Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/β-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/β-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/β-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3β at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3β was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of β-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating β-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the β-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.The protozoan parasite,  Trypanosoma cruzi, the causative agent of Chagas disease, is now considered a global health threat in all industrialized regions of the world. About 30% of infected individuals will develop cardiac pathology. The molecular mechanisms by which the parasite signals and infect host cells to cause pathology remain to be elucidated. We showed that host thrombospondin-1 (TSP1) is essential for T. cruzi infection since cells deficient in TSP1 are poorly infected by the parasite. In this study, we challenged mouse heart endothelial cells from wild type and TSP1 KO mice and evaluated the importance of TSP1 in Wnt/β-catenin signaling, β-catenin/YAP colocalization and how it can be exploited to regulate parasite infectivity. The parasite activated Wnt/β-catenin signaling pathway and nuclear β-catenin/YAP colocalization. In TSP1 KO MHEC (absence of TSP1), the parasite induced a continuous increase in nuclear translocation and colocalization of β-catenin/YAP. These results suggest that during the early phase of T. cruzi infection, TSP1 regulates β-catenin/YAP interaction. Inhibition of the Wnt/β-catenin pathway in WT MHEC led to a significant reduction of parasite infectivity. The level of parasite infection of the pretreated WT cells was as low as that of TSP1 KO MHEC. Our studies show that the β-catenin pathway is an important therapeutic target for T. cruzi infection. Trypanosoma cruzi, the causative agent of Chagas heart disease is an obligate intracellular parasite that can infect all nucleated cells of the body. The disease, which was originally endemic in Mexico and other Latin American countries, is now present in all economically advanced countries of the world including the United States in particular due to modern globalization making it a new global health threat [Triatoma infestans, has now been shown to be transmitted by several other modes including the use of contaminated blood/blood products, needles, use of contaminated organs during organ transplant and transmission through the oral route during consumption of contaminated foods or drinks [h threat –5. The dr drinks .T. cruzi-infected women of childbearing age and congenitally infected infants among the Latin American migrants [T. cruzi trypomastigotes infect host cells and transform to replicative amastigotes within the infected cells. The amastigotes multiply and transform to invasive trypomastigotes, which are released to infect other cells. Some of the released trypomastigotes infect neighboring cells while others are transported in blood through the host’s vascular system to infect cells in distant parts of the body. During parasite transportation in the blood, invasive trypomastigotes interact with and potentially infect endothelial cells lining the internal surface of the vascular wall through which blood is transported.Vertical transmission from mother-to-child has now become a new research and public health policy challenge especially as there is a substantial population of migrants ,8. In admigrants ,10. DuriEndothelial cells lining the inner surface of the vascular system are surrounded by the extracellular matrix proteins, which interact with matricellular proteins. Matricellular proteins are extracellular matrix (ECM) proteins that interact with cells and other ECM components to regulate cellular behavior and ECM organization but are not part of the structural elements of the ECM –14. One T. cruzi. The parasite induces the expression of TSP1 in host cells, including primary human coronary artery smooth muscle cells, to facilitate cellular infection [T. cruzi calreticulin (TcCRT) on the surface of the parasite to facilitate cellular infection, which was inhibited in the presence of TcCRT monovalent Fab antibody and in the absence of TSP1 [T. cruzi cellular infection while RNAi knockdown or absence of TSP1 led to a significant decrease in cellular infection [We showed that TSP1 plays a very important role in the process of host cell infection by nfection . However of TSP1 . We also of TSP1 . We shownfection ,22.The Wnt signal transduction cascade plays important roles in cardiac pathophysiology and its dysregulation can lead to cardiac dysfunction, such as cardiac hypertrophy, fibrosis, arrhythmias, and infarction –26. In tWhen a Wnt ligand is present, the Wnt-on state, it binds to one of several Frizzled (Fz) receptors and the low-density lipoprotein (LDL) receptor–related protein (LRP)5/6 coreceptors to activate the canonical Wnt signaling pathway. The cytoplasmic tail of LRP is phosphorylated by GSK3β and CK1 and becomes activated ,33. The Drosophila melanogaster has now been extensively reported in mammalian cells. This pathway plays an important evolutionarily role in the regulation of organ size, cell proliferation, and apoptosis [The Hippo signaling pathway, which was originally identified in poptosis ,37. In mpoptosis –40. In cpoptosis ,41.T. cruzi upregulates the expression of TSP1, which is downstream of hippo signaling during the early phase of infection. However, the crosstalk between Hippo and Wnt signaling pathways and the role that TSP1 plays in that crosstalk during the early phase of T. cruzi infectivity remains unknown.Recently, multiple layers of complex crosstalk between Wnt signaling and YAP/TAZ activity have been reported in several different contexts, especially in linking fibrosis and cancer research . YAP/TAZT. cruzi and heart endothelial cells, TSP1 plays an important role in the nuclear colocalization/crosstalk between the Hippo effector molecule YAP, and the Wnt effector molecule β-catenin through an unknown mechanism. To delineate the significance of TSP1 expression on YAP and β-catenin nuclear colocalization during the early phase of infection, we challenged MHEC from wild type (WT) and TSP1 knockout (TSP1 KO) mice with T. cruzi. We evaluated the kinetics of Wnt signaling pathway-associated proteins, nuclear translocation of hippo effector molecule YAP, and its nuclear colocalization with β-catenin. Here we show that in the absence of TSP1 (TSP1 KO MHEC) the parasite induces an increase in the levels of β-catenin in the total cell lysate and in the nuclear compartment. This was accompanied by a significant increase in the nuclear translocation of YAP and its colocalization with β-catenin. In contrast, in the presence of TSP1, WT MHEC, we observed a decrease in the levels of nuclear translocated β-catenin and a consequent significant decrease in YAP/β-catenin nuclear colocalization.We hypothesize that during the early phase of molecular interaction between T. cruzi cellular infection, we preincubated WT MHEC with 20 μM endo-IWR 1 inhibitor, which increases axin2 protein expression, stabilizes the destruction complex and promotes β-catenin phosphorylation. The pretreated cells were then challenged with the parasite to evaluate alterations in infectivity. We observed that inhibition of β-catenin pathway with endo-IWR 1 significantly reduced the level of T. cruzi infectivity. The level of T. cruzi infection in the endo-IWR1 inhibitor treated