do these branches have? Is there any crosstalk between the different branches of the UPRmt, resulting in an extensive signaling network? What is the precise nature of the UPRmt-induced transcriptional program, and what roles do these transcripts play? Is there a signaling node connecting the UPRmt, UPRER and HSR that regulates mitochondrial, ER and cytoplasmic homeostasis? The answers to these questions require further research.As an indispensable cytoprotective mechanism, activation of the UPRng cells . It is wmt has emerged as a protective response in various human diseases, including neurodegenerative diseases [mt is associated with the progression of familial and sporadic AD [mt was observed in the brains of APP/PS1 transgenic mice and SHSY5Y cells treated with Aβ [mt attenuated cognitive impairment and decreased deposition of Aβ in an AD model [mt is important for the maintenance of mitochondrial proteostasis and provides potential targets for AD therapy.The UPRdiseases . Alzheimdiseases . Mitochodiseases . Severalradic AD , 205. Ac with Aβ . Recentl with Aβ . More imAD model . Therefomt is activated in many types of tumors. The UPRmt promotes the development of cancer and boosts its progression through various mechanisms. Cancer cells utilize signaling molecules and transcriptional products in the UPRmt, such as ATF5, HSP60 and ClpP, to promote their proliferation, growth, invasion and metastasis. Therefore, targeting components of the UPRmt may be a potential, reliable, and effective method for the treatment of cancer [mt and cancer needs further interpretation. Can the UPRmt be activated only by mitochondrial dysregulation, or can cancer cells also promote constitutive activation of the UPRmt ? Which oncogenes and tumor suppressor genes interact with the components of the UPRmt in cancer? Although the role of the UPRmt in cancer biology has been clarified through many years of research, more hidden mysteries are still awaiting exploration.Accumulating evidence indicates that the UPRf cancer , 208. Fo


Dysfunction of these signaling molecules leads to the development of pulmonary hypertension (PH), atherosclerosis, and other vascular diseases. Features of PH include vasoconstriction and pulmonary artery (PA) remodeling, which can result from abnormal proliferation, apoptosis, and migration of PA smooth muscle cells (PASMCs). These responses are mediated by increased Rieske iron–sulfur protein (RISP)-dependent mitochondrial ROS production and increased mitochondrial Ca2+ levels. Mitochondrial ROS and Ca2+ can both synergistically activate nuclear factor κB (NF-κB) to trigger inflammatory responses leading to PH, right ventricular failure, and death. Evidence suggests that increased mitochondrial ROS and Ca2+ signaling leads to abnormal synthesis of ketones, which play a critical role in the development of PH. In this review, we discuss some of the recent findings on the important interactive role and molecular mechanisms of mitochondrial ROS and Ca2+ in the development and progression of PH. We also address the contributions of NF-κB-dependent inflammatory responses and ketone-mediated oxidative stress due to abnormal regulation of mitochondrial ROS and Ca2+ signaling in PH.Mitochondria are important organelles that act as a primary site to produce reactive oxygen species (ROS). Additionally, mitochondria play a pivotal role in the regulation of Ca Oxidative stress and ROS signaling in PASMCs are involved in PA vasoconstriction and remodeling and, therefore in the development and progression of PH . It is wand rats . In thisgnaling. . In addignaling. . Furthergnaling. ,124,125.gnaling. . On the gnaling. . 2+ concentration ([Ca2+]i) is crucial to maintain the vascular tone. At rest, basal [Ca2+]i is tightly regulated to be around 100 nM. After cellular stimulation with a vasoconstrictor agonist, such as norepinephrine, endothelin, vasopressin, etc., [Ca2+]i increases reaching values between 500 nM and 1 mM [The homeostasis of intracellular Caand 1 mM . 2+]i in PASMCs [2+ leak and increases [Ca2+]i in PASMCs (2+-activated K+ (BKCa) channels in VSMCs. These channels as well as RyRs are essential proteins in mediating vascular smooth muscle tone.Dr. Wang’s research group and other investigators have demonstrated that ROS facilitates the dissociation of FKBP12.6 from ryanodine receptor 2 (RyR2) to activate the channel ,130,131.n PASMCs ,132. Mosn PASMCs , thereby2+ uniporter (MCU) regulates mitochondrial Ca2+ (mitCa2+) allowing Ca2+ uptake. We have reported that Ca2+ release mediated by hypoxic or RyR simulation evokes an improved performance in the activity of MCU. The increased MCU leads to the generation of mitROS dependent on ROS, provoking a positive feedback mechanism to potentiate hypoxia-initiated mitROS in PASMCs [2+]i i and vasoconstriction [Mitochondrial Can PASMCs . This fiCs [2+]i . Mitochochannels . Expressls of PH ,136 and ls of PH . Inhibittriction . Glutaminolysis is a mitochondrial process responsible for obtaining cellular energy from the deamination of glutamine to glutamate by glutaminase (GLS1) . SubsequKetone bodies, or simply ketones, are highly polar molecules produced by β-oxidation of fatty acids in the mitochondria of the liver cells. However, these molecules may be produced by enterocytes, astrocytes, and kidney ECs to a lesser extent ,26,143. Ketone’s metabolism is divided in ketogenesis and ketolysis. Ketogenesis, which mainly occurs in perivenous hepatocytes, produces three molecules: acetone, acetoacetate, and β-HB , which rOn the other hand, throughout ketolysis, acetoacetate and β-HB are used as an energy source by the mitochondria of several extrahepatic tissues ,154. Β-H2 consumption i) is crucial to maintain the vascular tone. At rest, basal [Ca2+]I is tightly regulated to be around 100 nM. After cellular stimulation with a vasoconstrictor agonist, such as norepinephrine, endothelin, vasopressin, etc., [Ca2+]i increases, reaching values between 500 nM and 1 mM [Emerging evidence indicates that ketone bodies, and in particular β-HB, at low concentrations, potentially contribute to ameliorating endothelial and vascular function in metabolic disease, while elevated concentrations of ketone bodies as observed in diabetic ketoacidosis contribute to the diabetic vasculopathy and diabetic vascular complications. In mammals, β-HB decreases the senescence associated secretory phenotype (SASP) and the senescence of vascular cells. The homeostasis of intracellular Caand 1 mM . 2+ signaling have been shown to contribute and aggravate pulmonary hypertension. Thus, anti-ROS therapies targeting implicated proteins such as RISP should be investigated as novel alternatives for the treatment of this ailment. On the other hand, ketone bodies seem to offer a protection against oxidative stress damage in vitro and in vivo; however, they also may exert anti-inflammatory or pro-inflammatory roles in the cardiovascular system, and further research is needed to understand their different roles. Collectively, a better comprehension of the unique roles of RISP-dependent mitochondrial ROS and their specific interactions with mitochondrial Ca2+ signaling, NF-κB-mediated inflammatory responses, and ketone-associated oxidative stress can significantly improve our understanding of the molecular pathogenesis of PH and associated RVF. This new knowledge may also help to develop and implement innovative therapies in the treatment of PH and other vascular diseases.Mitochondria are involved in essential cellular regulatory and homeostatic process in the cardiovascular system and particularly in VSMCs. Mitochondrial dysfunction has been widely associated with several diseases including PH. These organelles exert a strict control of ROS and ketones production. Uncontrolled ROS generation and its crosstalk with Ca


