FLT3 and TP53. Our data indicate that hematological cells with elevated MCL1- and MEK-protein levels are most sensitive to the combined treatment with S63845 and trametinib. MCL1- and MEK1/2-protein expression may be valid biomarkers for treatment response to S63845 and trametinib, respectively. Amplification and overexpression of the myeloid cell leukemia differentiation protein MCL1 and the murine double minute protein MDM2 have been reported in various human tumors as well as hematological malignancies including acute myeloid leukemia (AML). While MCL1 is an anti-apoptotic member of the BCL-2 family proteins, MDM2 is an important cellular inhibitor of the p53 tumor suppressor. The key oncogene in AML is the FLT3 growth factor receptor gene. FLT3 signaling pathways including the MAPK cascade (RAS-RAF-MEK-ERK) are highly active in AML cells, leading to induced protein translation and cell proliferation as well as reduced apoptosis. Consequently, combined administration of MCL1-, MDM2-, and MEK-inhibitors may present a promising anti-leukemic treatment strategy. Here, we assessed the MCL1-antagonist S63845, the MDM2-inhibitor HDM201, and the MEK1/2-inhibitor trametinib as single agents and in combination in a variety of AML cell lines and mononuclear cells isolated from patients with hematological malignancies centered on myeloid leukemia, some lymphatic leukemia, as well as some lymphomas, for their ability to induce apoptosis and cell death. We observed a considerably varying anti-leukemic efficacy of the MCL1-inhibitor S63845 and the MEK1/2-inhibitor trametinib. Hematological cells with susceptibility to the single compounds as well as to the combined treatment were defined by elevated MCL1- and MEK-protein levels, independent of the mutational status of  Tumor cells have evolved mechanisms to prevent induction of apoptosis. In hematological malignancies including acute myeloid leukemia (AML) tumor cells prevent apoptosis by elevated expression of anti-apoptotic proteins of the BCL2 family. The induced myeloid leukemia cell differentiation protein 1 (MCL1), an anti-apoptotic member of the BCL2 family proteins, is often upregulated in AML cells, particularly at relapse [S63845 is a new selective MCL1-inhibitor with potential use across a number of hematological malignancies including myeloma, AML, and lymphoma [FLT3) gene. This growth factor receptor is often overexpressed in AML cells with a normal FLT3 gene [FLT3 mutation [The key oncogene in AML is the FMS-kinase 3 , OCI-AML3 , MOLM-13 , TP53wt), and MOLM-16  cell lines were supplied by the Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures. AML cells were grown in RPMI 1640  supplemented with 20% fetal bovine serum . We passaged the cells for a maximum of twelve times. OCI-AML2  and bone marrow mononuclear cells (BMMCs) were collected at the time of diagnosis before initiation of treatment. Mononuclear cells of 22 patients with hematological malignancies diagnosed and treated at the University Hospital, Bern, Switzerland between 2015 and 2018 were included in this study. Informed consent from all patients was obtained according to the Declaration of Helsinki, and the studies were approved by the local ethics committee of Bern, Switzerland, decision number #221/15. Mutational screening for AML cells were treated with compound diluent only (controls) or with the MCL1-inhibitor S63845 , the MDM2-inhibitor NVP-HDM201 , and the MEK1/2-inhibitor trametinib . Cell viability was determined using the MTT-based in vitro toxicology assay . For AML cell lines, four independent assays  with four measurements  per dosage were performed. For hematological patient samples two independent assays with three technical replicates per dosage were performed. Data were analyzed on GraphPad Prism software using Mann–Whitney tests and are depicted as average values with standard deviation on column graphs.Combination indexes were calculated on CompuSyn software , according to the method of Chou and Talalay ,17. For Imaging cytometry was done on the NC-3000 cell analyzer  with reagents supplied by ChemoMetec. To determine induction of cell death, apoptotic cells were stained with AnnexinV-CF488A conjugate  in AnnexinV buffer and Hoechst 33342 (10 μg /mL) for 15 min at 37 °C, followed by several washes. Three independent assays with two technical replicates per dosage were performed. Data were analyzed on Graphpad Prism software using Mann–Whitney tests and are depicted as column graphs with average values and standard deviation. ® 680LT goat anti-mouse IgG . Membranes were scanned on a LI-COR Odyssey Infrared Scanner . Bands were quantified on Image Studio software. Three independent assays were performed. One representative blot is depicted. Total protein extracts were prepared by cell lysis in radioimmunoprecipitation assay buffer (RIPA lysis). A total of 100 μg total protein extracts were separated on polyacrylamid gel by electrophoresis (PAGE), transferred to a nitrocellulose membrane and incubated with mouse anti-phospho-S6 (Ser235/236) cupk43k , mouse anti-actin IgG , followed by IRDyeProtein extraction was done according to standard protocol. In short, cell pellets were lysed in RIPA buffer. GAPDH, FLT3, MCL1, MEK1, and MEK2 protein levels were determined with double-antibody Sandwich ELISA . FLT3, MCL1, and MEK1/2 values were normalized with GAPDH values. Two independent assays with three technical replicates were performed per sample. Statistical analysis was performed using averaged normalized values and the Mann–Whitney test or linear regression on GraphPad Prism software. Data are depicted as scatter plots with median values, or as XY graphs with linear regression.FLT3-ITD and FLT3 wildtype, NPM1 mutant and wildtype, as well as TP53 mutant and wildtype genes. To determine the sensitivity of AML cells to S63845, HDM201, and trametinib, AML cell lines were studied in in vitro cytotoxicity assays. The cell lines covered the major molecular AML subtypes characterized by AML cells lines varied considerably with respect to S63845, HDM201, and trametinib sensitivity . MOLM-13Synergistic effects on cell viability were detected in the combination treatments with S63845 and trametinib in OCI-AML3 and MOLM-13 cells and in the combination treatments with S63845 and HDM201 in OCI-AML2, OCI-AML3, and MOLM-13 cells with combination indexes of 0.2 to 0.5. Synergism was calculated to be strong in OCI-AML3 cells for the combination of 100nM S63845 and 10nM trametinib or 100nM HDM201 with combination index values of 0.2 to 0.3. In direct comparison trametinib had hundredfold greater potency compared to HDM201, as 1nM trametinib and 100nM HDM201 had the same effect on MOLM-13 cells. Hence, the later studies in malignant cells of hematological patients were focused on the combination treatment with trametinib and S63845.To determine whether the loss of cell viability was due to induction of apoptosis, AML cell lines were studied in cytometric assays. The number of apoptotic cells was determined by annexin V staining. Apoptosis was induced in MOLM-13 and OCI-AML3 cells treated with S63845 and trametinib A,B. S638In summary, these data suggest that treating AML cells with the MCL1-inhibitor S63845 and the MEK-inhibitor trametinib leads to reduced levels of phospho-Ser-RPS6 protein, to inhibition of translation, and induction of apoptosis, as summarized in FLT3 wildtype and FLT3-ITD, TP53 wildtype and mutated, de novo, and secondary AML (FLT3 wildtype NK-AML (AML 1–4), the FLT3-ITD positive NK-AML (AML 5–8) appeared to be more susceptible to trametinib  A. Cytotodary AML C. The thametinib D, and leametinib E. The suametinib F. The AMp = 0.0005) (p = 0.0004). MEK1 protein levels ranged from 0.002 to 0.16 MEK1/GAPDH, while MEK2 protein levels ranged from 0.001 to 0.02 MEK2/GAPDH. Elevated MEK levels were present in cells susceptible to 50nM trametinib, with median values of 0.09 versus 0.006 MEK/GAPDH (p = 0.0006)  A–C. AML  0.0006) D–F. AML  0.0006) G–I. TherThe aim of this study was to identify subsets of patients with hematological malignancies susceptible to the combined treatment with the MCL1 antagonist S63845 in combination with the MDM2-inhibitor HDM201 or the MEK-inhibitor trametinib. The three inhibitors were assessed as single agents and in combination in a variety of AML cell lines. In direct comparison trametinib had a hundredfold greater potency compared to HDM201, as 1nM trametinib and 100nM HDM201 had the same effect on MOLM-13 cells. Hence, the studies in malignant cells of hematological patients were