WT MHEC was as low as the level of T. cruzi infectivity of TSP1 KO MHEC. Our data advances the understanding of the molecular interactions occurring between endothelial cells and T. cruzi in the presence and absence of TSP1 during the early phase of T. cruzi infection and pathogenesis. Furthermore, our data also show that inhibition of the β-catenin pathway is a potential prophylactic/therapeutic target for the prevention of T. cruzi infection.To further evaluate the significance of these effector molecules in the process of The animal studies were carried out in accordance with the protocol number 150514PN093 approved by the Institutional Animal Care and Use Committee (IACUC) of Meharry Medical College.2. The beads either fell off, get digested or diluted out as we expand the initial EC isolation. To confirm that these cells are EC, we examined the expression of two endothelial cell specific markers, PECAM-1 and VE-cadherin, by FACS analysis. Nearly 100% of wild type and TSP1 KO MHEC expressed high levels of these markers on their surface. Early passage cultures of WT and TSP1 KO MHEC were used in our studies.MHEC were isolated and maintained as previously described for retinal EC . Briefly2.Rat heart myoblasts (RHM) were maintained in DMEM containing glutamax (Life Technologies), 10% heat inactivated fetal bovine serum (Life Technologies), 1% Penicillin/Streptomycin (Life Technologies), 1% Non-essential amino acid and multivitamin (Life Technologies). RHM were grown in humidified tissue culture incubator at 37°C and 5% COT. cruzi trypomastigotes clone MMC 20A (Tulahuen strain) were maintained in DMEM containing glutamax (Life Technologies), 10% heat inactivated fetal bovine serum (Life Technologies), 1% Penicillin/Streptomycin (Life Technologies), 1% Non-essential amino acid and multivitamin (Life Technologies) at 37°C and 5% CO2. RHM monolayers (80% confluency) infected with T. cruzi trypomastigotes clone MMC 20A (Tulahuen strain) were fed with fresh complete medium daily. Released pure population of highly invasive T. cruzi trypomastigotes were harvested as previously described [7 parasites/ml for use in our experiments. Transgenic T. cruzi trypomastigotes clone MMC 20A (Tulahuen strain) overexpressing green fluorescent protein (GFP–T. cruzi) were similarly generated [T. cruzi trypomastigotes at a ratio of 10 parasites per cell. Cells challenged with the parasites were incubated for different time points 1, 2, 3 and 6 hours, respectively. At the end of each time point, parasites were washed with 1x DPBS  and the cells were either stored at -80°C or processed immediately for western blot analysis. The biological replicates of each time point were prepared in independent T75 flasks for use in the western blotting experiments or independent sets of 6 well culture plates containing coverslip coated with 1% gelatin for use in confocal immunofluorescence microscopy assays. Mock-infected (medium only) MHEC were used as control for each time point. For fluorescence microscopy, infection assays were similarly performed on 6 well plastic culture plates. NucBlue Live ReadyProbes Reagent (Thermofisher Cat #R37605) was added to the cells as a nuclear stain, 2 drops/mL of media, for 20 minutes and washed, essentially as described by manufacturer. Cells were also incubated with CellMask Orange Actin Tracking Stain (Thermofisher Cat #A57247) at 1:1000 dilution for 40 minutes to stain actin and washed essentially as described by the manufacturer. The stained cells were visualized using the Keyence All-in-One Fluorescence Microscope BZ-X710 and analyzed with BZ-X Analyzer software.Rat heart myoblasts (RHM) used for the generation of invasive escribed –49. The enerated ,50. For T. cruzi trypomastigotes at the different time points  and the parasites were washed off with DPBS . Control and parasite challenged cell monolayers were lysed in RIPA buffer (Life Technologies) containing protease inhibitor cocktail set III at 1:100 , phosphatase inhibitor cocktails 2 and 3 at 1:100 each . Whole cell lysates (20 μg/well) were separated by SDS-PAGE using 4–15% gradient polyacrylamide gels and transferred onto nitrocellulose membranes (Life Technologies). The membranes were incubated in Intercept TBS Blocking Buffer  followed by incubation with appropriate primary antibody diluted at 1:1000 at 4°C overnight on a shaker; mouse anti-Wnt-5a monoclonal antibody , mouse anti-GAPDH monoclonal antibody , rabbit anti-β-Catenin antibody , rabbit anti-phospho-GSK-3β (Ser9) antibody , rabbit anti-GSK-3β antibody . The blots were washed and incubated with the corresponding IRDye secondary antibody (LI-COR 800CW or 680RD anti-mouse/ anti-rabbit) in blocking buffer containing 0.01% Tween 20 for 1 hour at room temperature. The blots were washed and scanned using the infrared fluorescence detection Odyssey Imaging System (LI-COR Biosciences) to visualize the bound antibody. Housekeeping GAPDH signal was used for normalization of loading differences. Total GSK-3β signal was used for normalization of the level of phospho-GSK-3β (Ser9). For evaluation of the levels of YAP and β-catenin in the nuclear compartment by immunoblotting, parasite challenged and time matched control MHEC cells were washed in cold PBS and resuspended in hypotonic buffer  containing protease inhibitor cocktail set III and phosphatase inhibitor cocktails 2 and 3 at 1:100 each followed by addition of 10% NP-40. The cells were vortexed centrifuged for 10 minutes at 3000 rpm at 4°C. Cytoplasmic extraction was done twice and the nuclear pellet was resuspended in the cell extraction buffer  containing protease inhibitor cocktail set III and phosphatase inhibitor cocktails 2 and 3 at 1:100 each. The lysed nuclear fraction was centrifuged for 30 minutes at 14,000g at 4°C. The supernatant nuclear fraction was used in our assays. Nuclear lysates (20 μg/well) were separated by SDS-PAGE using 12% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with Intercept TBS Blocking Buffer (LI-COR Biosciences) followed by incubation with the appropriate primary antibody (1:1000) at 4°C overnight on a shaker; rabbit anti-β-Catenin antibody , rabbit anti-GSK-3β antibody , mouse anti-HDAC  and mouse anti-GAPDH monoclonal antibody . The blots were washed and incubated with the corresponding IRDye secondary antibody (LI-COR 800CW or 680RD anti-mouse/ anti-rabbit) in blocking buffer containing 0.01% Tween 20 for 1 hour at room temperature and scanned using the Odyssey Imaging System (LI-COR Biosciences) to visualize the bound antibody. The HDAC signal was used for normalization of loading differences and GAPDH signal was used to check for cytoplasmic contamination of the nuclear extract. Each experiment was done in biological triplicates and quantitation of band intensity was performed by densitometry using Image J. Statistical analysis of each parameter for the T. cruzi treated groups compared with non-treated control groups  using student’s t-test or one-way ANOVA (non-parametric) with Newman-Keuls post-hoc test. The difference was considered statistically significant if *P< 0.05, **P< 0.01 and ***P< 0.001 vs control.MHEC (WT or TSP1 KO) monolayers were challenged with T. cruzi trypomastigotes Tulahuen strain clone MMC 20A (10 parasites per cell) were incubated with the MHEC for different times 1, 2, 3 and 6 h. The parasites were washed off with DPBS. The cells were fixed with 4% paraformaldehyde for 5 min at room temperature and washed with 1x DPBS. Fixed cells were perforated with 0.1% Triton-X100 in TBS for 5 min and blocked with 3% BSA-PBS for 30 min at