FLT3 mutations are the most frequently identified genetic alterations in acute myeloid leukemia (AML) and are associated with poor clinical outcome, relapse and chemotherapeutic resistance. Elucidating the molecular mechanisms underlying FLT3-dependent pathogenesis and drug resistance is a crucial goal of biomedical research. Given the complexity and intricacy of protein signaling networks, deciphering the molecular basis of FLT3-driven drug resistance requires a systems approach. Here we discuss how the recent advances in mass spectrometry (MS)-based (phospho) proteomics and multiparametric analysis accompanied by emerging computational approaches offer a platform to obtain and systematically analyze cell-specific signaling networks and to identify new potential therapeutic targets.  Over the last decades, molecular biology has witnessed tremendous technological and scientific breakthroughs. This progress has advanced our understanding of cell molecular functioning. We are now aware that signals sensed by a cell do not necessarily propagate in linearly. Signaling networks are rather assemblies of intricate highly connected modules controlling key biological processes in a context-dependent manner. It has also become clear that diseases are often due to perturbation of molecular networks, rather than consequences of single or few gene dysfunctions. In this context, the analysis of networks rewiring in healthy and diseased conditions is critical to understand the properties of the system and to identify new therapeutic strategies. However, because of their complexity and size, the dynamic characterization of signaling networks in health and during disease onset and progression often turns out to be a daunting task ,2,3.Different computational strategies have been developed to model signaling networks and predict their behavior upon environmental or genetic external or internal  perturbations. Most of these approaches take advantage of high throughput proteomic or transcriptomic data. Although both data-types are necessary to build models faithfully mimicking a system behavior, mass spectrometry (MS)-based proteomics is unique for this scope as it enables to measure both the concentrations and the activation levels of thousands of proteins in different conditions . Over reAcute myeloid leukemia (AML) is characterized by a dysfunction of the hematopoietic process resulting in the perturbation of the balance between stem cell proliferation and differentiation. In the last decade, genomic studies identified many different genetic alterations in AML patients, revealing the complexity and heterogeneity of this cancer type ,9. ThankFLT3 belongs to the class III receptor tyrosine kinase (RTKs) group of receptors, characterized by an N-terminal extracellular region consisting of five immunoglobulin-like domains, a juxtamembrane domain (JMD) followed by two tyrosine kinase domains (TKDs). In physiological conditions, FLT3 is mainly expressed in hematopoietic stem cells (HSC) and B-cell progenitors. Its expression decreases during hematopoietic differentiation, with the exception of a subpopulation of monocytes that maintain high levels of FLT3 expression after differentiation . In the Over the past years, several systems of classification of AML patients have been proposed. The European Leukemia Net (ELN) risk stratification guidelines recommended to stratify patients into three groups with different inferred clinical outcome: favorable, intermediate and adverse. This classification system is based on cytogenetic and molecular criteria ,16,17. AAmong the 1540 AML patients, about 500 carry FLT3 mutations. Indeed, it has been estimated that about 30% of AML patients have their FLT3 gene mutated at diagnosis . FLT3 muthe TKD1 beta-sheet1 (amino acids 610 to 615) in 24.6% of FLT3-ITD positive AML patientsin the nucleotide binding loop (NBL) (amino acids 616 to 623) in 2% of FLT3-ITD positive AML patientsin the TKD2 beta-sheet2 (amino acids 624 to 630) in 1.3 % of FLT3-ITD positive AML patients.ITDs are characterized by in-frame duplications 3 to 1236 bp in length, mostly in exons 14 and 15, which correspond to the juxtamembrane domain (JMD). The crystal structure of autoinhibited FLT3 leads to direct evidence of how the JMD region may exert its autoinhibitory effect on the catalytic activity of FLT3. More specifically, the insertion of ITD sequences disrupts the autoinhibitory interaction between the juxtamembrane domain and the kinase domain, allowing FLT3 to switch from the inactive to the catalytically active conformation in absence of the FLT3 ligand . Such coInterestingly, these observations are clinically relevant as the ITDs in the kinase domain have been associated to unfavorable patient prognosis in terms of complete remission, relapse-free survival and overall survival. It has been observed that the ITD size is also extremely variable from patient to patient and that the ITDs in the JMD and TKD regions are highly variables in length ranging from a few to hundreds of nucleotides ,24,25,26Given the importance of FLT3 mutations in AML onset and development, over the past decade, therapeutic protocols based on FLT3 inhibitors have been tested in clinical trials more or less specific kinase inhibitors have been tested either in monotherapy or in combination with chemotherapy. Protocols based on first-generation inhibitors, in monotherapy, are often clinically inefficient possibly because of low potency and selectivity. Thus, these inhibitors are commonly used in combination with standard chemotherapy  ,28. In rOther than the efforts to develop targeted therapies to block or attenuate the activity of the FLT3 receptor, increasing evidence have shown that cancer cells treated with tyrosine kinase inhibitors (TKIs) tend to acquire additional genetic alterations to escape inhibition. Although some patients obtain great benefit from the addition of FLT3 inhibitors  to standard chemotherapy , a signiMutations in the FLT3 receptor have been associated with the development of innate or acquired resistance. For example, Linardopoulos’s team generated resistant cells by long-term exposure of FLT3-mutant human cell lines with selective inhibitors . The resulting resistant cells harbored an additional mutation (D835Y) in the tyrosine kinase domain on the gene with the FLT3-ITD (+) allele. Consistently, the acquired FLT3 D835Y mutation has been recently identified in relapsed FLT3-ITD positive AML patients . These oWorth to mention, as in other types of leukemia, AML cells harboring pre-existing resistance-mediating mutations can be selected by chemotherapeutic or TKIs treatment. Interestingly, FLT3-ITD mutation has been recognized as a relapse-related genetic marker . Here weThe reduced sensitivity of TKD-ITD cells is still poorly understood. It is clear, however that the molecular mechanisms underlying the different sensitivity cannot be simply explained by the hyperactivation of the usual culprits, such as AKT, STAT5 and ERK1/2 b. The TK2 beads affinity purification . The resulting high content phosphoproteomic datasets allow to obtain a comprehensive overview of a cell phosphoprotein profile. Additionally, the described workflow enables to simultaneously process multiple samples, allowing to characterize the cell phosphoproteome in a time and space-resolved manner.In principle, MS-based phosphoproteomics is the best possible technology to elucidate how signaling pathways are rewired in AML cells harboring the different ITD mutations ,43,44. HDifferent network-based approaches have been developed to obtain from phosphoproteomic measurements information about signaling pathway modulation in disease. It has emerged over the years that diseases are often caused by genetic alterations that trigger a significant rewiring of the protein interaction network rather than by the simple alteration of the activity of a single protein. Thus, the characterization of a disease state, diagnosis and prognosis requires a proteome wide characterization of protein activities. As phosphorylation is the main post translational mechanism modulating protein activity, MS based phosphoproteomics plays a prominent role in the characterization of the modules of the signaling network that are altered in a disease condition. Phosphoproteomics-based network medicine applies high-throughput phosphorylation profiling in the classification of cancers, therapy planning and prediction of drug response . In a reHere we review this approach and discuss its application to elucidate signaling networks rewiring in drug resistant and sensitive FLT3-positive AML cells. The strategy takes advantage of SIGNOR, a manually curated database capturing more than 26,000 causal interactions. Every entry is SIGNOR is a binary, signed and directional interaction between proteins or other biological entities , supported by literature evidence. In addition, entries are linked to additional metadata such the molecular mechanism involved in the regulation  and, when available, the amino acid position of post-translational modifications (PTM). At the data of writing SIGNOR stores approximately 9800 regulatory phosphorylation reactions and information for 8800 phosphorylated sites ,59. The (a)Network template: the file should contain a table with at least five columns listing the source nodes, the target nodes, the causal effects (up- or downregulation) and the information about the amino acid position of the phosphorylated site as well as the amino acid sequence context of the phosphosite (sequence window). In our case, this file is the complete list of causal interactions available from the SIGNOR database.(b)Node experimental attributes: a table containing the protein expression levels in specific experimental conditions, as revealed by MS-based proteomic experiments.(c)Edge attributes: a table listing the phosphorylation level of the regulatory phosphopeptides involved in each activation/inactivation reaction, as revealed by the MS-based phosphoproteomic experiment.