focused on the combination treatment with trametinib and S63845. Hematological cells characterized by elevated MCL1-protein levels were susceptible to S63845 treatment, while cells with elevated MEK protein levels were susceptible to trametinib. Consequently, susceptibility to the combination treatment with S63845 and trametinib was greater in hematological cells with elevated MCL1 and MEK levels. Thus MCL1 and MEK protein expression levels in the leukemic cells may be response predictors in combination treatments with S63845 and trametinib. The significance of the in vitro susceptibilities would have to be corroborated in a clinical phase I study, as effective drug concentrations may be different in vivo.The molecular mechanisms underlying the correlation of MCL1- and MEK1-protein levels and response to S63845 and trametinib may be related to the phenomenon of oncogene addiction, where the tumor relies on a dominant oncogene or mechanism for growth and survival, so that inhibition of this specific oncogene/pathway is sufficient to halt the neoplastic phenotype ,23. The FLT3 appear to be susceptible to FLT3- and MDM2-inhibitors, in particular the FLT3 inhibitor midostaurin and the MDM2-inhibitor HDM201 [FLT3-ITD positive AML, a combination of FLT3- and MDM2-inhibitor may be a justified first line therapy, which could be followed by a second line therapy with a combination of MCL1- and MEK-inhibitor. Different combinations of targeted therapies emerge to be suitable for the treatment of specific subsets of AML patients. While NK-AML cells with mutated r HDM201 , NK-AML r HDM201 . Here weThe MCL1 inhibitor S63845 and the MEK inhibitor trametinib may be effective therapeutic compounds in acute myeloid leukemia. Here we found that the MCL1-inhibitor S63845 and the MEK-inhibitor trametinib specifically target primary cells of acute myeloid leukemia with elevated MCL1- and MEK-protein levels. MCL1- and MEK1/2-protein expression may be valid biomarkers for treatment response to S63845 and trametinib, respectively.


The cancer metabolic reprogramming allows the maintenance of tumor proliferation, expansion and survival by altering key bioenergetics, biosynthetic and redox functions to meet the higher demands of tumor cells. In addition, several metabolites are also needed to perform signaling functions that further promote tumor growth and progression. These metabolic alterations have been exploited in different cancers, including acute myeloid leukemia, as novel therapeutic strategies both in preclinical models and clinical trials. Here, we review the complexity of acute myeloid leukemia (AML) metabolism and discuss how therapies targeting different aspects of cellular metabolism have demonstrated efficacy and how they provide a therapeutic window that should be explored to target the metabolic requirements of AML cells. Acute myeloid leukemia (AML) is the most frequent acute leukemia in adults, with a median age at diagnosis of around 68 years. It is an aggressive disease, requiring an urgent accurate diagnosis and a multifaceted, timely, and effective therapeutic approach in order to achieve disease remission and even long-term cure . DespiteThe strategies used by malignant cells to escape the mechanisms of homeostasis control are appreciably varied, with metabolic reprogramming playing a key role in the final outcome. Metabolic reprogramming is considered a hallmark of cancer and, basically, consists of providing a rewired metabolism to support high metabolic demands for the elevated rate of growth and proliferation. Indeed, to maintain tumor proliferation, expansion, and survival, a reprogramming of metabolism must be performed to satisfy key bioenergetics, biosynthetic and redox functions of malignant cells. In addition to the altered metabolism, several metabolites have a signaling function, promoting tumor growth and progression . NumerouNutrient addiction is considered a hallmark of cancer and this metabolic reprogramming has two fundamental aspects: first, the rapid production of ATP and intermediates for the synthesis of nucleotides, amino acids, lipids, and redox elements (NADPH); second, the reset of the nutrient-sensing signaling pathways operating in physiological conditions. In addition, to support fast cell division, the tumor cells must generate enough energy and escape from cell cycle checkpoints control. The Warburg effect is a well characterized metabolic phenotype of tumor cells, which consists of increased glycolytic flux for the rapid ATP generation per unit time, instead of producing ATP through oxidative phosphorylation (OXPHOS)  Figure . Below, Enhanced glycolysis has been observed in AML cell lines and in human primary AML blasts , while bThe high glycolytic profile has been shown to be important for chemotherapy response, having been found to confer greater resistance to apoptosis induced by All-Trans-Retinoic-Acid (ATRA) and arsenic trioxide in primary blasts and AML cell lines, besides having an impact on the clinical prognosis . IncreasMetabolic reprogramming in cancer cells occurs far beyond the Warburg Effect ,20. The Glutaminolysis supplies nitrogen for protein and nucleotide synthesis and is necessary to maintain the redox state through the production of NADPH . It is rLipid metabolism dysregulation has been established as an important metabolic phenotype in malignant cells. It is overly reported that the various processes inherent to lipid metabolism, such as fatty acid synthesis and fatty acid oxidation (FAO) are altered in many cancers ,31. In A2 and NADH, which are then oxidized to generate ATP via OXPHOS , leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG) , especially in patients with normal cytogenetics, and that IDH2 mutation improves OS particularly in patients with intermediate-risk AML [In the last decade, mutations in two key metabolic enzymes, cytosolic and mitochondrial isocitrate dehydrogenases , have been identified in myeloid malignancies ,39. IDHspatients ,41. Thes relapse . IDH mute (2-HG) , which i in IDH2 ,45. It hpatients . Nonetherisk AML .As mentioned above, multiple signaling pathways control metabolic reprogramming of leukemic cells including PI3K/AKT, mTOR, and the AMP-activated protein kinase (AMPK). This is a complex network hub of signaling proteins and kinases functioning as sensors and regulators of response to environmental stimuli which govern cell growth, proliferation, and survival. These pathways integrate the availability of nutrients, mitogenic growth factors, cellular energy levels, and stress with the biosynthesis of three major classes of macromolecules . These signaling pathways have been reported to regulate normal hematopoiesis, reviewed in , but aremTORC1 is an important anabolic kinase in the regulation of normal hematopoiesis which is inhibited when amino acids and oxygen levels are decreased. Insofar, mTORC1 suppression contributes to HSCs maintenance by repressing mitochondrial biogenesis. Being HSCs in the hypoxic environment of the bone marrow, they are dependent on glycolysis for their energy needs more than of OXPHOS, so mTORC1 suppression is vital for their homeostasis ,54. mTORFLT3, mutations in the c-KIT gene, increased production of angiogenic factors  and angiopoietins) and increased expression of IGF-1 growth factor [Constitutive activation of mTORC1 has been systematically identified in patients’ myeloid blast cells. There are several mechanisms responsible for this activation, namely, activating mutations in the receptor tyrosine kinase h factor . Furtherh factor . mTORC1 h factor ,59. In sh factor ,63,64,65ATG10 and ATG12, GABARAPL2 and MAP1LC3B, or GABARAP, respectively [ATG5 increases the progression and aggressiveness of an AML model [ATG5 in primary AML cells resulted in impaired engraftment of human cells in immunodeficient NSG mice [Curiously, similar to the signaling pathways controlling this catabolic degradative process, dysregulation of autophagy has been extensively described in AML with both tumor-suppressor and -promoter functions ,66,67,68ectively . AccordiML model . A recenNSG mice .We have previously addressed the relationship between signaling pathways and autophagy in the context of AML cell lines . The reshttps://clinicaltrials.gov).New drugs targeting metabolism in AML have been developed and studied in preclinical and clinical trials. These drugs target, among other, glycolysis, amino acids and fatty acid metabolism, signaling pathways, autophagy, metabolic enzymes , and epigenetic modifiers. Importantly, a clinical