room temperature. For evaluation of nuclear translocation, slides were incubated with rabbit anti-β-Catenin monoclonal antibody diluted 1:100  and phalloidin  in 1% BSA-PBS at 4°C overnight. The cells were washed with 1% BSA-PBS and re-probed with goat anti rabbit IgG secondary antibody conjugated with Alexa flour 647  in wash buffer for 1 h. They were further washed and mounted with mounting medium containing DAPI (Life Technologies) to stain the nuclei. For colocalization assays, the fixed, perforated and blocked cells on the slides were incubated with a mixture of antibodies containing mouse anti-YAP monoclonal antibody diluted 1:100 (Santa Cruz Biotechnology cat no. sc-271134), rabbit anti-β-Catenin (D10A8) monoclonal antibody  diluted1:100, and phalloidin  at 4°C overnight. The cells were washed and the bound primary antibodies were detected using the corresponding secondary antibodies cocktail; goat anti-mouse Alexa fluor 488 and goat anti-rabbit Alexa fluor 647 antibodies diluted 1:1,000 in 1% BSA-PBS for 1 h at room temperature, washed and mounted with mounting media containing DAPI to stain the nuclei. The coverslips with treated cells were transferred on to glass slides and sealed. Stained slides were analyzed using the Nikon A1R confocal microscope located at the Centralized Core Facility at Meharry Medical College. Cellular and nuclear intensities including Pearson’s correlation coefficient for colocalization of signals were determined by imaging software NIS Elements AR Analysis version 5.20.02 64-bit.MHEC were seeded on glass coverslip coated with 1% gelatin in 6-well culture plate. T. cruzi infection, we preincubated MHEC in the presence or absence of 20 μM endo-IWR1 inhibitor, challenged them with GFP–T. cruzi and evaluated the level of cellular infection by reading the fluorescence intensity of GFP parasites in the cells with a fluorimeter and confocal microscopy assays. For the fluorometric assays, WT MHEC monolayers growing on 1% gelatin precoated 48-well plates were pretreated with or without 20 μM of endo-IWR1 inhibitor for 72 hours. The preincubated WT MHEC were challenged with GFP- T. cruzi parasites at a ratio of 10 parasites per cell. The challenged cells received fresh complete medium daily for 72 h. At the end of 72 h, the cells were washed with 1x DPBS  and the GFP intensity was read. Fluorescence intensities were recorded at an emission of 485/20 and excitation of 528/20 using a microplate reader  and compared to similarly treated control cells not challenged with the transgenic parasites. Each biological replicate was prepared in triplicates. TSP1 KO MHEC challenged with GFP–T. cruzi in the absence of the inhibitor were treated similarly. The average mean fluorescence intensity of infected MHEC replicates with or without inhibitor treatment compared to their respective uninfected controls were calculated and plotted using the graphpad prism software package.To evaluate the importance of Wnt signaling pathway in the process of endo-IWR1 for 72 h. The cells were then challenged with GFP–T. cruzi at a ratio of 10 parasites per cells and fed with fresh complete medium daily for 72 h. The cells were washed with 1x DPBS . Live cells were stained with NucBlue Live ReadyProbes Reagent CellMask, Orange Actin Tracking Stain as described by the manufacturer (Thermofisher) and visualized using the Keyence All-in-One Fluorescence Microscope BZ-X710 and analyzed with BZ-X Analyzer software located at the Centralized Core Facility at Meharry Medical College.For the fluorescence microscopy assays, WT MHEC seeded on 6 well plastic culture plates coated with 1% gelatin were pretreated with or without 20 μM T. cruzi treated groups. Statistical analysis was done using Student’s t-test or one-way analysis of variance (ANOVA) for multiple groups of data followed by a Newman-Keuls test. P-value strength increases with number of asterisks P<0.05 (*), P<0.01 (**), and P<0.001 (***). Statistical analyses were performed using GraphPad Prism .Data from at least three biological independent experiments are expressed as mean ± SEM. Statistical comparisons were made between controls and T. cruzi and parasite induced pathogenesis. Others have suggested that the Wnt signal transduction cascade also plays important roles in cardiac pathophysiology. We next determined the importance of TSP1 expression and regulation of Wnt/β-catenin signaling proteins and their associated signaling cascade during the early phase of T. cruzi infection. We challenged WT and TSP1 KO MHEC with T. cruzi for different lengths of time  and determined the levels of Wnt-5a by immunoblot assays. We found that when WT MHEC were challenged with T. cruzi, the levels of Wnt-5a protein decreased significantly at 1 h , and 2 h  compared with control. The significant decrease in the levels of Wnt-5a remained almost constant at 3 h  and at 6 h  time points compared with control  during the early phase of T. cruzi infection, we challenged WT and TSP1 KO MHEC with T. cruzi for different times  and analyzed the levels of phospho-GSK-3β (Ser9) using immunoblot assays. We found that when WT MHEC were challenged with T. cruzi, the levels of p-GSK-3β showed a continuous decrease from 1 h (0.84±0.14) to a minimum at 2 h  followed with an increase at 3 h  and 6 h  compared with control, which all lower than the control (We found that  control .T. cruzi.When TSP1 KO MHEC (in the absence of TSP1) were challenged with the parasite, we observed a significant decrease in the levels of p-GSK-3β (Ser9) at 1 h  followed by an increasing trend from 2- to 6 h that was maximum at 2 h  compared with control . These rT. cruzi significantly increased the levels of Wnt-5a and p-GSK-3β in the absence of TSP1. Phosphorylation of GSK-3β, a component of the destruction complex in the presence of the Wnt signaling ligand, leads to the stabilization of β-catenin. We next determined whether increased levels of Wnt-5a and p-GSK-3β lead to an increase in the level of stable β-catenin during the early phase of T. cruzi infection. We challenged WT and TSP1 KO MHEC with T. cruzi for different lengths of time  and evaluated the levels of stable β-catenin using immunoblot assays. When WT MHEC were challenged with T. cruzi, the levels of unphosphorylated β-catenin showed a general decreasing trend from 1 h , to a minimum at 2 h , followed by a gradual increase which was significant at 6 h  compared with control  and evaluated the levels of nuclear translocation of the downstream Wnt effector molecule β-catenin using confocal microscopy. Our data show that when WT MHEC were challenged with T. cruzi trypomastigotes, the level of β-catenin nuclear translocation significantly decreased at all time points from 1–6 h with a sinusoidal characteristic as shown by the decrease in mean nuclear fluorescence intensity of the fluorophore in several fields. The mean nuclear fluorescence intensity of β-catenin showed a significant decrease from 1 h ; 2 h  and continued to 3 h  and finally 6 h  compared with control  and continuously to a minimum at 3 h  before increasing at 6 h  showing a sinusoidal characteristic. The relative levels of nuclear YAP decreased to a significant minimum at 3 h  and remained low compared with the control . We determined the levels of nuclear co-localization of the downstream Wnt effector molecule, β-catenin, with YAP by measuring the mean Pearson’s correlation coefficient of both proteins in the nucleus using confocal microscopy. In the presence of TSP1 in WT MHEC, we observed that the mean Pearson’s correlation coefficient of