(d)Cytoscape software installed. For the scope of this example, we used default options. Alternatively, a plethora of adds on applications developed to visualize and analyze networks and omics data are made available at the Cytoscape App store .To reproduce the workflow, the following material is required:These are the steps required to obtain a FLT3 specific-causal network.https://signor.uniroma2.it/downloads.php accessed on 24 April 2021).Download the complete list of interactions from the Download all data section of the SIGNOR database  ≥ 0 to remove unconnected nodes.Use the “style” tab to modify the layout of the network, e.g., the size of the nodes to reflect protein expression level c.Import phosphoproteomic data as attribute of the edges, using the 15mer sequence in SIGNOR as key (see field SEQUENCE).Use the “style” tab to modify the visual properties of the edges. Use arrow style to show effect and directionality and modify color according to phosphoproteomic data d.The result of this pipeline is a signed directed graph that includes only proteins (nodes) expressed in a specific context, as per proteomic data, and where edges represent phosphorylation events at specific sites detected by phosphoproteomics.The resulting network still lacks a crucial piece of information as the activation level of each node is not annotated. Converting phosphorylation data into qualitative information on protein activity is fundamental to interpret signaling network rewiring. Phosphorylation of one or more residues in the activation loop is the most common mechanism to regulate kinase activity. Thus, for many signaling proteins, it is possible to estimate activity from the phosphorylation levels of its regulatory sites. However, there are also pitfalls to consider. Although annotation of the relation between site phosphorylation and kinase activity is annotated in databases such as SIGNOR and PhosphoSitePlus ,59,60, iThe resulting network offers a static snapshot of how the FLT3-ITD mutations impact signaling network and determine a different sensitivity to chemotherapeutic treatments. The goal is that of obtaining a predictive network which can be used as a framework to infer potential therapeutic targets to revert drug resistance. Boolean approaches have been widely applied to obtain context-specific predictive signaling network models . HistoriThe CNO software takes as input a prior knowledge network (PKN) and a training data set. The PKN could be either downloaded from the many pathway or model databases or curated from causal databases as we have described for the FLT3-ITD specific signaling networks e. The trTo obtain the training dataset, it is necessary to select a panel of proteins, often dubbed “sentinel proteins”, whose activity can be monitored under different perturbation conditions. System perturbations can be obtained by treating cells with small-molecule inhibitors and/or cytokines, whereas the activity of the sentinel proteins is analyzed by experimental approaches such as the Luminex xMAP sandwich assay, which permits to measure multiple protein concentrations or modifications in up to 96 samples in a single run.In conclusion, this approach enables to obtain dynamic networks describing the different response to perturbations in sensitive and resistant cells.Only few studies addressed the impact of FLT3-ITD TKD mutations on patients’ survival and therapy resistance. These studies have contributed to raise new fundamental and applied questions: (i) How do the TKD-ITD mutations alter the structure of the FLT3 receptor? (ii) Can the size and the position of ITD trigger the activation of different signalling pathways? (iii) Can the different ITD localization be considered as a patients’ stratification feature? (iv) Which is the role of co-mutations of AML patients in the TKD-ITD resistance? We have described here a general strategy to address such questions. Understanding the molecular changes occurring in a perturbed system provides the means to build predictive models and to set a rational basis to formulate new hypothesis. This approach can also have a translational impact as the optimized model may be used to test new strategies to revert the drug resistance phenotype in AML ITD patients. AML patients carry a complex mutational landscape which can be explored by taking advantage of the recent technological advances in single-cell techniques. Over the last years, mass cytometry has played a crucial role in the single cell characterization of AML patient-specific signalling ,74,75,76


In addition, marked changes in the ryanodine receptors and Ca2+-pump ATPase have been shown to cause Ca2+-release and depress Ca2+ accumulation in the sarcoplasmic reticulum as a consequence of oxidative stress. Such alterations in sarcolemma and sarcoplasmic reticulum are considered to cause Ca2+-handling abnormalities, which are associated with mitochondrial Ca2+-overload and loss of myofibrillar Ca2+-sensitivity due to oxidative stress. Information regarding the direct effects of different oxyradicals and oxidants on subcellular organelles has also been outlined to show the mechanisms by which oxidative stress may induce Ca2+-handling abnormalities. These observations support the view that oxidative stress plays an important role in the genesis of subcellular defects and cardiac dysfunction in heart disease.It is now well known that oxidative stress promotes lipid peroxidation, protein oxidation, activation of proteases, fragmentation of DNA and alteration in gene expression for producing myocardial cell damage, whereas its actions for the induction of fibrosis, necrosis and apoptosis are considered to result in the loss of cardiomyocytes in different types of heart disease. The present article is focused on the discussion concerning the generation and implications of oxidative stress from various sources such as defective mitochondrial electron transport and enzymatic reactions mainly due to the activation of NADPH oxidase, nitric oxide synthase and monoamine oxidase in diseased myocardium. Oxidative stress has been reported to promote excessive entry of Ca Furthermore, various antioxidants have been observed to exert beneficial effects in improving cardiac function and attenuating heart disease [2+-handling in cardiomyocytes in chronic myocardial infarction. Although oxidative stress is known to modify different proteins in subcellular organelles by affecting cardiac gene expression and various signal transduction mechanisms as well as by activating different proteases, no effort is made to deal with these issues in this article. On the other hand, some evidence is presented in this review to show that oxyradicals and some oxidants exert direct effects on subcellular organelles, and thus may explain the role of oxidative stress in the development of subcellular abnormalities, Ca2+-handling defects and cardiac dysfunction in heart disease.Several clinical and experimental investigations have shown that oxidative stress plays a critical role in the pathogenesis of heart disease ,11,12,13 disease ,29,30,312O2) and hypochlorous acid (HOCl) and/or reduction in the activities of endogenous antioxidants such as superoxide dismutase, glutathione peroxidase and catalase [It is now well known that the occurrence of oxidative stress is mainly a consequence of excessive formation of reactive oxygen species (ROS) including superoxide radicals and hydroxyl radicals as well as oxidants such as hydrogen peroxide  are transmembrane proteins, which are involved in the generation of ROS by transferring electrons from NADPH to molecular oxygen, and serve as signaling molecules for inducing cardiac hypertrophy, apoptosis, fibrosis and heart failure ,67,68. Roverload . Studiesoverload , and in overload . Apocynioverload . Furtheroverload .2O during the process of oxidative breakdown of catecholamines and serotonin. It should be noted that the levels of plasma catecholamines and serotonin are elevated in different types of heart disease and these may partly generate ROS during their degradation by MAO. In fact, MAO-A has been shown to play a key role in the development of acute and chronic heart diseases [Another major source of ROS production is a monoamine oxidase (MAO), which participates in degradation of neurotransmitters such as norepinephrine, epinephrine and dopamine as well as serotonin ,80. Thisdiseases . Exposurdiseases . Furtherdiseases . It is adiseases , and itsdiseases . Furtherdiseases .2+-handling abnormalities in diverse cardiovascular disorders [2+-handling abnormalities in heart disease [2+-handling abnormalities and cardiac dysfunction in heart disease are shown in Extensive research over the past several decades has revealed that oxidative stress may be a major mechanism for the genesis of cardiac dysfunction, subcellular defects and Caisorders ,88,89,90isorders ,92,93,94isorders ,42,43,44 disease ,97,98,992+ in cardiomyocytes by promoting the entry of Ca2+ upon affecting the sarcolemmal membrane as well as by inducing changes in Ca2+-release and Ca2+-uptake activities in the sarcoplasmic reticulum under different pathological conditions [2+ -handling associated with mitochondrial Ca2+-overload, depression of mitochondrial function, generation of oxidative stress, activation of proteases, fragmentation of DNA and impaired cardiovascular function [2+-sensitivity due to prolonged oxidative stress and Ca2+-handling abnormalities is considered to explain cardiac dysfunction as a consequence of myofilament derangements and myofibrillar degeneration [2+-stimulated ATPase activity due to the modification of myosin gene expression as a consequence of both oxidative stress and Ca2+-handling abnormalities in cardiomyocytes [It is now well