trial, still in the recruitment phase, aims to determine the global metabolic adaptations that occur following exposure to standard chemotherapeutic agents using peripheral blood mononuclear cells from AML patients (NCT02581917). Below, a brief summary of the main approaches targeting metabolism in AML is presented , IDH305, AG-881, BAY-1436032, and FT-2102 are some of the IDH1 inhibitors under investigation . Ivosidee events . In concIDH1/2 and in the epigenetic modifier enzymes—DNMT3A and TET2—were detected in more than 70% of AML patients [TP53 mutations, constituting a possible therapeutic option in cases with poor prognoses [Mutations in patients . All of patients ,103. It rognoses . In factrognoses .Guadecitabine is a new-generation hypomethylating agent with improved uptake of decitabine, the active metabolite, into the DNA of rapidly dividing cancer cells . DecitabIDH1- and IDH2-mutant primary human AML cells were more sensitive than IDH1/2 wild-type cells to venetoclax, which was due to mitochondrial electron transport chain alterations that lowered the threshold of apoptosis induced by this BCL-2 antagonist [An important therapeutic strategy in AML is the combination of venetoclax  with lotagonist . In viewtagonist .Due to the relevance of PI3K/AKT, mTOR, and AMPK on AML pathogenesis, these signaling pathways present appealing therapeutic targets. In a murine model in which primary leukemic cells were co-cultured with stroma, PP242, a mTOR inhibitor, simultaneously showed inhibitory activity against mTORC1 and mTORC2 as well as an antagonism against the stromal-induced survival signal, by suppressing expression of CXC chemokine receptor type 4 (CXCR4) . This efA potential mechanism for PI3K activation in AML might implicate increased VEGF levels . EnhanceAMPK pathway has also been implicated in hematological malignancies, mainly as a tumor suppressor axis ,120,121,Although several studies have demonstrated beneficial effects of drugs targeting signaling pathways in preclinical models of AML, results of clinical trials are either modest, or disappointing ATG5, ATG7, or the ULK1-interacting partner Fip200 diminished normal HSCs activities, promoted a pre-leukemic phenotype and, consequently, impaired survival of these mice [ATG5 or ATG7 are required for the efficient initiation of AML associated with MLL (mixed lineage leukemia)-AF9 fusion gene, while autophagy is no longer required for the maintenance of established AML or LSCs functions in secondary xenotransplantation experiments [ATG5 or ATG7 in a murine MLL-ENL (eleven nineteen leukemia) AML model decreased the number of functional LSCs, increased activation of mitochondria and ROS levels in these cells, and extended survival of leukemic mice [Decreased mTOR activity as well as nutrient or growth factor deprivation increase autophagy, therefore constituting an essential survival pathway . Autophaese mice ,69,127. eriments ,130,131.mic mice . These amic mice .TP53 mutations [Recently, it was also shown that autophagy suppression mediated by receptor tyrosine kinase effectors-mTORC1 signaling stabilize mutant FLT3 in AML, while an increase in autophagy was achieved through receptor tyrosine kinase inhibition and led to FLT3 depletion . In contutations .Altogether, these contradictory data suggest a highly complex, context-dependent role for autophagy in leukemic transformation and LSCs properties in AML . To furtAML is a very heterogeneous and complex group of diseases and, despite advances in therapeutic strategies, there is still a high rate of resistance to the standard treatment. Accordingly, it is necessary to find innovative ways of approaching and treating patients with AML. Age, performance status, white blood cell count, secondary disease, cytogenetic risk, and the mutational state are incorporated in the predictive models of resistance . HoweverThe results reviewed above show that energy metabolism is a fundamental process that should be considered in AML therapy. Depending on the balance between glycolysis and OXPHOS, the amino acid requirements of AML cells also change, and this could be useful for metabolic targeting. Importantly, lipid metabolism is emerging with great therapeutic potential given the alterations found in AML cells. Nevertheless, the knowledge of the complexities of lipid metabolism is still scarce, and probably this is one of the reasons why no current clinical trials are found which feature drugs targeting this axis of metabolic reprogramming. Summing up, more preclinical and clinical studies have to be performed in order to establish the best metabolic target (or combination of targets) that is better suited to affect LSCs sparing HSCs. Hopefully, the current preclinical and clinical ongoing studies will shed light on the yet unknown aspects of metabolic reprogramming in AML.In this review, we intended to provide an updated look at AML therapy; this new perspective will, eventually, allow age constraints that current therapies still have to be overcome and, in addition, facilitate the prediction of better results in refractory cases in the near future.


It is shown that rigidity‐dependent FAK and Ca2+ signals in human mesenchymal stem cells (hMSCs) are selectively activated at detergent‐resistant membrane (DRM or rafts) microdomains during the cell–matrix adhesion process, with minimal activities at non‐DRM domains. The rigidity‐dependent Ca2+ signal at the DRM microdomains is downregulated by either FAK inhibition or lipid raft disruption, suggesting that FAK and lipid raft integrity mediate the in situ Ca2+ activation. It is further revealed that transient receptor potential subfamily M7 (TRPM7) participates in the mobilization of Ca2+ signals within DRM regions. Thus, the findings provide insights into the underlying mechanisms that regulate Ca2+ and FAK signals in hMSCs under different mechanical microenvironments.The dynamic regulation of signal transduction at plasma membrane microdomains remains poorly understood due to limitations in current experimental approaches. Genetically encoded biosensors based on fluorescent resonance energy transfer (FRET) can provide high spatiotemporal resolution for imaging cell signaling networks. Here, distinctive regulation of focal adhesion kinase (FAK) and Ca Even less is known about the spatial organization of activation patterns of FAK and Ca2+ at the plasma membrane, which is structurally organized into two functional microdomains called detergent‐resistant membrane (DRM) and non‐DRM regions.2+ is crucial for the initiation of FAK and Ca2+ signals.Cell adhesion to the surrounding matrix starts with the binding of integrin receptors in the plasma membrane to ECM proteins. Binding to matrix proteins such as fibronectin and collagen leads to integrin clustering and subsequent downstream assembly of both mechanical structures and chemical signaling complexes, including adaptor proteins, cytoskeletal components, catalytic signaling proteins, and secondary messengers.The DRM, known as lipid rafts membrane, is enriched with sphingolipids and cholesterol, whereas non‐DRM domains lack these lipid compositions.2+ biosensors to investigate the regulation of these two signals during cell–matrix adhesion process in hMSCs seeded on substrates with different stiffness. Through live single cell imaging, we first demonstrate that rigidity‐dependent FAK and Ca2+ signals are selectively enriched at detergent‐resistant membrane (DRM) microdomains in a concerted manner, but not at non‐DRM during this adhesion process. Furthermore, we report that transient receptor potential subfamily M7 (TRPM7) is involved in the regulation of matrix rigidity‐dependent Ca2+ signals at DRM microdomains, which is mediated by the functional FAK and the integrity of lipid rafts.In this study, we take advantage of membrane‐targeting fluorescent resonance energy transfer (FRET)‐based FAK and Ca22.1Figuren = 15, ***P < 0.001) , but not on 0.6 kPa gel (n = 10)     Figure2a–c. I2.32+ signals also play an important role in cell adhesion and spreading.2+ signals could be activated at plasma membrane microdomains. To perform this study, two distinct types of FRET‐based Ca2+ biosensor were engineered to tether at DRM and non‐DRM microdomains using Lyn and Kras peptide sequences, respectively , suggesting that similar Ca2+ influx occurs at these two different membrane microdomains DRM versus non‐DRM regions with ionomycin treatment. Interestingly, the Ca2+ increase during cell–matrix adhesion process selectively occurred only at the DRM microdomains in a