β-catenin and YAP nuclear colocalization remained steady from 1–3 h before showing a significant decrease at 6 h  compared with control , which were significantly less infected  was significantly less than WT MHEC (23.75±0.58), supported by the microscopy data , we showed that MEF generated from TSP1 KO mice were significantly less infected compared to MEF from WT mice supporting the importance of TSP1 in the process of cellular infection [T. cruzi infection and cardiovascular health including the functioning of vascular cells like endothelial cells [T. cruzi research.We showed that during the early phase of infection, parasite . Additionfection . The upral cells ,53. Furtal cells . ElucidaT. cruzi infection [T. cruzi infection of bone marrow derived macrophages [T. cruzi and heart endothelial cells, TSP1 plays an important role in the nuclear crosstalk between the Hippo effector molecule, YAP and the Wnt effector molecule, β-catenin, and that disruption of this suggested crosstalk will affect parasite cellular infectivity.We recently showed that TSP1 plays an important role in the dysregulation of the hippo signaling pathway during the early phase of nfection . In addirophages . Howeverin vitro, we challenged MHEC from WT and TSP1 KO mice with invasive T. cruzi trypomastigotes Tulahuen strain clone MMC20A for different times. We evaluated the activation of Wnt/β-catenin pathway, the molecular colocalization of Wnt/Hippo signaling pathway effector molecules, and how its disruption could affect the early phase of T. cruzi infection. Our data showed that the parasite turns on the Wnt signaling pathway in MHEC in a TSP1 dependent manner. Specifically, in the absence of TSP1, the levels of the Wnt ligand, Wnt-5a, which activates the Wnt signaling pathway was significantly increased at 1, 2 and 3 h time points while it remained toned down and steady in the presence of TSP1 , the parasite induced nuclear colocalization of β-catenin and YAP Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Movie(MOV)Click here for additional data file.


Mounting an appropriate ventilatory response to exercise is crucial to meeting metabolic demands, and abnormal ventilatory responses may contribute to exercise-intolerance (EX-inT) in heart failure (HF) patients. We sought to determine if abnormal ventilatory chemoreflex control contributes to EX-inT in volume-overload HF rats. Cardiac function, hypercapnic (HCVR) and hypoxic (HVR) ventilatory responses, and exercise tolerance were assessed at the end of a 6 week exercise training program. At the conclusion of the training program, exercise tolerant HF rats (HF + EX-T) exhibited improvements in cardiac systolic function and reductions in HCVR, sympathetic tone, and arrhythmias. In contrast, HF rats that were exercise intolerant (HF + EX-inT) exhibited worse diastolic dysfunction, and showed no improvements in cardiac systolic function, HCVR, sympathetic tone, or arrhythmias at the conclusion of the training program. In addition, HF + EX-inT rats had impaired HVR which was associated with increased arrhythmia susceptibility and mortality during hypoxic challenges . Finally, we observed that exercise tolerance in HF rats was related to carotid body (CB) function as CB ablation resulted in impaired exercise capacity in HF + EX-T rats. Our results indicate that: (i) exercise may have detrimental effects on cardiac function in HF-EX-inT, and (ii) loss of CB chemoreflex sensitivity contributes to EX-inT in HF. Current pharmaceutical approaches to HF treatment are effective in delaying disease progression; however, the 5-year mortality rate is approximately 50%9. Exercise training (EX) has been shown to be an effective non-pharmacological therapeutic adjunct in treatment of HF19, that results in improvements in cardiac function, quality of life, and survival. These beneficial effects are frequently associated with improvement in cardiac autonomic imbalance and normalization of abnormal chemoreflex function19, both of which are associated with lower survival rates in HF6. However, these beneficial effects rely on the ability to tolerate EX, which is not always a given in patients with HF20.Heart failure (HF) is a global public health problem characterized by autonomic abnormalities and impaired cardiac function21. It is one of several important indicators in the diagnosis of patients with HF22. The precise mechanisms underlying EX-inT in HF are not fully understood; however, it has been proposed that EX-inT may be linked to reductions in perfusion of working muscles and consequent decreases in oxygen supply. Theoretically, this is a result of impaired ability to increase cardiac output to working muscles which is compounded by persistent sympathoexcitation and reduced vasodilatory mechanisms in vascular beds regulating muscle perfusion23. Most of the experimental support for this hypothesis comes from studies examining reduced ejection fraction HF, which is indeed, characterized by reductions in blood flow to muscle secondary to heart damage24. However, it is worth noting that patients with preserved ejection fraction HF, who often do not have hemodynamic compromise at rest also experience EX-inT.Exercise intolerance (EX-inT) is defined as impairment of the ability to perform physical activity and is characterized by decreased exercise and functional capacities25. Previous studies have shown that aberrant chemoreflex function contributes to autonomic dysfunction, abnormal breathing patterns, and cardiac dysfunction in HF8. However, to date no studies have comprehensively addressed the role of chemoreflex function in EX-inT in HF with preserved ejection fraction (HFpEF). Reductions in chemoreflex gain could potentially have an adverse impact on exercise tolerance in HF due to inadequate pulmonary ventilation during exercise. In the present study, we aimed to determine the prevalence of EX-inT in a volume-overload HF model (which lacks the confounding effect of reduced blood flow)26 and the extent to which aberrant chemoreflex function contributes to EX-inT in this model.In order to meet the metabolic demands of exercise, increases in ventilation are required in addition to increases in and re-distribution of cardiac output. Peripheral chemoreflexes are one of several important homeostatic mechanisms that contribute to increases in pulmonary ventilation during exercise. Early evidence of this, came from studies in patients who had undergone bilateral denervation of the peripheral chemoreceptors (i.e. carotid bodies) for treatment of bronchial asthma. In these people, carotid body resection resulted in a significant reduction in exercise hyperpneaEDD) , LV end-systolic diameter (LVESD) , LVED volume (LVEDV) , LVESV , LV ejection fraction (LVEF)  or LV fractional shortening (LVFS)   were noted between groups.The timeline of the experiments is shown in Fig. ESD  , compared to HF + Sed rats   , LVESD (p = 0.69), LVEDV (p = 0.85), LVESV (p = 0.38), LVFS (p = 0.43) and LVEF (p = 0.38)  Fig. D,F and LHF + EX-inT rats showed a significant increase of cardiac hypertrophy compared to HF + EX-T animals Table .T amplitude or Rf. Accordingly, no changes in respiratory cycle duration or peak flows were found between groups (Table Resting respiratory parameters (in normoxia) are shown in Table ps Table .Table 2Rmax (p = 0.82), dP/dtmin (p = 0.86), dV/dtmax (p = 0.62), dV/dtmin (p = 0.36), TauW (p = 0.48) were not different between groups (Table Cardiac parameters including SV (p = 0.28), SW p = 0.73), LVESP (p = 0.92), dP/dt, LVESP . In contrast, HF + EX-inT rats