known that oxidative stress increases the intracellular Canditions ,91. Expofunction . The losneration ,100. Theneration . Prolongneration . Failing2+ through the sarcolemmal membrane has been shown to occur for the development of Ca2+-handling abnormalities in heart failure due to myocardial infarction, the exact contribution of Ca2+-influx and Ca2+-efflux mechanisms has not been fully established [2+-channels, which are associated with beat-to-heat Ca2+-influx through sarcolemma, was decreased but the activity of sarcolemmal Ca2+-pump, which is involved in Ca2+-efflux, was unaltered in the failing heart [+-K+ ATPase in the infarcted heart has indicated that the Ca2+ entry in cardiomyocytes may increase indirectly through the Na+-Ca2+ exchange mechanism. In fact, the sarcolemmal Na+-Ca2+ exchange activity was also decreased, which may impair Ca2+-efflux (via the reverse mode) and contribute in the development of Ca2+-handling abnormalities in failing cardiomyocyte [+-K+ ATPase activity by cardiac glycosides has been shown to cause abnormal Ca2+-cycling and impair mitochondrial energetics in guinea pig cardiomyocytes [2+-entry through the sarcolemmal membrane by increasing its permeability due to lipid peroxidation, changes in the sarcolemmal phospholipid composition by other mechanisms cannot be overlooked. Particularly, different oxyradical generating systems have been shown to decrease phospholipid N-methylation, which is known to determine the membrane fluidity [2+-handling abnormalities in cardiomyocytes [While excessive entry of extracellular Caablished . In thisng heart ,91. On tomyocyte . Furthermyocytes . Althougfluidity . Furthermyocytes .2+ [2+-handling abnormalities associated with oxidative stress are known to induce mitochondrial Ca2+-overload and impair mitochondrial function for the production of energy [2+-handling whereas a loss of mitochondrial Ca2+ uniporter has been reported to trigger arrhythmias possibly by affecting the Ca2+-handling function of the sarcoplasmic reticulum [2+ on a beat-to-heat basis, is known to play a major role in Ca2+-handling in cardiomyocytes, and has been indicated to serve as a critical target for oxidative stress. Both oxidative stress and nitrosative stress have been demonstrated to promote Ca2+-leak by modifying ryanodine receptors in the sarcoplasmic reticulum in various types of failing hearts [2+-pump ATPase and ryanodine receptors in heart failure are considered to occur due to oxidative stress as these were corrected by an antioxidant, edaravone, as well as by carvedilol (due to its antioxidant properties) [It needs to be emphasized that mitochondria is not only a major source for the production of cellular ATP but is also a major contributor for the production of oxyradicals in cardiomyocytes, and these processes are regulated by the intracellular concentration of Ca2+ ,108. Ca2f energy ,110,111.eticulum ,113. It g hearts ,115,116.perties) ,118.2+-handling abnormalities and subsequent cardiac dysfunction, it is not clear whether these effects of oxidative stress on subcellular organelles are of any direct or indirect nature. In order to gain some information in this regard, sarcolemmal membranes, sarcoplasmic reticulum, mitochondria and myofibrils were isolated from control hearts; these subcellular organelles were incubated with different oxyradical generating systems and oxidants for 30 min and their activities were determined. The results in 2O2 and hydroxyl radical generating mixture depressed Na+-K+ ATPase and Na+-Ca2+ exchange activities. These changes were associated with increased malondialdehyde (MDA) content and reduced sulfhydryl groups (SH-groups). The effects of superoxide radicals were attenuated by superoxide dismutase (SOD), the effects of H2O2 were attenuated by catalase and those of hydroxyl radicals were reduced by mannitol [2+ uptake activity, Ca2+-stimulated ATPase activity and Mg2+-ATPase activity were also depressed by superoxide radicals, H2O2 and hydroxyl radicals and these effects were attenuated by their scavengers, SOD, catalase and mannitol, respectively  was increased by superoxide radicals, H2O2 and hydroxyl radicals  as well as depression in the sarcolemmal Na+-K+ ATPase and Na+-Ca2+ exchange systems due to oxidative stress. Furthermore, oxidative stress promotes the release of Ca2+ (due to defect in ryanodine receptors) and depresses Ca2+-uptake (due to changes in Ca2+-pump ATPase) in the sarcoplasmic reticulum. Such Ca2+-handling alterations in both sarcolemma and sarcoplasmic reticulum can be seen to induce mitochondrial Ca2+-overload and depress the process of energy production. Prolonged Ca2+-handling abnormalities in diseased myocardium will also cause derangements of myofilaments and loss of myofibrillar Ca2+-sensitivity. Taken together, all these defects in subcellular organelles can be seen to result in cardiac dysfunction due to the development of oxidative stress in heart disease. It should be emphasized that it is not our intention to exclude the participation of several other pathogenic factors in the pathogenesis of cardiac dysfunction but the information in this article provides evidence in support of the concept that oxidative stress may induce cardiac dysfunction due to subcellular defects and Ca2+-handling abnormalities in heart disease.An in-depth analysis of the literature regarding the pathophysiology and pharmacotherapy of different types of cardiovascular diseases has revealed that oxidative stress is one of the most critical factors, which is involved in the pathogenesis of cardiac dysfunction. It is also evident that the occurrence of oxidative stress represents a disbalance between the excessive formation of oxyradicals and the activities of antioxidant defense mechanisms. Furthermore, it has been demonstrated that the generation of oxyradicals in diseased myocardium is a consequence of defects in the mitochondrial electron transport system as well as the by-product of reactions involving different enzymes including NADPH oxidase, xanthine oxidase, nitric oxide synthase and monoamine oxidase. Although the contribution of each source to produce oxidative stress is not clear, it seems that the involvement of each source for the generation of oxyradicals may differ from one disease to the other and may be specific for the stage and type of heart disease. In addition, oxidative stress has been shown to produce a wide variety of changes in various metabolic pathways and signal transduction systems for the induction of fibrosis, necrosis and apoptosis as well as the depression of cardiac gene expression and activation of different proteolytic enzymes in heart disease. However, alterations in subcellular organelles such as sarcolemma, sarcoplasmic reticulum, mitochondria and myofibrils may be more related to the development of Ca


Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca2+, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca2+, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [3H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca2+-binding site to sensitize RyR1 to Ca2+. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca2+. However, enhanced RyR1 activation occurred in the presence of Ca2+, AMPPCP, and Caf. In the absence of Ca2+, Na+ inhibited [3H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca2+-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca2+, Na+ is predicted to inhibit RyR1 by interacting with the Ca2+-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca2+-binding site, and conversely, Ca2+ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca2+, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca2+-, ATP-, and Caf-binding sites.Ryanodine receptor type 1 (RyR1) releases Ca Rhologies . Studiesractions , 6.2+-, ATP-, and Caf-binding sites, and a large cytoplasmic shell serving as a target for calmodulin, protein kinases, and redox active species regulating RyR1 via long-range allosteric mechanisms  domain and the C-terminal domain (CTD), Caf binds at the interface of S2S3 and the CTD, and ATP binds at the junction of thumb and forefingers (TaF), S6c, and the CTD domains  together reduced channel open probabilities and restrained open channel states in cryo-EM . Furthermore, in the presence of ATP, the binding of Caf breaks interactions between CTD and S2S3 domains and rearranges Ca2+-binding pocket for favorable Ca2+ binding. This suggests that the binding of ATP and Caf modulates the Ca2+-binding site and affect Ca2+ sensitivity of RyR1 , a nonhydrolyzable ATP analog, and Caf. The structures of Ca2+-, ATP-, and Caf-binding sites were resolved using cryo-EM micrograph densities of purified RyR1 in the presence of 0.5 M NaCl . To determine the effects of Na+ on RyR1 activity, [3H]ryanodine binding to RyR1 was compared in 0.5 M choline Cl or 0.5 M NaCl media. Computational methods using cryo-EM densities suggest that RyR1 is regulated through a network of allosteric interactions involving the Ca2+-, ATP-, and Caf-binding sites.In the present study, we employed biochemical and molecular dynamics (MD) simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca5 M NaCl . Our stuing site . We repo2+activation site of RyR1 