rigidity‐dependent manner, but with undetectable signals at non‐DRM regions . This pattern of calcium regulation during adhesion process is similar to that of FAK activation.Similar to FAK, Caly Figure3a,b. T2+ signals are differentially mobilized at different plasma membrane microdomains during the process of cell–matrix adhesion in hMSCs. It is possible that there is a high level of buffer proteins such as calmodulin specifically localized at the non‐raft regions, which can bind to and neutralize free Ca2+ diffused from other subcellular domains, for example, DRM and cytosolic organelles. This is consistent with earlier reports that there are different compositions of membrane proteins at different microdomains during cell adhesion processes, including Ca2+ channels, integrins, and FAK.We have previously reported that the membrane targeting motifs should not affect the function of fused FRET biosensors.2.42+ mobilization and FAK activation during the adhesion process, both of which are dependent on the magnitude of matrix rigidity. In contrast, these two signals were poorly activated at non‐DRM regions regardless of matrix rigidity. To further examine whether Ca2+ signals are correlated with FAK signals, we took advantage of two FAK mutants, FAK NT  and FAK KD (a kinase dead mutant). These mutants were cotransfected into cells expressing the calcium biosensor Lyn‐D3cpv. As shown in Figure2+ signaling activity at DRM microdomains . Pharmacological inhibitor PF228 or ML‐7 also decreased Ca2+ mobilization within the DRM region . These results indicate that Ca2+ mobilization at the DRM region is regulated by FAK during the cell–matrix adhesion process.In this study, we found that within DRM microdomains, hMSCs displayed higher Ca2+ mobilization. One possibility is that FAK directly affects Ca2+ channels at DRM regions. Even though FAK is mostly interacting with integrins at the plasma membrane, it is possible that FAK can physically interact with mechanosensitive membrane channels.2+ channels indirectly in an integrin‐dependent manner. In fact, FAK modulates different types of voltage‐gated Ca2+ channels at the plasma membrane via integrins.2+ influx through the plasma membrane has also been shown to closely correlate with the activation of high affinity β2 integrin and subsequent adhesive signals due to their dynamically coupled events.The physiological interpretation of these phenomena requires further investigation to understand detailed mechanisms with regard to how FAK regulates Ca2.52+ mobilization at these local regions during the cell–matrix adhesion process, by depleting cholesterol with MβCD. As shown in Figure n = 5–7, ***P < 0.001), we found that MβCD treatment caused significant inhibition of FRET changes in Lyn‐D3cpv calcium biosensor, while a mutant of caveolin‐1 (Cav1S80E) did not affect the Ca2+ mobilization at DRM microdomains . These results suggest that cholesterol at DRM microdomains is crucial in Ca2+ mobilization during cell–matrix adhesion process, which is not dependent on the caveolin‐1 function.It is reported that cholesterol, along with caveolin, plays an important role in signal transduction at DRM microdomains.2+ influx under mechanical stretch,2+ influx. While our previous study has shown that cholesterol disruption by MβCD treatment inhibited FAK activation at DRM region upon PDGF stimulation,Although it is known that caveolae disruption prevents Ca2.62+ channels at DRM regions is different from that of non‐DRM regions. For example, TRPC3 and TRPC6 are dominantly expressed at non‐DRM regions, but other TRPC channels are expressed throughout the plasma membrane.2+ permeable channel, which is known to dominantly accumulate at DRM microdomains, but not at non‐DRM regions.2+ signals. Indeed, previous studies support the note that TRPM7 and integrin‐FAK complex may be closely interrelated. For example, TRPM7 regulates focal adhesions by controlling m‐calpain,2+ mobilization at DRM microdomains during the cell–matrix adhesion process. In our previous reports, we have shown that the delivery of TRPM7 siRNA clearly suppressed the expression of TRPM7 in hMSCs.2+ mobilization at the DRM region, with nontargeting (NT) siRNA (control group) having minimal effects  . Our finding hence suggests that TRPM7 is a crucial regulator of matrix rigidity‐dependent Ca2+ mobilization at DRM microdomains during the adhesion process. The colocalization of TRPM7 with Lyn, a DRM marker, as well as with paxillin and p‐FAK (Tyr397), provides additional supporting evidence that TRPM7 locally associated with FAK and integrin/focal adhesion complex . As such, a putative connection between FAK and TRPM7 mediated by CSK‐actomyosin may contribute to the overall control of Ca2+ mobilization.The distribution of TRP Ca32+ and FAK signaling specifically occurring within DRM microdomains in hMSCs on hard and soft matrix at the initial stage of cell–matrix adhesion  originated from Lyn kinase to the N‐terminus of the cytosolic‐FAK biosensor. Kras‐FAK biosensor was also constructed by insertion of a non‐raft‐targeting motif: a prenylation substrate sequence from Kras (KKKKKKSKTKCVIM) to the C‐terminus of the cytosolic‐FAK biosensor.Escherichia coli, and in pcDNA3.1 plasmid for the expression in mammalian cells. As FAK mutants, the kinase‐dead FAK with its kinase domain mutated (FAK KD) and the N‐terminal tail (containing 1–400 amino acids) of FAK (FAK NT) were used in this study.2+ biosensors based on FRET were generated in the same manner. The plasmids Lyn‐D3cpv and Kras‐D3cpv were constructed by fusion of a raft‐targeting motif: the myristoylation and palmitoylation sequence from Lyn kinase (MGCIKSKRKDNLNDDGVDMKT) to the N‐terminus of the D3cpv and a non‐raft‐targeting motif (KKKKKKSKTKCVIM) to the C‐terminus of the D3cpv.Cell Culture and Chemicals: hMSCs  were purchased from Lonza and maintained in mesenchymal stem cell growth medium  containing 10% fetal bovine serum (FBS), 2 × 10−3m l‐glutamine, 100 U mL−1 penicillin, and 100 µg mL−1 streptomycin in a humidified incubator of 95% O2 and 5% CO2 at 37 °C. The DNA plasmids were transfected into the cells  by using Lipofectamine 2000 or LTX  reagent according to the product instructions. PF228, ML‐7, methyl‐beta‐cyclodextrin (MβCD), and naltriben methanesulfonate hydrate were purchased from Sigma Aldrich .RNA Interference Assays: Small interfering RNA (siRNA) sequences targeting human TRPM7 (ON‐TARGETplus SMARTpool siRNA) and nontargeting control sequences were designed by Dharmacon RNAi Technology . Cotransfection of 1–2 µg siRNA specific for TRPM7 (L‐005393‐00) or a nontargeting pool (D‐001810‐10‐05) along with the plasmid DNA was conducted according to the product instructions.Bis‐acrylamide‐PA Gel Fabrication: The fabrication of PA hydrogel with a defined modulus of elasticity (E or stiffness), a characteristic of the ECM can be a useful technique to study the interactions of cells with their mechanical microenvironment. Such matrix substrate from PA gels can be created by simply changing relative concentration of acrylamide and bis‐acrylamide.−1, sulfosuccinimidyl 6(4′‐azide‐2′‐nitrophenyl‐amino) hexanoate, Pierce) was used. For adhesion via integrins, 200 µL of a 0.1 mg mL−1 fibronectin solution  was incubated overnight with the PA gel at 37 °C.Image Acquisition and Microscopy: FRET sensor‐transfected cells were incubated for 1 h on 1% agarose dishes in a humidified incubator of 95% O2 and 5% CO2 at 37 °C to maintain in a state of suspension after detachment with 4 × 10−3m EDTA in phosphate buffered saline (PBS). During imaging process, the cells were maintained with MSCGM in a chamber, which was designed to provide the constant humidified air containing 5% CO2, 10% O2, and 85% N2. The 37 °C degree of temperature throughout the samples in the chamber was maintained by a controlled heater (Nevtek ASI 400). Nikon Eclipse Ti inverted microscope with a cooled charge‐coupled device (CCD) camera was used for the image acquisition under perfect focus system (PFS) which allows for minimizing any focal change during cell–matrix adhesion. The pixel‐by‐pixel ratio images of FRET/ECFP were quantified after background subtraction in fluorescence intensity images of FRET and ECFP. The emission ratio images were computed and quantified by the MetaFluor software, and presented in the intensity modified display (IMD) mode. The following filter sets were utilized in the imaging experiments: dichroic mirror (450 nm), excitation filter for ECFP (420/20 nm), emission filter for ECFP (480/40), and FRET emission filter (535/25 nm).Immunofluorescence (IF) Staining and Confocal Microscopy: IF staining was carried out on hMSCs seeded on 40 kPa substrate. Fixation was performed with 4% paraformaldehyde in PBS for 10 min at room temperature, followed by washing three times with PBS. Fixed cells were permeabilized with 0.1% Triton‐X100  in PBS for 15 min and blocked with 3% BSA  in PBS for 1 h at room temperature. The cells were then stained with primary antibodies, mouse anti‐TRPM7 , and rabbit anti‐phospho‐FAK  overnight at 4 °C and secondary antibodies, FITC‐conjugated anti‐mouse IgG  and CFL 555 conjugated anti‐rabbit IgG  at room temperature for 1 h. After washing three times with PBS, the samples were mounted in VECTASHIELD medium containing DAPI  and stored at 4 °C in the dark. Confocal images were acquired using a Leica laser scanning confocal microscope (TCS‐SP8) with a 40× Plan‐Apo objective/numerical aperture (NA) 1.40.Statistical Analysis: All statistical data were expressed as the mean ± standard error of the mean (s.e.m.). Statistical evaluation was performed by unpaired t‐test and one‐way ANOVA using Graphpad Prism 6.0 software to determine the statistical differences between groups. A significant difference was determined by the P‐value (<0.05).Y.W. is a scientific co‐founder of Cell E&G Inc. D.‐H.K is a co‐founder and scientific board member of NanoSurface Biomedical Inc. However, these financial interests do not affect the design, conduct, or reporting of this research.SupplementaryClick here for additional data file.


In failing hearts both electrophysiological and structural remodeling, including fibroblast proliferation, contribute to changes in Ca2+ handling which promote the appearance of Ca2+ alternans . Ca-alt in turn give rise to repolarization alternans, which promote dispersion of repolarization and contribute to reentrant activity. The computational analysis of the incidence of Ca2+ and/or repolarization alternans under HF conditions in the presence of fibroblasts could provide a better understanding of the mechanisms leading to HF arrhythmias and contractile function disorders.Heart failure (HF) is characterized, among other factors, by a progressive loss of contractile function and by the formation of an arrhythmogenic substrate, both aspects partially related to intracellular Cain silico the mechanisms leading to the formation of Ca-alt in failing human ventricular myocytes and tissues with disperse fibroblast distributions. The contribution of ionic currents variability to alternans formation at the cellular level was analyzed and the results show that in normal ventricular tissue, altered Ca2+ dynamics lead to Ca-alt, which precede APD alternans and can be aggravated by the presence of fibroblasts. Electrophysiological remodeling of failing tissue alone is sufficient to develop alternans. The incidence of alternans is reduced when fibroblasts are present in failing tissue due to significantly depressed Ca2+ transients. The analysis of the underlying ionic mechanisms suggests that Ca-alt are driven by Ca2+-handling protein and Ca2+ cycling dysfunctions in the junctional sarcoplasmic reticulum and that their contribution to alternans occurrence depends on the cardiac remodeling conditions and on myocyte-fibroblast interactions.The goal of the present study was to investigate It can thus be concluded that fibroblasts modulate the formation of Ca-alt in human ventricular tissue subjected to heart failure-related electrophysiological remodeling. Pharmacological therapies should thus consider the extent of both the electrophysiological and structural remodeling present in the failing heart. The pacing cycle length (PCL) threshold was determined by computing a restitution curve starting at a PCL of 1000 ms and reducing the cycle length until loss of capture was observed. Each simulation was run until steady-state was reached. The last two APs and CaTs were used to measure AP duration from maximal upstroke to 90% of repolarization (APD+ current (INa), late Na+ current (INaL), transient outward K+ current (Ito), L-type Ca2+ current (ICaL), rapid delayed rectifier K+ current (IKr), slow delayed rectifier K+ current (IKs), inward rectifier K+ current (IK1), Na+/ K+ ATPase current (INaK), Na+/Ca2+ exchange current (INCX), SR Ca2+ uptake via SERCA pump (JSERCA), SR Ca2+ release flux via RyR (Jrel), SR Ca2+ leak (Jleak), and Na+ background current (INab). Four basic cellular models were considered with different baseline conditions and/or coupling to build the populations: normal myocyte coupled to normal myocyte (N-N), normal myocyte coupled to fibroblast (N-Fb), failing myocyte coupled to failing myocyte (HF-HF), and failing myocyte coupled to fibroblast (HF-Fb). Populations of 300 different human AP models were analyzed and compared to study the role of ion channel variation in alternans generation.After determining pacing threshold in the baseline models, populations of models were built and simulations were carried out at a fixed low PCL in which alternans tend to occur. Populations of models consisted of different cellular models built after varying the selected parameters related to ionic currents of the baseline model, mimicking biological inter-individual variability. We modified 13 maximal ion channel conductances and fluxes simultaneously using random scale factors obtained from a normal distribution of mean equal to 1 and standard deviation (σ) equal to 0.15, so that 95% (±2σ) of the parameters varied in the range ±30% of their baseline values. The selected variables affect the conductances of the fast Na2+ alternans ratio JSR  was very similar for all four groups, but the normal myocytes (N-tissue and N-Fb-tissue) had the lowest values of [Ca2+], while HF (HF-tissue and HF-Fb-tissue) had the highest Ca2+ levels of the interval covered by the different models.Differences were found in CaT amplitude of the alternans and no alternans groups of the different populations. Since intracellular Ca]JSR see . The lin90 and [Ca2+]i peak for two consecutive beats (blue for even and red for odd beats) in a representative case from each population, quantified in the central nodes of the tissue along the longitudinal axis (x). APD-alt patterns are long-short (L-S) and Ca-alt patterns are large-small (L-S). When a long APD is concomitant with a large CaT (same color) there is positive V-Ca coupling or electromechanical concordance, otherwise there is electromechanical discordance. Consecutive dots with the same color show that alternans between adjacent myocytes are in phase , while changes in color indicate a change in alternans patterns between consecutive myocytes . Spatial concordance in APD alternans is predominant in the four populations because all the myocytes are in phase following the same pattern in APs. This can be seen in both N-tissue  due to myocyte-fibroblast interactions.In addition to spatial differences, Since fibroblasts seem to contribute to a more chaotic spatial distribution of Ca-alt, the whole tissue is represented in Electrically discordant alternans are the most arrhythmogenic type, but concordant alternans often precede discordant alternans and play an important role. Ca-alt help in the identification of APD-alt, as they are concomitant, but also indicate dysfuntional contraction behavior. Discordant Ca-alt could aggravate the situation by increasing the number of myocytes that do not contract synchronously.2+ alterations and reveals how Ca2+ impairment in HF or fibroblast interaction influences the development of cellular alternans and how they appear in cardiac tissue. Our main findings are as follows: (i) Ca2+ mechanisms, involving SERCA, ICaL, and NCX, are responsible for the occurrence of cellular alternans by modulating Ca2+ cycling to threshold levels; these mechanisms act differently in normal and failing mocytes, (ii) the degree of Ca2+ impairment, given by [Ca2+]JSR, determines the myocyte susceptibility to Ca-alt development, (iii) fibroblasts induce discordant Ca-alt in normal cardiac tissues, (iv) the high number of alternans in failing tissue with myocyte-fibroblast interaction is reduced due to exacerbated Ca2+ impairment, and (v) discordant Ca-alt are more prone to be translated into discordant APD-alt in failing tissue with fibroblasts than in normal tissue with fibroblasts. Fibroblasts and HF can therefore promote alternans because they alter Ca2+ dynamics, deteriorate contractility and increase the risk of cardiac arrhythmia. Restoring Ca2+ dynamics could thus potentially be used to prevent alternans