showed a similar maximum bradycardic response to propranolol compared to HF + Sed rats  . HF + EX-inT rats showed no change in arrhythmia incidence compared to HF + Sed animals Fig. C,D.0: 289.5 ± 21.3 vs. 254.2 ± 19.4 and 237.8 ± 6.6 µl, HF + EX-inT vs. HF + EX-T and HF + Sed rats, respectively) . Notably, chemoreflex-induced cardiac arrhythmias were blunted in HF + EX-T rats. Indeed, arrhythmia incidence was ~ threefold lower in HF + EX-T animals compared to HF + Sed animals    and hypoxic (HVR: FiO2 10%) ventilatory responses, respectively. HVR and HVCR assessed before the onset of the EX-program (2 weeks post-HF surgery) was not statistically differences between groups .Responses to central and peripheral chemoreflex stimulation were evaluated through the hypercapnic   Fig. A,B, but 04) Fig. B. In con04) Fig. B.Figure E/ΔFiO2% [p = 0.03], HF + EX-inT vs. HF + EX-T and HF + Sed) , minute ventilation (Ve), expiratory time (Te), total respiratory time (Ttot), peak inspiratory flow (PiF) and peak expiratory flow (PeF) responses to hypoxia compared to HF + EX-T and HF + Sed animals  Fig. A,C. In aThe effects of CBA on training times are displayed in Fig. In the present study we found that: (i) EX-inT was present in 39% of high-output HF rats; (ii) EX-inT was not related to the initial degree of cardiac failure nor to HCVR/HVR prior to the onset of EX in HF rats; (iii) HF + EX-inT rats had similar degrees of autonomic dysfunction, arrhythmia incidence, and cardiac systolic dysfunction compared to HF sedentary rats; (iv) EX-inT results in a greater degree of cardiac diastolic dysfunction in HF; (v) lower HVR in HF + EX-inT was associated with increased incidence of cardiac arrhythmias and higher mortality during hypoxic challenge; and (vi) ablation of peripheral chemoreceptors in HF + EX-T rats was sufficient to induce a phenotype similar to EX-inT animals. Taken together, our data suggest that decreased peripheral chemoreflex gain contributes to EX-inT in HF. Based on our findings it is plausible that reductions in chemoreflex gain in a subset of HF patients may lead to EX-inT, further abnormalities in cardiac function, and potentially predispose to severe arrhythmogenesis and increased mortality risk during moderate hypoxia exposure.22. HF patients typically have reduced exercise and functional capacity and experience dyspnea during daily activities27. Interestingly, intolerance to physical exercise is observed in both types of HF (i.e. reduced and preserved ejection fraction)27. Current theories on exercise intolerance center on an inability to adequately increase cardiac output during exercise and reductions in muscle blood flow secondary to reduced EF and potentially abnormalities in muscle metabolism20. In addition to cardiac, vascular, and metabolic responses to exercise, respiratory adjustments to exercise are just as crucial to maintaining homeostasis and therefore exercise tolerance.Intolerance to exercise is a well-recognized characteristic of HF26. We showed that volume-overload HF rats display exercise intolerance independent of the initial degree of cardiac failure. With respect to respiratory control, we observed that these animals displayed decreased HVR after training despite the fact that HVR was similar between all groups prior to the onset of the EX-program. Therefore, it is reasonable to hypothesize that exercise intolerance in volume-overload HF stems in part from a reduction in peripheral chemoreflex gain. To test this assumption, we eliminated the peripheral chemoreflex in HF rats that were previously proven to be exercise tolerant. These HF + EX-T rats underwent bilateral ablation of the carotid bodies after 6 weeks of EX. Notably, we found that HF + EX-T rats showed a significant loss in EX performance at 2 weeks post-ablation. Indeed, total training times in HF + EX-T rats that underwent ablation were not significantly different from the values obtained in HF + EX-inT rats. This result strongly supports a role for peripheral chemoreceptors in contributing to exercise tolerance in HF. Based on our findings, the precise mechanisms by which peripheral chemoreceptors contribute to exercise tolerance in volume-overload HF, a model of HF with no reductions in EF, warrants further investigation.In the present study, we used a volume-overload HF model to study the contribution of chemoreflexes to exercise tolerance in the absence of reduced EF28. Whether or not EX results in improvements in diastolic function in HF is still controversial. A meta-analysis performed by Pandey et al.29 showed that preserved ejection fraction HF patients did not experience significant improvements in diastolic function following EX29, and our previous work showing that EX training did not improve diastolic dysfunction in high-output HF rats1 is consistent with these findings. The results of the present study in HF + EX-T rats confirm and extend our previous observations by illustrating the detrimental effects of EX in EX-inT HF rats. We found that EX intolerance in HF rats was associated with worse diastolic function after completing an EX protocol. This result suggests that in some HF populations with no compromise in ejection fraction physical exercise may be detrimental. Interestingly, this concern has previously been discussed as an important factor in disease progression and decompensation in human HF independent of its etiology20.Impairment of systolic and diastolic function has been observed in patients with HF and in animal models of HF31. It has been shown that endurance EX in HF patients results in minor improvements in systolic function32, however in these studies no effort was made to distinguish between EX tolerant vs. intolerant patients32. In contrast, we previously reported that EX improves systolic function in HF rats1. It is worth noting that in the present study we found similar beneficial effects of EX on cardiac contractility, but that this was restricted to EX tolerant HF animals. Indeed, no beneficial effects of EX on cardiac contractility were observed in HF + EX-inT rats. For future studies to accurately assess the beneficial effects of EX on cardiac function in HF it is important to identify and differentiate the proportion of the patient population that do not tolerate EX.Similar to what is known about the effects of EX on diastolic function in HF, evidence regarding the beneficial effects of EX on systolic function is limited and controversial33. Our previous work and that of others has shown that volume-overload HF rats exhibit autonomic imbalance characterized by heightened sympathetic activity34. It has been proposed that autonomic imbalance, mainly sympathoexcitation, contributes to EX intolerance in HF by reducing muscle blood flow along with increases in peripheral vascular resistance23. Importantly, volume-overload HF with preserved ejection fraction is a particularly useful model for examining the effects of EX intolerance in the absence of overt reductions in cardiac output. We found that EX was an effective means to reduce cardiac sympathetic tone in HF rats that tolerate EX. This is in agreement with our previous observations1; but, this research also extends our previous findings by showing that EX has no salutary effect on sympathetic activity in HF rats with EX intolerance. In this study, cardiac chronotropic responses to propranolol from HF + EX-inT rats were undistinguishable from those in in sedentary HF rats. Whether this lasting heightened sympathetic activity in HF + EX-inT group contributes to the EX intolerant phenotype in