by Ca2+ and the combined action of ATP and Caf. However, it is not known how ATP or Caf in the absence of the other coactivator modulates the structure of the Ca2+-binding site. In the present study, the Ca2+-dependent regulation of RyR1 was determined in the absence and presence of Caf, AMPPCP, and the presence of both ligands, where enhanced binding correlates with an increased open channel probability (Po) in lipid bilayers . At 25 μM Ca2+, the combined addition of AMPPCP/Caf significantly increased [3H]ryanodine binding, whereas RyR1 activity did not increase in the presence of AMPPCP or Caf in 0.5 M NaCl compared with 0.5 M choline (C).The addition of AMPPCP/Caf to 0.5 M NaCl media increased ryanodine, and protease inhibitors. Bmax value of [3H]ryanodine binding was determined by incubating crude membrane isolates for 20 to 24 h at 24 °C in 0.6 M choline Cl containing 20 mM imidazole, pH 7.0, 0.15 mM Ca2+, 20 nM [3H]ryanodine, and protease inhibitors. Nonspecific binding was determined in the presence of 10 μM unlabeled ryanodine. Amounts of bound [3H]ryanodine were determined using a filtration assay . Differences between samples were analyzed using two-way ANOVA followed by Holm–Sidak method. p < 0.05 was considered significant.Free Ca2+, ATP, and Caf), we performed MD simulations on cryo-EM structures of RyR1 in different ligand-bound states (5TB0), Ca2+-only bound RyR1 , ATP- and Caf-bound RyR1 , Ca2+-, ATP-, and Caf-bound closed RyR1 , and Ca2+-, ATP-, and Caf-bound open RyR1  as starting structures to simulate the dynamics of RyR1 in various ligand-bound states. Furthermore, these systems will also serve as control systems to understand the ligand site conformations and pore dynamics in the presence or absence of modulators. To mimic the RyR1 + ATP/Caf system in 0.5 M NaCl concentration, we manually docked Na+ ions to Ca2+-binding sites of RyR1 + ATP/Caf. We placed the Na+ ion in such a way that the side chains of Glu-3893, Glu-3967, and Thr-5001 could induce electrostatic interactions with the Na+ ion. We referred this system throughout the article as RyR1 + ATP/Caf/Na+. To understand the individual role of ATP and Caf on Ca2+-binding site, we generated RyR1 + ATP and RyR1 + Caf conformations by removing Caf and ATP molecules, respectively, from RyR1 + ATP/Caf system (PDB: 5TAP). Furthermore, we generated RyR1 + ATP/Ca2+ and RyR1 + Caf/Ca2+ systems by removing Caf and ATP, respectively, from RyR1 + ATP/Caf/Ca2+ (open) system. The details of MD system configurations, experimental conditions, pore status, and abbreviations used for each system are summarized in To understand the mechanism of coregulation and synergy between the RyR1 activators (Cad states  11). Br. Br2+, Ahttps://swissmodel.expasy.org/)  (https://www.gromacs.org/) (cgenff_charmm2gmx.py script provided in CHARMM Web site (http://mackerell.umaryland.edu/charmm_ff.shtml#gromacs)  . Loop opdeller/)  to refindeller/) . A snapsdeller/)  was emplcs.org/) . The appgromacs) . The equbarostat . Long-rabarostat . The simdi is the distance between backbone atom i and its reference position, and N is the total number of atoms.RMSD of protein residues is a common measure to estimate the dynamics of protein structures. The mean square displacement of protein backbone atoms with respect to their initial positions estimates the RMSD of protein residues in a simulation trajectory as shown in the following equation:gmx sasa tool to estimate the SASA of Ca2+-binding site with respect to simulation time (gmx sasa evaluates SASA by rolling and tracing the path of a sphere with r = 1.4 Å along the van der Waals surface of Ca2+-binding site. The SASA of all four Ca2+ sites across respective MD trajectories was averaged and compared for meaningful data extraction.We used the ion time . BrieflyThe data that support the findings of this study are available from the corresponding author, V. R. C., upon reasonable request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe yet rare inherited arrhythmia disorder. The cornerstone of CPVT medical therapy is the use of β-blockers; 30% of patients with CPVT do not respond well to optimal β-blocker treatment. Studies have shown that flecainide effectively prevents life-threatening arrhythmias in CPVT. Flecainide is a class IC antiarrhythmic drug blocking cardiac sodium channels. RyR2 inhibition is proposed as the principal mechanism of antiarrhythmic action of flecainide in CPVT, while it is highly debated. In this article, we review the current progress of this issue. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare inherited arrhythmia syndrome characterized by bidirectional or polymorphic ventricular tachycardia (VT) provoked by emotional stress and/or physical activity. Clinical phenotypes include catecholamine-associated syncope and a characteristic pattern of bidirectional VT in the absence of structural heart disease . The priCPVT has been mainly related to mutations in genes encoding the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2), which can be identified in 60–70% of CPVT patients . RyR2 an2+ fluxes between the intracellular compartment and the extracellular space in cardiac myocytes is crucial for normal excitation-contraction (EC) coupling . The cytosolic Ca2+ sharply from 150 nM to 1 μM activates the contractile apparatus. Then, the elevated cytosolic Ca2+ promptly resumed to 150 nM during the diastolic phase to ensure regular relaxation properties of the myocytes. The majority of Ca2+ is reuptake into the SR by Ca2+ ATPase isoform 2a (SERCA2a). The remaining Ca2+ is extruded into the extracellular fluid via a forward-mode Na+/Ca2+ exchanger (NCX).The delicate balance of Cacoupling . During 2+ handling in the SR, enhancing the open probability of RyR2 and leading to spontaneous Ca2+ release events during the diastolic period. Under adrenergic stress, Ca2+ overload in the SR can facilitate abnormal Ca2+ leakage during relaxation. Elevated intracellular Ca2+ levels during the diastolic period would activate the forward mode of NCX, which extrudes Ca2+ in exchange for Na+ with a stoichiometry of 1:3, generating a net inward current. The transient inward current (Iti) produces delayed afterdepolarizations (DADs) and causes triggered activity once it reaches the threshold of the Na+ channel. Taken together, the molecular pathophysiology of arrhythmia occurrence in CPVT involves two critical steps: (1) spontaneous Ca2+ release from SR during the diastolic period, which could be exaggerated by adrenergic stimulation and (2) triggered activity activated by Iti, which is induced by spontaneous Ca2+ release.The mutations of RyR2 and CASQ2 disrupt normal CaThe insufficient protection of β-blockers in CPVT has been reported . Almost 2+ leak events from RyR2 are the essence of molecular arrhythmogenic mechanism in CPVT. Theoretically, direct RyR2 blockers are promising mechanism-based therapies for CPVT. Tetracaine, a sodium channel blocker, is a RyR2 blocker that effectively inhibits Ca2+ leak from SR. Thus, Knollmann and his collaborators screened the RyR2 inhibiting effects of clinically available sodium channel blockers in a lipid bilayer study and found that flecainide reduced the duration of RyR2 channel openings, but not its closed channel duration. They then tested the effects of flecainide on CASQ2 knockout mice. Intraperitoneal administration of flecainide completely suppressed exercise-induced VT in vivo, and incubation with flecainide significantly ameliorated the spontaneous Ca2+ release from SR induced by isoproterenol in isolated myocytes. In contrast, without RyR2 blocking action in the lipid bilayer, lidocaine did not show therapeutic effects in vivo and in vitro. Therefore, they propose that the underlying antiarrhythmic mechanism of flecainide in CPVT attributes to its RyR2 blockade but not its intrinsic sodium channel inhibiting action.The abnormal Ca2+ wave with IC50 12.8 μM in permeabilized ventricular myocytes. The blocking action of flecainide is use-dependent, suggesting that RyR2 activity determines the potency and efficacy of flecainide. Given flecainide’s blocking features of the sodium channel and RyR2 channel, it is challenging to dissect the antiarrhythmic mechanisms of flecainide in CPVT. Subsequently, the Knollman group performed a series of experiments to reinforce the concept. 