and improve cellular contraction and cardiac function, although the differences between normal and failing tissues underline the need to consider HF when developing new pharmacological treatments.Mechanical and electrical alternans, which are known to be bidirectionally coupled, may compromise cardiac function. This study shows that alternans have their origin in Ca2+ cycling (as in our case) myocytes-fibroblast coupling promotes alternans because the alternans onset is then driven by Ca2+ cycling instabilities [2+ dynamics, which also oscillates from beat to beat, is one of the most important factors in alternans research. The bidirectional coupling between membrane voltage and intracellular Ca2+ explains why APD-alt and Ca-alt occur concomitantly [90 and Ca2+. However, there is still controversy about the origin and underlying causes of these fluctuations, due to the Ca2+ dependence of ion channels, which also control Ca2+ cycling [Although cardiac alternans usually occur at fast pacing rates, the threshold for the onset of fluctuations can vary between individuals. It is also a characteristic outcome in heart disease. The first cellular theory for cardiac alternans was based on the relationship between APD alternans and the APD restitution slope . Alternabilities . In factmitantly , as can  cycling ,47–49.2+ subsystem at high frequencies (PCL< 300 ms). They also occur in a 100-endo-cell strand, and pacing rates faster than 230 ms cause 2:1 block [In the present computational study, in which the ORd model was used , the alt:1 block , which a:1 block . In tissOur first simulations at different PCLs determined that Ca-alt were easier to identify than APD-alt and so this was used as the criterion to detect cellular alternans. In previous studies, AP amplitude alternans have also been used to explain T-wave alternans instead of APD variations and were able to predict malignant arrhythmias in patients with systolic dysfunction . However2+-handling proteins . This agrees with the findings of Zhou et al. [Kr was low, but not to other parameters, since the alternans were measured as differences in AP and CaT duration. As a novelty, we introduced fibroblast coupling to normal and failing myocytes and then compared them to myocyte-myocyte coupling. The results indicated that although the mechanisms driving alternans in the different populations were similar they had opposite effects on normal conditions and HF.Introducing natural electrophysiological variability into ionic channel conductances and transporters provides a population of coupled cells with different electrophysiological characteristics. The analysis of the model variants prone to alternans revealed the electrophysiological mechanisms that promote these fluctuations, i.e. the Cau et al.  and Walmu et al.  for non-u et al.  associat2+ cycling [2+-handling proteins help to maintain normal Ca2+ cycling in myocytes to ensure contractility and their disruption can lead to impaired Ca2+ dynamics in the different cell compartments. This has been linked to beat-to-beat Ca2+ oscillations [2+ content [2+ reuptake and Ca2+ release explains the genesis of Ca-alt [2+ into the sarcoplasmic reticulum (SR), is involved in alternans-prone myocytes, while SERCA overexpression may possibly suppress cardiac alternans, as has been shown by Cutler et al. [2+ uptake into the SR [2+ cycling improves although it still remains at failure levels and can lead to alternans. These levels are mainly determined by junctional SR Ca2+ maximal content. Normal myocytes can also reach these failing Ca2+ levels when mechanisms like ICaL, SERCA, NCX, and INaK are altered, after which Ca-alt may occur. With reduced ICaL, Ca2+-induced Ca2+ release (CICR) tends to fail, high NCX contributes to intracellular Ca2+ imbalance by extruding Ca2+ out of the cell, and the higher INaK could indirectly facilitate inward NCX.CaMK appears to play an important role in alternans generation as it controls Ca cycling ,52. Thisllations , includi content , which af Ca-alt ,31. We fr et al. . Howevero the SR –56. Our 2+ cycling model integrated in the AP model could explain how Ca2+ sparks can lead to cellular alternans due to the randomness, refractoriness and recruitment properties of the couplon network [2+ cycling model, such as the one formulated in the ORd model, RyRs cannot show refractoriness, which could explain why we found that, unlike other parameters, changes in RyR hardly affected CaT. However, Tomek et al. [2+ release through RyR can also affect vulnerability to alternans. We therefore believe that incomplete JSR recovery is the factor that affects Ca2+ release and generates alternans [Many experimental studies have also highlighted the role of RyR in alternans generation due to RyR refractoriness and a failure in the CICR process ,29,57. C network . Howeverk et al.  showed tlternans .2+ ions [2+ coupling, which reduces APD-alt [Regarding the spatiotemporal patterns in cardiac tissue, we found that alternans entail different degrees of risk that depend on individual conditions. Electrical propagation runs smoothly in normal tissue, which explains the tissue’s spatial and electromechanical concordance. However, we found that cellular uncoupling in HF may promote the formation of alternans, in agreement with Hammer et al. , who fou2+ ions  in compa APD-alt . Electro APD-alt  when pas APD-alt , the use2+-handling proteins is the main cause of Ca-alt in HF, which is then translated to APD-alt. Although we found spatially concordant alternans in HF tissue, they could become discordant at lower PCL, as was shown in [2+ impairment and reduced CV, which may contribute to the positive APD-alt and Ca-alt coupling. Failing myocytes become susceptible to Ca-alt with unstable Ca2+ dynamics provided that CaT is not very depressed, and the resultant repolarization alternans can be high. If the tissue properties were to trigger spatial discordance, these alternans could become arrhythmogenic.Electrophysiological remodeling of Cashown in . There ishown in . It is d2+ dynamics in HF could not improve cardiac function but did contribute to Ca-alt development and its associated effects. The conclusion is therefore that mechanisms that restore Ca2+ instabilities produced by fibroblasts and/or HF help to recover normal myocyte electrophysiological activity and avoid heart problems only if they are applied to specific heart conditions.Arrhythmogenic activity in cardiac tissue can have a non-electric origin if Ca-alt is the initial cause. In contrast to normal hearts, the attempt to restore Ca2+ cycling pathways in non-myocytes because of the lack of data related to cardiac fibroblasts, and this could contribute to Ca2+ dynamics in myocytes and therefore to developing alternans. Myofibroblasts, the contractile fibroblast activated form, arise in response to injury and present a different phenotype from fibroblasts. However, as no specific electrophysiological models of these contractile cells have been developed to date, in the present study we used the same mathematical fibroblast model in both normal conditions and HF, in spite of the fact that fibroblast electrophysiology may be altered in response to HF. We implemented the most critical of the different configurations for myocyte-fibroblast coupling, in which fibroblasts form conduction pathways and act as an electrotonic load. Representing fibroblasts as the same size as myocytes in a continuous mesh still has certain limitations.Mathematical models can help in the mechanistic analysis of alternans onset and complement experimental findings, although computational results may be limited by a number of uncertainties. Only one fibroblast model and one configuration for myocyte-fibroblast coupling were considered. The electrophysiological fibroblast model is thought to be more accurate, as it represents the ion currents in the fibroblasts, but using a different model (eg. passive fibroblast) or variations in the parameters could change the results. In our simulations in particular the fibroblast model does not take into account the potential CaAnother limitation was the scarcity of human electrophysiological data, which would have been very useful to validate our findings. Our results were thus compared to animal models, including some species with a different electrophysiology to that of humans.Despite its limitations, this study provides information that plausibly explains the response of myocytes to HF and fibroblast coupling in humans and can thus be used as a guide in the design of HF treatments.