HF requires further study.Autonomic dysfunction characterized by sympatho-vagal imbalance is a hallmark of HF in humans and is faithfully reproduced in animal models of HF29 and are often associated with increased cardiac sympathetic tone35. In our previous studies we showed that volume-overload HF rats had a higher incidence of cardiac arrhythmias than healthy control rats and that EX training resulted in significantly lower occurrence of these arrhythmias8. In this study we extend our previous findings with the observation that EX results in no beneficial effect on cardiac arrhythmias in HF rats with EX intolerance. It is important to note that HF + EX-inT rats do not experience improvements in cardiac sympathetic tone as observed in HF rats that tolerate EX. Previously, we reported that cardiac arrhythmias in volume-overload HF rats are driven primarily by sympathetic activation3, therefore it is plausible that the lack of effects of EX on cardiac arrhythmias in HF + EX-inT rats was related, at least in part, to the inability of EX to reduce sympathoexcitation. However, we cannot rule out the possibility that cardiac remodeling occurred following EX training in HF + EX-inT rats which contributed to an arrhythmic substrate.Cardiac arrhythmias are a major contributor to mortality in patients with HF35. Previously we found that volume-overload HF is associated with enhanced central chemoreflex gain and autonomic dysfunction, but that these animals did not exhibit changes in peripheral chemoreflex gain8. In the present study we showed that EX in HF + EX-T rats significantly reduced central chemoreflex gain without changing peripheral chemoreflex gain. Therefore, it is possible that reductions in central chemoreflex gain in HF + EX-T rats following EX contribute to the improvements we observed in autonomic and cardiac function. In contrast, EX in HF + EX-inT rats had no discernable effect on central chemoreflex gain compared to HF sedentary rats. Surprisingly, we found that peripheral chemoreflex gain was attenuated after EX in HF + EX-inT rats. This finding is of note in part because when exposed to moderate hypoxia, HF + EX-inT rats had increased incidence of cardiac arrhythmias and higher mortality compared to HF + Sed animals. It is plausible that a diminished hypoxic ventilatory response compromises cardiac oxygen supply during hypoxic challenge in HF + EX-inT animals triggering lethal cardiac arrhythmias.It has been proposed that chemoreflex-mediated sympathoexcitation is a major contributor to the progression of HF independent of its etiology36, but the extent to which this affects exercise tolerance is unclear. It has been proposed that carotid body chemoreceptors act not only to maintain arterial blood gas homeostasis, but that they also serve an important role as metabolic sensors. Recently, Chang et al.36, showed that carotid bodies respond to increases in extracellular lactate and can elicit a ventilatory response to physiologically relevant lactate concentrations. It is widely known that plasma lactate levels increase during acute exercise (of sufficient intensity), therefore, it is possible that peripheral chemoreceptors modulate the ventilatory adjustments and tolerance to EX in HF. In the case of a decrease in peripheral chemoreflex gain (as we observed) during higher intensity exercise, a loss in the sensitivity to metabolic by-products by peripheral chemoreceptors would adversely affect ventilatory responses to EX and potentially contribute to EX intolerance.Whether a decrease in peripheral chemoreflex gain affects tolerance to EX in HF has not been previously addressed. It has been shown that CB ablation in humans results in a significant reduction of exercise hyperpneaSince our HF + EX-inT animals displayed decreased peripheral chemoreflex gain we decided to determine if reducing peripheral chemoreflex gain (via carotid body ablation) would produce a phenotype switch in HF animals that had previously demonstrated good tolerance for EX. Notably, ablation of the carotid bodies in HF + EX-T rats did indeed result in a phenotype switch, transforming the exercise tolerant animal into an intolerant one. Taken together, our observations suggest that reductions in peripheral chemoreflex gain may play an important role in determining EX tolerance in volume-overload HF. Measuring ventilation during exercise in rodents is very difficult, however future studies that address the precise role of peripheral chemoreceptors in EX ventilation in HF would add important insight to our findings.37 and Edgell et al.38 noted an influence of CB on cardiovascular regulation at rest and during exercise in HFrEF patients but did not find that CB inhibition had any beneficial effect on EX tolerance. The implications of these studies are unclear as the relative chemosensitivity of these patients was not quantified, and not all HFrEF patients have altered chemoreflex function. Furthermore, HFrEF patients with altered chemoreflex function are more likely to have enhanced chemosensitivity/HVR, whereas our findings in a high output model of HF is of reduced HVR. It is possible that a reduced HVR is a product of pulmonary mechanical restraint on ventilation, however we did not observe any evidence of significant changes in pulmonary ventilation between groups or lung wet-to-dry weight ratios. As mentioned previously our studies were not designed to address this question, but they provide no evidence to support it.In addition to our hypothesis regarding reductions in chemosensitivity, there are certainly other factors which may contribute to EX intolerance in HF. Previous studies have shown right heart to pulmonary circulation uncoupling and ventilatory inefficiency in HF patients with both reduced and preserved ejection fraction. As our study did not directly address this hypothesis, our data do not support or refute this possibility. With that said, our data showing that CB ablation in formerly EX tolerant HF animals results in EX intolerance do lend strong support to the notion that CB plays a role in EX tolerance in some settings. This does not preclude the potential contribution of changes in ventilatory efficiency or uncoupling of right heart and pulmonary circulation. While some studies have looked at the role of the CB in controlling hemodynamic function and EX performance during HF, these were performed in HF patients with reduced EF. Given the distinct changes in chemoreflex function that occur in different HF etiologies (preserved vs. reduced EF) a direct comparison between these studies is difficult. Interestingly, Collins et al.39 are present. In this study we used volume overload to create a high-output model of HF, but we feel caution is required when trying to extrapolate our findings to human HF41. Further validation is required before generalizing our results to the clinical setting. Other limitations of the present study include the use of anesthesia during invasive measurements of cardiac function and autonomic tone. This has the potential to impact our results due to the well-known cardiovascular effects of anesthetics. However, it is worth noting that the exact same anesthetic agent and depth of anesthesia were used in all three experimental conditions. So, any effect of anesthesia on our measurements would be expected to be similar across experimental conditions. Also, in our study we did not assess right ventricular (RV) function. Since alterations in pulmonary vascular mechanics and right heart pressure due to congestion could affect exercise performance, a