2+ release after the acute administration of flecainide in isolated cardiac myocytes.Flecainide is a hydrophilic sodium channel blocker with a pKa of 9.3. At pH 7.4, only 1% of flecainide is neutral and is available for diffusion across the membrane of myocytes . The int+/− mouse model. Flecainide significantly reduced ventricular arrhythmias induced by adrenaline and caffeine in vivo. In isolated intact RyR2R4496C+/− myocytes, flecainide did not affect Ca2+ transient amplitude, decay, or SR Ca2+ content. In permeabilized RyR2R4496C+/− myocytes, flecainide did not alter the frequency of spontaneous Ca2+ sparks. In contrast, when the dosage of flecainide reached 6 μM, the upstroke of action potential was blunted significantly at the pace of 5 Hz (2+ transients (SCaTs) elicited by isoproterenol  could reduce spontaneous Ca2+ release events under their experimental conditions. After inactivation of the sodium channel using the voltage-clamp approach, flecainide could not reduce Ca2+ waves. Therefore, they proposed that Na+ channel blockade by flecainide could reduce Na+ influx into cardiac myocytes, resulting in the enhancement of Ca2+ efflux through NCX and decrease of Ca2+ in the vicinity of the RyR2 channels, ultimately reducing the frequency of spontaneous Ca2+ release events. In the HEK293 cell line expressing hRyR2, which is devoided of Na+ channels in the cellular membrane, Bannister et al. reported that flecainide did not affect spontaneous Ca2+ release events  was used in intact or permeabilized myocytes to elucidate antiarrhythmic mechanisms. Flecainide has a narrow therapeutic window between the effective dose and the dose that can produce adverse toxic effects. The target range for flecainide concentration is 0.5–2.4 μM in the clinical practice . Despite2+ overload, and are detrimental to CPVT  of RyR2 mutations has been identified as a new clinical entity, termed cardiac ryanodine receptor calcium-release syndrome (CRDS), which is characterized by ventricular fibrillation and sudden death. However, it does not manifest ventricular tachyarrhythmias during stress testing . CRDS is2+ dysregulation in cardiac myocytes and help develop a more specific therapeutic strategy for CPVT.Flecainide has a significant impact on the clinical management of patients with CPVT. Efforts have been made to explore the underlying mechanisms of flecainide therapy for CPVT. There is a hot debate regarding the effects of flecainide on RyR2. Understanding the mechanisms of flecainide in CPVT will improve our knowledge of CaYR and NL defined the theme of review. YL wrote the manuscript. XP, RL, XW, and XL took part in preparing the manuscript. YR, NL, RT, CM, and RB prepared and reviewed the manuscript before publication. All authors confirmed that they have read and approved the manuscript and they have met the criteria for authorship.This work was supported by the National Science Foundation of China  and Beijing Municipal Natural Science Foundation (grant no. 7192051).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TLR3 mRNA expression, and 1,25D3 treatment significantly reduced its expression. In addition, 1,25D3 decreased the expression of IL-6, IFN-β1, CCL2, FN1 and COL1A1 induced by polyI:C in BSMCs. The regulatory effect of 1,25D3 treatment on polyI:C-stimulated BSMCs was further confirmed at protein levels. Our findings suggest that vitamin D3 attenuates TLR3 agonist-induced inflammatory and fibrotic responses in BSMCs and support the clinical relevance of vitamin D3 supplementation in patients with viral infections having chronic respiratory diseases, such as asthma and COPD.Toll-like receptor 3 (TLR3) activation by viral infections plays a key role in promoting inflammatory immune responses that contribute to pulmonary fibrosis in chronic inflammatory respiratory diseases. Vitamin D3 has been shown to be beneficial to patients with asthma and chronic obstructive pulmonary disease (COPD) through its anti-inflammatory and anti-fibrotic properties. Smooth muscle cells are one of the major contributors to airway remodeling in asthma and COPD. We therefore aimed to investigate the effect of vitamin D3 treatment on viral-induced TLR3 responses in Bronchial Smooth Muscle Cells (BSMCs) as a mechanism contributing to pulmonary fibrosis in asthma and COPD. Primary BSMCs from patients with asthma (n=4), COPD (n=4), and healthy control subjects (n=6) were treated with polyinosinic: polycytidylic acid (polyI:C), TLR3 agonist in the presence or absence of vitamin D3 . Here we report the mRNA expression and protein levels of pro-inflammatory and pro-fibrotic markers  among BSMCs groups: asthma, COPD, and healthy controls. We show that at the baseline, prior to polyI:C stimulation, asthma and COPD BSMCs presented increased pro-inflammatory and pro-fibrotic state compared to healthy control subjects, as measured by quantitative PCR and immunoassays (ELISA/Flow Cytometry. Ligation of TLR3 by polyI:C in BSMCs was associated with increased  Pulmonary fibrosis is a complex process that involves activation of several pattern recognition receptors (PRRs) and an interplay of many cell types that contribute directly or indirectly to airway remodeling. Increased thickness of the airway smooth muscle (ASM) layer due to hypertrophy and hyperplasia of ASM cells is a feature of airway remodeling in both asthma and chronic obstructive pulmonary disease (COPD) , 2. Bronvia TRIF results in the upregulation of multiple pro-inflammatory cytokines, such as interleukin-6 (IL-6), IL-1β, tumor necrosis factor-alpha (TNF-α) and chemokines. The chemokine (CXC motif) ligand 10 (CXCL10) and the chemokine (CC motif) ligand 2 (CCL2), encoding monocyte chemoattractant protein 1 (MCP-1), are potent inflammatory mediators involved in inflammatory immune cell migration. These pro-inflammatory mediators have been recently described in the context of viral infections  before and after exposing the BSMCs to polyI:C, either alone or combined with 1,25D3 treatment. Our results suggest that vitamin D3 may play a protective role in the development of exacerbations due to viral infections, by its ability to regulate TLR3 responses in BSMCs.Although TLR3 has a major role in viral-induced immune responses, excessive TLR3 inflammatory responses may play a key role in promoting exacerbations and fibrosis in asthma and COPD. Therefore, in this study, we investigated the implications of vitamin D3 in offsetting pro-inflammatory and pro-fibrotic responses induced by TLR3 agonist, polyI:C in BSMCs isolated from asthmatic, COPD, and healthy control (non-smokers and smokers) subjects. We hypothesized that 1,25D3 treatment downregulates polyI:C-induced pro-inflammatory and pro-fibrotic responses in BSMCs. We assessed the mRNA expression of For asthma experiments, Bronchial Smooth Muscle Cells (BSMCs) from non-asthmatic (n=3) and asthmatic (n=4) subjects were purchased from a commercial source . These cells were chosen from subjects who were age-matched and non-smokers. For COPD experiments, BSMCs were obtained from the Hospital Research Institute of the University of Manitoba, as previously described . All pro2/37˚C incubator. For experiments, the cells were seeded into 12- or 6-well plates at a cellular density of 1 x 105 cells and 2 x 105 cells, respectively. At ~ 80% confluence, the cells were serum-starved overnight in FBS-free DMEM. BSMCs were then stimulated for 24 hours with 5 µg/ml of polyinosinic:polycytidylic acid (polyI:C)  alone or combined with 1,25D3 (100 nM)  treatment. Both reagents were added simultaneously to the cells. Ethyl alcohol (EtOH) (0.1%) vehicle was added to unstimulated BSMCs and used as control for the experiments. After stimulation, the cells were processed for RNA extraction and cell-free culture media were collected and frozen for ELISA experiments.BSMCs from passages two to five were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)  supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin in a humidified 5% CO-ΔΔCT method was applied to determine the relative fold change in gene expressions in samples treated with polyI:C alone or polyI:C-1,25D3-stimulated BSMCs from asthma/COPD relative to unstimulated BSMCs from healthy controls, after normalization to the housekeeping gene, Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH), like previously described . Real time-PCR primers are listed in Cell-free supernatants from polyI:C alone or polyI:C-1,25D3-stimulated BSMCs were harvested and stored for further cytokine measurements. The following ELISA kits: Human CCL2/MCP-1 DuoSet DY279-05 (R&D Systems), Human Fibronectin (FN1) DuoSet ELISA DY1918-05 (R&D Systems), Human IFN-beta DuoSet ELISA DY814-05 (R&D Systems), and Human IL-6 ELISA MAX Deluxe  were used. Assay procedure was followed according to the manufacturer’s protocol. The limits of detection for all ELISAs’ kits were in picogram range (> 7.8 pg/ml), except for fibronectin 1 for which the limit of detection was > 0.1 ng/ml. The absorbance was read at 450 nm (corrections 570 nm) in a microplate reader (Epoch Spectrophotometer System).5 cells were pre-incubated with Live/Dead dye (1/1000) for 15 minutes at room temperature. Cells were then washed with phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde (PFA) for 10 minutes. After PBS washing, the cells were immunolabelled for 1 h at room temperature with anti-human type I collagen-FITC (1/200) diluted in permeabilization buffer . Then, the cells were washed with PBS and kept on ice while they were analyzed by flow cytometry . EtOH (0.1%) treated and unstained cells served as control samples. Compensation beads  were stained with anti-human type I collagen-FITC or Pacific Blue Mouse IgG1 isotype control  and used as compensation controls, according to the manufacturer`s instructions. For each sample, 1×105 single cell events were acquired and analyzed by FlowJo 10.0.7 software. Live-cells gating strategy to analyze type I collagen positive cells is shown in To quantify intracellular type I collagen produced in BSMCs, the anti-human type I