Despite its significance, the mechanisms that maintain the cochlear lymphatic Ca2+ concentrations within a certain range are not fully clarified. We investigated the functional expression of calcium-sensing receptor (CaSR), which plays a pivotal role in sensing extracellular Ca2+ concentrations for feedback regulations. Western blotting for CaSR revealed an approximately 130-kDa protein expression in cochlear tissue extracts and immunohistochemical analysis revealed its expression specifically in type I fibrocytes in the spiral ligament, fibrocytes in the supralimbal and limbal regions, the epithelium of the osseous spiral lamina, and the smooth muscle cells of the spiral modiolar arteries. Ca2+ imaging demonstrated that extracellular Ca2+ increased the levels of intracellular Ca2+ in CaSR-expressing fibrocytes in the spiral ligament, and that this was suppressed by the CaSR inhibitor, NPS2143. Furthermore, hearing thresholds were moderately elevated by intracochlear application of the CaSR inhibitors NPS2143 and Calhex231, across a range of frequencies (8–32 kHz). These results demonstrate the functional expression of CaSR in the cochlear perilymphatic compartment. In addition, the elevated hearing thresholds that are achieved by inhibiting CaSR suggest this is a required mechanism for normal hearing, presumably by sensing perilymphatic Ca2+ to stabilize Ca2+ concentrations within a certain range. These results provide novel insight into the mechanisms regulating Ca2+ homeostasis in the cochlea and provide a new perspective on cochlear physiology.Maintaining Ca In addition, perilymphatic Ca2+ concentration is also low (~1.3 mM) compared with that of the plasma  secretion and in turn, regulating serum Ca2+ homeostasis  is a G protein-coupled receptor that detects Ca2+ imaging studies, 4-week-old Wister/ST rats (100–130 g) were used.All experiments involving animals were performed using procedures approved by the Institutional Animal Care and Use Committee of Nagoya City University . Five-week-old adult male Wistar/ST rats (150–180 g) were purchased from Japan SLC Inc., Shizuoka, Japan. For CaRRID:AB_2071589) in 5% normal goat serum and 5% skim milk in Tris-buffered saline. Protein bands were visualized by applying a horseradish peroxidase (HRP)-conjugated secondary antibody , and were detected using a chemiluminescent reagent . Kidney tissues were used as a positive control. Anti-ß-actin antibody  was used as an internal loading control. The blot was visualized and bands were imaged using an Amersham Imager 600 and its associated software .For western blotting, cochleae and whole kidneys from 5-week-old adult male Wistar/ST rats were snap frozen and stored at −80°C until protein extraction. Tissues were homogenized in ice-cold RIPA lysis buffer , after which 30 μg of homogenates were fractionated by SDS-PAGE and transferred by electrophoresis onto a PVDF membrane . Membranes were blocked with 5% skim milk  and 5% normal goat serum . Immunoblotting was performed using a mouse monoclonal anti-CaSR antibody  for 1 h at room temperature. After rinsing with PBS, sections were mounted with ProLong Gold antifade reagent  and stained with DAPI (Life Technologies). For CaSR staining, incubation without primary antibody was used as a negative control (Frozen sections were treated with Tris-EDTA buffer (pH 9.0) for antigen retrieval. After rinsing with PBS, slides were preincubated overnight at 4°C in 10% normal goat serum and 0.1% Triton-X dissolved in PBS, followed by incubation with the monoclonal anti-CaSR antibody  in 10% normal goat serum and 0.1% Triton-X in PBS overnight at 4°C. After rinsing with PBS, the sections were incubated with goat secondary antibodies conjugated to Alexa 594 . Rats were euthanized by cervical dislocation with ketamine (100 mg/kg) and xylazine (10 mg/kg); the temporal bones were dissected in dissection solution . Subsequently, the apical turn was dissected with the spiral ligament in fresh dissection solution, and rat cochlear tissue for Ca2+ imaging was processed for recording as previously described ; for the Ca2+-free HEPES-buffered solution, 2.2 mM CaCl2 was removed and 1 mM EGTA was added to normal HEPES-buffered solution. CaSR-immunoreactive fibrocytes were located lateral to the stria vascularis and medial to the fibrocytes lining the otic capsule. Therefore, to focus on CaSR-labeled fibrocytes, the responses of fibrocytes in the center of the layer between the two sides of the spiral ligament were recorded.To determine the functionality of CaSR expressed in fibrocytes of the spiral ligament, we performed CaFive-week-old rats were anesthetized and placed on a heating pad  to maintain body temperature. Using an aseptic technique, a postauricular incision was made on the right side, and a small hole was made on the otic capsule. A glass capillary needle with a tip diameter of approximately 100 μm was inserted into the round window membrane and either 4 μl of vehicle (DMSO) or CaSR inhibitor solution was applied at the following concentrations: NPS2143, 2 μM ; Calhex231, 0.1 μM . Solutions were injected into the perilymphatic space with a microinjector  at a speed of 0.133 μl/min over 30 min, which is a feasible protocol for inner ear preservation  recordings, tone-burst stimuli were digitally generated by System II  according to the manufacturer’s instructions, and presented in a closed field. The duration of tone burst stimuli was 0.1 ms, and four different frequencies were applied . Auditory thresholds were obtained for each stimulus by reducing the sound pressure level (SPL) in 10-dB steps for rough measurements and 5-dB steps when approaching the threshold to determine the precise threshold to identify the lowest sound pressure at which an ABR wave could be visually detected, which was found to be a maximum intensity of 100 dB SPL and a minimum intensity of 15 dB. This was done by comparing ABR patterns with two or three suprathreshold ABRs displayed successively on the screen. The pharmacological effects of each inhibitor were evaluated 60 min after finishing the injection to allow diffusion into the cochlea .Unless otherwise noted, all data are presented as mean ± standard error of the mean. Differences between two groups were evaluated using Student’s CaSR transcripts were previously detected by reverse transcription-PCR (RT-PCR) analysis of the spiral modiolar artery, the expression of which was much lower than that observed in the kidney , we used a 2.2-mM Ca2+ solution to achieve an extracellular Ca2+-induced increase in intracellular Ca2+ levels . Considering that this extracellular solution has a relatively low Ca+ levels . In fibr+ levels , analysi ± 0.069 , while operiment . In addie manner .2+ concentrations could be inhibited by the CaSR-selective inhibitor NPS2143 . This inhibition by NPS2143 was reversible, as calcium concentrations increased to 0.320 ± 0.082 (73% recovery) when the inhibitor was washed out of the solution . These results confirm that this injection protocol preserves hearing and is in line with previous reports —that is, marginal cells of stria vascularis and interdental cells  are located directly behind cells that intensely express plasma membrane Ca2+ homeostasis in the syncytia of connective tissue. For example, the TRP channels involved in Ca2+ transport in absorptive epithelial cell membranes have also been identified in the connective tissue system and its adjacent lesion. The Ca2+-selective TRP channels TPPV5 and TRPV6 were identified in the stria vascularis and in the spiral ligament, respectively  NPS2143 and Calhex231 had a moderate impact on hearing, inducing 20–30 dB hearing loss throughout the frequency range examined. One possibility for this hearing impairment is the inhibition of CaSR signaling, which suppresses the entry of extracellular calcium into the connective tissue system as suggested by the results of imaging. This is similar to human mesangial and renal tubular cells where CaSR activation suppresses Ca2+ concentrations in lymphatic fluids and its physiological significance in normal hearing.In summary, our results provide an understanding of the feedback mechanism that exists to regulate CaThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.All experiments involving animals were performed using procedures approved by the Institutional Animal Care and Use Committee of Nagoya City University .AI contributed to the conception and design of the study. TM and AY organized the database. AI, TM and HY performed the statistical analysis. AI wrote the first draft of the manuscript. HY wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.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.