full study on RV function is needed to determine the extent to which this plays a role in exercise intolerance in this model. We did not find significant changes in lung wet-to-dry weight ratio in EX-inT HF rats, but we cannot rule out the possibility that subtle changes in pulmonary mechanics may have adversely affected RV function. Additionally, our experimental design did not allow for measurement of ventilation or other cardiovascular and metabolic parameters during EX sessions. Furthermore, we did not test whether the animals displayed different peripheral chemoreflex sensitivity prior to HF induction which may, or may not, have influenced EX performance. Furthermore, our study cannot discern whether changes in HVR precede or coincide with EX-inT in volume overload HF. Therefore, future studies probably at early timepoints will be required to clarify the precise moment at which HVR and EX-inT develops in HF condition. Nevertheless, previous studies have shown little interindividual variability in chemoreflex drive in control conditions (before HF)35. Therefore, we consider it highly unlikely that initial differences, if any, in chemoreflex sensitivity influenced our results. Additional studies addressing cardiorespiratory responses before and during acute exercise will provide additional insights into EX intolerance in HF.It is important to acknowledge the limitations of the chosen experimental model of HF. In this model, as with many other HF models, none of the common comorbidities that are typically associated with development or progression of HF Exercise intolerant HF rats display low peripheral chemoreflex gain and worse cardiac function than exercise tolerant HF rats. Interestingly, ablation of peripheral chemoreceptors in exercise tolerant HF rats resulted in transformation of exercise-tolerant animals to exercise-intolerant animals. Our data suggest that lower peripheral chemoreflex sensitivity contributes, at least in part, to exercise intolerance in volume-overload HF rats.Forty male Sprague–Dawley rats (250 ± 20 g) were used in these experiments. All experiments were performed 8-weeks following induction of HF. In accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Guía para el Cuidado y Uso de los Animales de Laboratorio from CONICYT, all animals were kept at controlled room temperature under a 12 h light/dark cycle with ad libitum access to food and water. All experimental protocols were approved by the Ethics Committee for Animal Experiments of the Pontificia Universidad Católica de Chile (#170710022) and were performed according to the ARRIVE Guidelines. All experiments were performed in the Laboratory of Cardiorespiratory Control in the Department of Physiology of the Pontificia Universidad Católica de Chile. At the end of the experimental period all animals were humanely euthanized via anesthetic overdose .42. From inT animals, n = 6 rats were assigned to plethysmography experiments and n = 5 rats, were allocated to anesthetized preparation at week 8. From tolerant animals, n = 7 rats were allocated to anesthetized experiments, n = 6 rats were assigned to plethysmography experiments. Finally, n = 4 rats from HF + EX-T rats were used for CBA experiments  or sedentary  conditions. Sedentary animals were assigned to plethysmography experiments (n = 6) and echocardiography and cardiac function experiments (n = 6). Exercise tolerance was evaluated during the 60 min training session duration. Then, based on training times, rats that completed all training sessions  were assigned to the HF + EX-T n = 17) group and those who failed to complete or showed less than 50% of completion of the 60 min session time (n = 11) were classified as HF + EX-inT group an45. Briefly, under anesthesia (2% isoflurane/ 98% O2) a laparotomy was performed and an anastomosis was created between abdominal aorta and inferior vena cava vessels using 1.20 × 40 mm needle (BD Precision Glide). The opening in the aorta was closed using tissue adhesive . The abdomen was then closed in layers. Post-operative management consisted of administration of 5 mg enrofloxacin (s.c.), 1 mg ketoprofen (s.c.), 5 ml saline solution (i.p.) and 2% lidocaine hydrochloride jelly . Success rates for this surgical approach range from ~ 80 to 90%. The inclusion criteria for allocation to the HF group included: concomitant increases in EDV and SV (~ twofold increase/each), and the presence of preserved EF45.Volume overload was used to induce HF as described previously1. Briefly, rats ran on a motor-driven treadmill  at low speed (10 m/min), low % grade (0%), and for a short duration (10 min/day) during the first 2 weeks of the training program. Then, intensity and duration were gradually increased to 25 m/min at 10% grade for 60 min/day until they completed 6 weeks of training. Regardless of whether HF rats were classified as EX-T or EX-inT, all rats exercised for 6 weeks. Exercise intolerance was defined as the inability to complete one full training session . Exercise intolerance was assessed 2 weeks after initiation of the exercise program. Soleus to body weight ratio was calculated in each animal to estimate effectiveness of EX as previously described1.The EX-training protocol was similar to previous studies46. Echocardiography was repeated in all groups at week 8 post-HF induction after completing EX or Sed protocols. Briefly, under anesthesia  rats were placed in supine position and scanned in M-mode with an echocardiograph , using a 12-MHz electronic transducer. Images were obtained from the left parasternal short-axis views of the left ventricle (LV) at the level of papillary muscles47. Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured. Subsequently the left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), ejection fraction (LVEF) and fractional shortening (LVFS) were calculated. Animals were classified as high-output HF if they exhibited a minimum of a 2.5-fold increase in SV and LVEDV, as previously described8.To assess left ventricular function, echocardiography was performed at 2 weeks post-HF surgery and again at the end of the protocol (8 weeks post HF surgery), as previously described50. In addition, another catheter was inserted in the jugular vein for bolus calibration48. Before LV placement of the PV catheter, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MABP), pulse pressure (PP) and heart rate (HR) were determined. Systolic and diastolic cardiac function were determined by single beat analysis53. After an equilibration period (25-min), PV loops were recorded, and LV hemodynamic parameters were calculated using 10–15 successive PV loops. PV loop parameters were left ventricular end-systolic pressure (LVESP) and left ventricular end-diastolic pressure (LVEDP). Load-dependent cardiac function parameters were determined by means of the calculation of dp/dtmax and dp/dtmin. Load-independent systolic cardiac function was determined by calculating the slope of end-systolic pressure–volume relationship (ESPVR) from one cardiac cycle. Diastolic cardiac function was determined by volume at pressure 0, from one cardiac cycle. In addition, during PV-loop preparation cardiovascular response to hypoxia  and hypercapnia (FiCO2 7% CO2/93% O2) were evaluated. Volumes were calibrated using arterial blood by the cuvette calibration method and NaCl 30% i.v. bolus for determination of parallel conductance44. Data analysis was performed using the PV loop module of LabChart 8.0 software.Invasive assessment of left ventricular hemodynamic function was performed at the end of the