collagen antibodies conjugated to fluorescein isothiocyanate (FITC)  and Live/Dead Fixable Violet  dual staining was performed, according to the manufacturer`s instructions. Briefly, polyI:C alone and polyI:C-1,25D3-stimulated BSMCs were harvested using non-enzymatic cell stripper solution , centrifuged for 5 min at 500xg and 2 x 10p value > 0.05 was considered not statistically significant (ns). The level of significance was set at *p ≤ 0.05, **p < 0.01, and ***p < 0.001.One-way analysis of variance (ANOVA) coupled with Newman‐Keuls post-hoc tests were performed to assess statistical significance between groups. All results are presented as mean ± standard error (SE) from 2 independent experiments using GraphPad Prism 5 . A CYP24A1 and VDR were tested by qRT-PCR in polyI:C-stimulated BSMCs, with or without 1,25D3 for 24 hours. CYP24A1 gene expression was significantly upregulated upon addition of 1,25D3 to polyI:C-stimulated BSMCs, while polyI:C alone had no significant effect. The addition of 1,25D3 to polyI:C-stimulated BSMCs significantly induced CYP24A1 mRNA expression in asthma  (p < 0.01)  (CYP24A1 and VDR expression (p < 0.05) when 1,25D3 alone was added to the cells and this effect was observed at a higher extent in diseased BSMCs as compared to non-diseased BSMCs,  for 24 hours significantly induced mRNA expression of TLR3, in asthma  (p < 0.001)  (p < 0.05) (p > 0.05) on TLR3 mRNA expression in BSMCs, , polyI:C at 5 µg/ml was considered optimal and used to determine the pro-inflammatory and pro-fibrotic responses in BSMCs. Because 1,25D3 has anti-inflammatory and anti-fibrotic effects, we hypothesized that 1,25D3 treatment decreases the pro-inflammatory and pro-fibrotic responses in polyI:C-stimulated BSMCs. Therefore, BSMCs were stimulated with polyI:C (5 µg/ml) alone or in combination with 1,25D3 (100 nM) for 24 hours. Following stimulation, BSMCs were collected, and RNA was extracted. As shown in IL-6, IFN-β1, CCL2 compared to untreated cells and this effect was observed to a higher extent in asthma and COPD BSMCs (p < 0.05). When 1,25D3 was added to polyI:C-stimulated BSMCs, there was a significant decrease in mRNA expression of IL-6, IFN-β1, CCL2. In addition, we observed a higher extent of the anti-inflammatory effect of 1,25D3 in BSMCs from COPD, namely for IL-6   compared to BSMCs from COPD  than in COPD (p > 0.05). Although not statistically significant, we observed an increased overall mRNA expression of pro-inflammatory and pro-fibrotic markers at the baseline in unstimulated-BSMCs from asthma , as a mechanism contributing to pulmonary fibrosis in asthma and COPD. Polyinosinic: polycytidylic acid (polyI:C), a synthetic analog of viral double-stranded RNA, has been previously described in driving viral immunomodulatory response  and dermCYP24A1 when 1,25D3 was added to the polyI:C stimulated BSMCs, whereas polyI:C alone slightly altered its mRNA expression (CYP24A1) encodes 24-hydroxylase, an enzyme that regulates vitamin D metabolism through a negative feedback loop activation, thereby regulating its own metabolism  may have increased sensitivity to polyI:C than BSMCs from control groups.To gain a mechanistic insight into the effect of vitamin D3 on the activation and functionality of vitamin D receptors (VDR) in BSMCs, the effects of 1,25D3 supplementation of polyI:C-stimulated BSMCs was investigated. We observed a significant increase in the mRNA expression of tabolism . This reTLR3, which may indicate their ability to respond to innate immune stimuli and a possible role in viral-induced inflammatory exacerbations. Although TLR3 agonists are well known to stimulate type I interferons (IFN-α and IFN-β1), their activation also results in upregulation of a variety of NF-kB regulated pro-inflammatory cytokines and chemokines, including IL-6, IL-8, tumor necrosis factor alpha (TNF-α) and CCL2  Smad signaling, and the ECM turnover mechanisms in asthma and COPD , 36. Ourr levels .Study limitations. From the clinical data, patients with severe asthma or COPD are predisposed to severe lung injury and presented an increased risk of fibrosis compared to mild-to-moderate illnesses. The main limitation to this study is that BSMCs from COPD group were exclusively from subjects with mild COPD because of sample availability. However, it is also known that subjects with mild COPD presented underlying inflammation in the airways and are at increased risk of respiratory infections compared to healthy subjects , 38. AnoRecent clinical evidence identified COPD and asthma as comorbidities in COVID-19 infections, and patients with severe COVID-19 infection have extensive pulmonary fibrotic tissue, in addition to an enhanced inflammatory state –41. AlthIn conclusion, our findings demonstrated that TLR3 agonist polyI:C induce pro-inflammatory and pro-fibrotic responses in BSMCs and suggest their potential for deregulation of the pathways involved in fibrotic responses. Moreover, we have demonstrated that 1,25D3 by VDR-TLR3 dependent pathways, significantly reduced the pro-inflammatory and pro-fibrotic effects in polyI:C-stimulated BSMCs, indicating a protective function of 1,25D3 in patients with chronic respiratory conditions and viral infections. Further research is required to shed the light on the interplay between SARS-CoV-2, PRRs and other lung cell types, in the context of 1,25D3 supplementation as a potential antiviral adjuvant therapy in COVID-19 patients with asthma and COPD.All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by Human Research Ethics Board of the University of Manitoba. The patients/participants provided their written informed consent to participate in this study.MP performed experiments, analyzed the data, and drafted the manuscript. MG and AM contributed to data analysis and manuscript preparation. NJ participated in the revision of the manuscript. AH provided BSMCs from healthy smokers and COPD subjects and participated in the revision of the manuscript. SA advised throughout the study development and participated in the revision of the manuscript. QH contributed to the design of the experiments and participated in the revision of the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by the Richard and Edith Strauss Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Although mechanistic studies clarifying the molecular underpinnings of AML have facilitated the development of several novel targeted therapeutics, most AML patients still relapse. Thus, overcoming the inherent and acquired resistance to current therapies remains an unsolved clinical problem. While current diagnostic modalities are primarily defined by gross morphology, cytogenetics, and to an extent, by deep targeted gene sequencing, there is an ongoing demand to identify newer diagnostic, therapeutic and prognostic biomarkers for AML. Recent interest in exploring the role of circular RNA (circRNA) in elucidating AML biology and therapy resistance has been promising. This review discerns the circular RNAs’ evolving role on the same scientific premise and attempts to identify its potential in managing AML. Acute myeloid leukemia (AML) is characterized by clonal expansion of myeloid blasts in the bone marrow and peripheral blood. AML was originally classified based on the morphological appearance of the myeloid blasts , and subRecent advances in high-throughput sequencing demonstrated that the majority of mRNA ~ 75%) of the entire human transcriptome do not get translated into proteins and are called non-coding RNA (ncRNA) % of the , 7. AlteCircRNA is a single-stranded ncRNA which, due to a closed-loop structure secondary to a phosphodiester bond between the 3′ and 5′ ends, as well as the absence of a 3′ polyadenyl tail, has increased stability and a longer half-life compared to linear RNA . The samLong non-coding RNA (lncRNA) is another ncRNA produced during transcription by RNA polymerase II and is expressed in a tissue-specific manner . In contCircRNA, specifically, has been implicated in Alzheimer’s disease, diabetes mellitus, cardiovascular diseases, and many other conditions, including but not limited to cancer . AberranVIM positively correlated with circ-VIM expression. Though VIM expression’s prognostic significance was not assessed in this study, authors alluded to the possibility of circ-VIM as a tumor promoter.Multiple studies have explored the potential use of circRNAs in the diagnosis and prognostication of AML. Variable expression of specific circRNAs studied in different model systems have been found to be prognostic in AML. Yi et al. demonstrated upregulation of circ-VIM by means of reverse transcriptase and quantitative polymerase chain reaction (RT-qPCR) in AML patients’ bone marrow samples compared to healthy controls . Based oSimilarly, in another study, circ-Foxo3 was downregulated in de novo AML patients, and circ-Foxo3 expression positively correlated with Foxo3 gene expression . ROC anaFLT3 and NPM1 mutation status compared to four healthy controls [In another study, microarray-based analysis revealed 147 up and 317 downregulated circRNAs respectively in six newly diagnosed cytogenetically normal AML (CN-AML) patients with varying prognostic risk based on controls . FurtherMitofusin 2 gene on chromosome 1) and circ-ANAPC7 were upregulated in AML patients compared to controls with iron deficiency [Circ_009910  associated with poor prognosis, and 2 genes (PAPLN and NRXN3) whose overexpression correlated with better prognosis.More recently, whole-genome microarray comparing bone marrow mononuclear samples from extramedullary infiltrating (EMI) AML and non-EMI AML patients revealed 253 and 259 circRNAs to be up and downregulated, respectively, in EMI compared to non-EMI AML patients . The autFOXM1. High expression of circ-PTK2 was associated with shorter survival in AML patients, presumably due to its effect on leukemic cell proliferation and apoptosis.Another study demonstrated circ-PTK2 (hsa_circ_104700/hsa_circ_0005273) to be upregulated in AML bone marrow samples compared to healthy controls . KnockdoIn another study, microarray analysis performed on bone marrow samples from patients with acute lymphoblastic leukemia (ALL) and AML revealed differential expression of 10 specific circRNAs . For exaIn summary, circRNAs have potential utility in AML as a diagnostic and prognostic biomarker and as a means of monitoring disease status and therapeutic response. The role of circRNA measurement to monitor disease status in the context of commercially available clinical measurable disease (MRD) monitoring remains to be determined. As MRD influences AML treatment decisions, it creates more opportunities to study circRNA further in this setting.Apart from being a diagnostic/prognostic biomarker in AML, the role of circRNA in AML biology and pathogenesis has also been explored Table . There i, and accelerated tumor growth in vivo. Circ-DLEU2 upregulated PRKACB gene expression via sponging of miRNA-496. Note that PRKACB protein is the catalytic subunit of cyclic AMP-dependent protein kinase and is thus involved in regulating various cell signaling processes. While studies have reported the role of parent gene DLEU2 in leukemogenesis [DLEU2 expression and its effects were not investigated in this study.Although there are numerous factors contributing to leukemogenesis in AML, evading apoptosis seems to have a critical role . A recenogenesis , 43, DLEFLI-1 were found to be upregulated in AML patients [FLT3-ITD patients than in FLT3 wild-type (WT) patients and was associated with a poor prognosis. In Vitro, FLT3-ITD expression positively correlated with circ_0000370 expression, and quizartinib (a FLT3 inhibitor) downregulated circ_0000370 expression. Circ_0000370 regulated cell survival and apoptosis via increased S100A7A expression by sponging miR-1299. In a different study, Sun et al. demonstrated that circMYBL2  expression to be approximately five-fold higher in FLT3-ITD AML patients than in FLT3 WT patients [FLT3-ITD leukemic cells but not FLT3 WT cells. Further, circMYBL2-KD led to reduced FLT3 kinase expression, resulting in modulation of downstream signaling, like reduced phosphorylation of STAT5 (which in turn resulted in reduced expression of c-MYC). In addition, circMYBL2 enhanced translation of FLT3 kinase by facilitating the polypyrimidine tract-binding protein 1 (PTBP1) binding to FLT3 messenger RNA. In previous studies, FLT3-ITD mutation was associated with increased MCL1 and decreased p27/Kip1 expression, which led to enhanced apoptosis resistance [FLT3-ITD AML progression and was associated with improved survival in mice with quizartinib sensitive and resistant AML. Overall, these results suggest the effects of driver mutations like FLT3 ITD in AML biology may be mediated (or at least modulated) by altered expression of circRNAs (circ_0000370 and circMYBL2).In a study by Zhang et al., both circ_0000370 and its parent gene patients . Circ_00patients . CircMYBsistance , 47. CirRNF13) gene at chromosome 3q25.1, was significantly upregulated in pediatric AML patients. Circ-004136 sponges miR-142 and miR-29a and regulates AML cell proliferation. Another study also demonstrated upregulation of has_circ_0001346 (circRNF13) in AML patients [In another study, 273 and 296 circRNAs were up and downregulated respectively in pediatric AML patients compared to healthy controls . Circ-00patients . In thisGSK3beta (a tumor suppressor) are shown to be down-regulated in leukemia cells [GSK3beta promoter to recruit DNA demethylase TET1. The resultant upregulation of GSK3beta expression inhibits the Wnt/beta-catenin signaling pathway, leading to leukemia cell proliferation.Both the circRNA (hsa_circ_0121582) and its parent gene ia cells . Hsa_cirOther investigators have also demonstrated an association between circRNA and evasion of apoptosis. Hsa_circ_100,290 (encoded by parent gene SLC30A7) was upregulated in AML patients . Hsa_cirWhether altered expression of circRNA is sine qua non for AML leukemogenesis, or whether it is simply one of several potentially overlapping abnormalities affecting several steps in AML disease biology remains to be fully determined. Much of the work described herein suggests sponging miRNA and resultant alteration of target gene expression is a central function of circRNA as it relates to leukemogenesis, other mechanisms, including direct binding to the gene promoter regions, need to be further characterized, as well.NPM1 gene, followed a similar expression pattern as the parent NPM1 gene, independent of NPM1 mutation status. High expression of hsa_circ_0075001 associated with no or minimal blast maturation and down-regulation of Toll-like receptor signaling pathway.The historical classification of acute leukemia (FAB) relied on lineage and maturational features of blasts, as reflected by morphological features. A global evaluation of circRNA expression from healthy hematopoietic controls and AML patients identified hematopoietic differentiation-associated and AML subgroup-specific signatures . Hsa_cirSimilarly, in another study, whole-transcriptome profiling of 365 younger adults with CN-AML  identifiPrimary or secondary refractoriness to treatment  is a key limiting factor for optimal outcomes in AML patients. Genetic alterations leading to aberrant activation of drug resistance-related signal pathways is a well-known resistance mechanism in AML . ElucidaPAN3) in leukemogenesis. CircPAN3 was upregulated in relapsed/refractory compared to chemosensitive AML patients, though the parent gene expression did not follow a similar pattern. In cell lines, circPAN3 down-regulation was associated with restoring doxorubicin sensitivity, and in contrast, up-regulation was associated with doxorubicin refractoriness. The circPAN3-miR-153-5p/miR-183-5p-XIAP (X-linked inhibitor of apoptosis protein) axis was proposed as a mediator of drug resistance. Also, other investigators established circPAN3-mediated autophagy regulation via the AMPK/mTOR pathway as a resistance mechanism in AML cell lines [ANXA2 (Annexin A2) expression was not evaluated in this study, but it was proposed that Circ-ANXA2 might enhance transcription of annexin A2, which has a well-established role in leukemogenesis [Shang et al. studied circRNA expression patterns in doxorubicin (ADM)-resistant THP1 cells and ADM sensitive cells and demonstrated that 49 circRNAs were differentially expressed (35 up- and 14 down-regulated) in doxorubicin (ADM)-resistant THP1 cells compared to ADM sensitive cells . CircPANll lines . Similarll lines . Circ-ANll lines , 61. ANXogenesis , 63.Structural uniqueness of circRNA can be challenging to study these molecules and it is one of the reasons for being less investigated than other components of the transcriptome. Sequencing of ribosomal RNA (rRNA) depleted total RNA was traditionally used in earlier studies . Though The precise mechanism of regulation of circRNA expression in AML is not well studied. Except for fusion circRNAs, it is unclear if dysregulated circRNA expression is a primary event in leukemogenesis or an epiphenomenon. Also, most studies have used bone marrow samples, and only a few had peripheral blood samples. Correlative studies between bone marrow samples and peripheral blood are also lacking.There is very limited knowledge about the metabolism and transport of circRNA within and outside the cell. It is known that surplus circRNAs are transported out of the cell in exosomes . AlthougRelative expression of circRNA and the corresponding parent gene varies and thus may not be regulated in a uniform fashion across the entire genome. In a recent study, the expression of most circRNAs correlated with the expression of the parent gene in both AML patients and healthy controls, but there are instances of discordant expression , and alsAlthough KD circRNA studies represent a potential opportunity to assess the impact of circRNA expression on that of the parent gene, most studies reviewed herein did not evaluate the circRNA-KD effect on the parent gene expression , 53, 59.AML is a challenging, biologically heterogenous disease with an unmet need for novel diagnostic, therapeutic, and prognostic markers. In this review, we summarized the biological and clinical significance of circRNA in AML. The role ncRNAs, particularly circRNAs, in each of these clinical facets, as well as of leukemogenesis in general is an evolving horizon.