This review seeks to evaluate the notion that teaching, tutoring, role modelling, coaching and supervision may in fact all be part of the mentoring process. To evaluate this theory, this review scrutinizes current literature on teaching, tutoring, role modelling, coaching and supervision to evaluate their commonalities with prevailing concepts of novice mentoring.Recent studies have gone to great lengths to differentiate mentoring from teaching, tutoring, role modelling, coaching and supervision within Internal Medicine, published between 1st January 2000 and 31st December 2018. Braun and Clarke’s (2006) approach to thematic analysis was used to identify key elements within these approaches and facilitate comparisons between them.A three staged approach is adopted to evaluate this premise. Stage one involves four systematic reviews on one-to-one learning interactions in Stage two provides an updated view of one-to-one mentoring between a senior physician and a medical student or junior doctor to contextualise the discussion.Stage three infuses mentoring into the findings delineated in stage one.Seventeen thousand four hundred ninety-nine citations were reviewed, 235 full-text articles were reviewed, and 104 articles were thematically analysed. Four themes were identified – characteristics, processes, nature of relationship, and problems faced in each of the four educational roles.Role modelling, teaching and tutoring, coaching and supervision lie within a mentoring spectrum of increasingly structured interactions, assisted by assessments, feedback and personalised support that culminate with a mentoring approach. Still requiring validation, these findings necessitate a reconceptualization of mentoring and changes to mentor training programs and how mentoring is assessed and supported. This would see mentoring encompassing supervision’s role and occupying the outer most ring in Fig. Mentoring’s use of personalized holistic and longitudinal support throughout the mentoring process would require mentees to be taught, and provided with guidance as they apply their knowledge and skills, be assessed and provided with feedback and then re-evaluated before progressing to the next stage of the mentoring process. At each stage of the mentoring process which Krishna et al. (2019)  describementoring spectrum . The mentoring spectrum describes a range of educational practices contained under the aegis of mentoring beginning with role modelling on the left side of the spectrum and mentoring on the right. Beginning with role modelling, there is progressively complex interactions culminating with personalized attention in role modelling behaviours, attitudes and practices, teaching new skills and knowledge, coaching individual learner’s on different aspects of the skills they need, appraising their progress and providing feedback as they are supervised to complete their immediate goals within the project. Separating supervision from the more complex relationships seen in mentoring is the provision of personalized, timely, holistic and longitudinal support and the adaptation of the mentoring approach to accommodate the mentee’s needs, goals, circumstance and abilities.This suggests that role modelling, teaching, tutoring, coaching and supervision lie within a rum Fig. . The menMotivation of learnersOther features that evidence the notion of a mentoring spectrum include2.Learning RelationshipAll educational approaches are reliant upon the learner’s ability to observe, discern gaps in their ability, learn, reflect, weigh up considerations, be open to feedback and be accountable for their own learning. However, the approaches rely on increasing learner motivation moving from left to right of the mentoring spectrum.Moving from left to right along the mentoring spectrum also highlights increasing planning and structuring of the mentoring process. Improved structuring of educational interactions better supports learning relationships and nurtures more holistic and personalised educational relationships. Better learning relationships also facilitate better outcomes.3.Nurturing learning environmentLearning relationships also become more interactive moving from left to right in the mentoring spectrum. In role modelling, learners may not have an educational relationship with the tutor whilst learning relationships in mentoring are dynamic and enduring , 116–1194.Learning assessmentBuilding a learning relationship relies on the learning environment and moving from left to right of the mentoring spectrum sees learning environments becoming increasingly important to the quality and nature of the learning relationship. These learning environments also become more individualised and serve to nurture particular learning relationships within the larger educational environment. This is especially evident in supervision and mentoring , 91.4.LAssessments also play an increasing role moving from left to right of the mentoring spectrum. These assessments must be timely, appropriate and personalised and accompanied by open and frank discussions and personalized, appropriate, specific, timely, holistic, accessible and longitudinal feedback and support , 116–119The implication upon mentor training is significant. Acknowledging the roles to be played within the mentoring spectrum highlights the need for mentors to be trained in all these educational approaches.The unplanned and unconscious nature of role modelling and the need for balance between personalising and consistency within the mentoring approach both highlight the need for clear standards of practice, codes of conduct and practice guidelines (henceforth Codes of Practice or CoP). There must also be opportunities for mentee and mentors to align expectations and accept their responsibilities and roles and for mentees and mentors to be briefed on the prevailing goals and timelines of their respective educational projects and processes.The learners and tutors must also be appropriately matched to ensure that they have complementary working styles, learning approaches and personalities, goals and abilities , 121. ThAn overarching mentoring spectrum combining role modelling, teaching and tutoring, coaching, supervision, and mentoring has wide ramification upon how these educational approaches are employed.i.mentors-in-training to be trained and skilled on all these educational approaches and be mentored when applying these skill sets and competencies for each of the educational roles.ii.mentors and mentees to be briefed on CoPs, expectations on roles, responsibilities and expectations and effective oversight, assessment, and support provided by the host organization.iii.robust, longitudinal and holistic assessment processes in light of the changing nature of the mentoring process and the mentor’s roles and the presence of evolving mentoring relationships and different stages of the various aspects within the mentoring spectrum.iv.the host organization to take an active role in overseeing and providing personalized, appropriate, specific, timely, holistic, accessible and longitudinal financial and administrative support in running and overseeing the mentoring process given the diverse processes within the mentoring spectrum , 116–119v.that the mentoring process is sufficiently structured to accommodate for the inevitable changes in the mentoring process without breaching the CoP.vi.the need for a safe and nurturing working environment that will nurture trusting and enduring mentoring relationships that will not only enhance better role modelling when the mentee has established ties with the mentor but also facilitate discussions that extend beyond professional issues which will allow the provision of holistic support.vii.the need for an open and safe mentoring culture that allows open discussions, constructive feedback and frank discussions.The implications of the mentoring spectrum upon mentoring practice is vast and includes requiringThis review posits that these practices are interrelated is based on a number of novel yet unproven assumptions. Selecting only four of the many educational roles also limits the scope of understanding of the entire spectrum of educational roles in mentoring. In addition, the practices described in this review focus specific education settings, and draw from a particular definition of role modelling, teaching and tutoring supervision and coaching that may not be applicable in other education settings. Within the context of role modelling for example, there is no consideration of negative role modelling which limits the validity of the conclusions reached. In addition, many of the papers contextualized within the European and American healthcare system and training programs, limit their applicability to other educational and healthcare systems.The findings of this review not only suggest a new way of conceptualizing mentoring but also highlights the need for further study into the matching, pre-mentoring, mentoring relationship, mentoring evaluations, mentoring structure, the mentoring environment and mentoring culture. This theoretical concept though supported by data from novice mentoring processes will still need to be carefully studied and validated. One key area for further study must be the manner that mentees, mentors and the host organization interact (mentoring dynamics) given its influence upon all processes within the mentoring spectrum. Similarly, important is the design of effective assessment tools and policing of the mentoring process and the mentoring environment.However, we are confident that this new concept of mentoring will enhance the mentoring process and mentoring outcomes as medical education strives toward personalized medical education.