experimental period (8 weeks post HF induction). Rats were anesthetized with α-chloralose (40 mg/kg) and urethane (800 mg/kg), and then were intubated (16-g cannula). After this a 2F pressure–volume (PV) conductance catheter  was placed into the right carotid artery and advanced into the left ventricle8. Briefly, during PV-loop recording a bolus of propranolol was injected (1 mg/kg i.v.) and the maximal chronotropic response was quantified. Sympathetic tone was quantified as the change in heart rate (ΔHR) in response to propranolol.We determined cardiac sympathetic tone by measuring the maximum bradycardic response to propranolol, as previously described8. HR was derived from blood pressure dP/dt waveforms. Irregular heartbeats were visually inspected and counted as previously described54. Arrhythmias were defined as premature or delayed beats with changes greater than 3 standard deviations (SD) from the mean beat-to-beat interval duration8. Arrhythmia incidence was expressed as events/hour.Arrhythmia incidence was measure as previously described8. Briefly, unrestrained whole-body plethysmography was used to measure or calculate the following: tidal volume VT, respiratory frequency Rf, minute ventilation (VE), inspiratory time (Ti), expiratory time (Te), total respiratory time (Ttot), peak inspiratory flow (PiF) and peak expiratory flow (PeF). These were measured during stimulation of central chemoreceptors with hyperoxic hypercapnia  and during stimulation of peripheral chemoreceptors with poikilocapnic hypoxia . The hypercapnic ventilatory response (HCVR) was obtained by calculating the slope of the linear regression adjustment of the VE response following FiCO2 0.03% and 7%, as previously described8. The hypoxic ventilatory response (HVR) was obtained by calculating the slope of the linear regression adjustment of the VE response following FiO2 21% and 10% challenges, as previously described8. All recordings were made at an ambient temperature of 25 ± 2 °C. Data was calculated using ECG auto software 8.Chemoreflex function was assessed at 2 weeks post-HF induction and at the end of the experimental protocol (8 weeks post-HF induction). Central and peripheral chemoreflex sensitivity was assessed by allowing the rats to breathe a mixture of hypercapnic or hypoxic gas, respectively4. Briefly, rats were anesthetized with 2% isoflurane in O2. Under sterile surgical conditions, the CBs were exposed via a ventral incision on the neck, visually identified, and cryogenically destroy4. The effectiveness of this maneuver was confirmed by the lack of hypoxic ventilatory responses immediately after recovery from surgery4. Post-operative management consisted of administration of 5 mg enrofloxacin (s.c.), 1 mg ketoprofen (s.c.), 5 ml saline solution (i.p.) and 2% lidocaine hydrochloride jelly .After 6 weeks of exercise training the carotid bodies (CBs) were ablated as previously describedData were expressed as mean ± standard error of the mean (SEM). All data were subjected to Shapiro–Wilk normality test. Differences among groups were assessed using one-way ANOVA, followed by Holm-Sidak post hoc comparisons. An alpha of p < 0.05 was considered statistically significant. All analysis was performed with Prism version 8.4.0 .


Near-infrared (NIR) fluorescence imaging has been emerging as a promising strategy to overcome the high number of early esophageal adenocarcinomas missed by white light endoscopy and random biopsy collection. We performed a preclinical assessment of fluorescence imaging and endoscopy using a novel CXCR4-targeted fluorescent peptide ligand in the L2-IL1B mouse model of Barrett’s esophagus.Six L2-IL1B mice with advanced stage of disease (12–16 months old) were injected with the CXCR4-targeted, Sulfo-Cy5-labeled peptide (MK007), and ex vivo wide-field imaging of the whole stomach was performed 4 h after injection. Before ex vivo imaging, fluorescence endoscopy was performed in three L2-IL1B mice (12–14 months old)  by a novel imaging system with two L2-IL1B mice used as negative controls.Ex vivo imaging and endoscopy in L2-IL1B mice showed that the CXCR4-targeted MK007 accumulated mostly in the dysplastic lesions with a mean target-to-background ratio > 2. The detection of the Sulfo-Cy5 signal in dysplastic lesions and its co-localization with CXCR4 stained cells  by confocal microscopy further confirmed the imaging results.This preliminary preclinical study shows that CXCR4-targeted fluorescence endoscopy using MK007 can detect dysplastic lesions in a mouse model of Barrett’s esophagus. Further investigations are needed to assess its use in the clinical setting.The online version contains supplementary material available at 10.1186/s13550-021-00875-7. Barrett’s esophagus (BE), which is highly associated with gastroesophageal reflux disease (GERD), is a precursor lesion of esophageal adenocarcinoma (EAC). Its prevalence is estimated at 0.5–2% in the general population and at 5–15% in symptomatic GERD-patients , 4. Despλex,max/λem,max in PBS: 649/666 nm) and the improved tissue penetration compared with conventional near-infrared dyes such as ICG FC131 as radioligand. The 125I-labeled, chemically identical counterpart of MK007 showed a log P of − 1.83 ± 0.02 at pH 7.4 (PBS). In a competitive binding assay using Jurkat human T-cell leukemia cells and [125I]FC-131 as the radioligand, MK007 demonstrated high CXCR4 affinity with an IC50 of 10.2 ± 4.0 nM (n = 3).Given promising data obtained with MK007 in our preclinical mouse model, we performed preliminary in vitro studies on human cell lines. Competition binding studies (IC50) were performed  using JuThe increasing incidence of EAC and the poor survival of patients affected demand the development of novel strategies for early detection. Indeed, white light endoscopy and random biopsy collection have resulted in the miss diagnosis of more than 50% of EAC cases . Among oOur data on ex vivo imaging of the whole stomach and confocal microscopy after injection of Sulfo-Cy5 CXCR4-targeted peptide are consistent with our previous imaging of dysplastic lesions in IL1B mice , in whicAlthough we did not inject MK007 and a CXCR4-blocker in IL1B mice , we foun99mTc; based on the structure of MK007) for SPECT/CT imaging [Our proof-of-concept study also presents limitations: we performed endoscopy and imaging in a reduced number of mice and we did not perform further evaluation on human tissues, but rather focused on the murine CXCR4 receptor (target of this preliminary study) in a preclinical mouse of BE . Indeed, imaging  are enco imaging . Further imaging . Additio imaging  are highIn conclusion, our preliminary data show that dysplastic lesions can be detected by imaging using a CXCR4-targeted far-red-fluorescent probe. Further studies on excised human tissues and IL1B mice < 12 months are needed to evaluate the potential of CXCR4-targeted endoscopy and imaging for the identification of pre-dysplastic lesions in patients with BE.Additional file 1. Methods, Peptide synthesis, Determination of lipophilicity (log P), Determination of CXCR4 affinity. Figure S1. Evaluation of MK007 6 hours post-injection and MK007 fluorescence signal. Figure S2. Ex vivo imaging and confocal microscopy of main mouse organs after injection of MK007.


Epoxy resins are thermosets with interesting physicochemical properties for numerous engineering applications, and considerable efforts have been made to improve their performance by adding nanofillers to their formulations. MXenes are one of the most promising functional materials to use as nanofillers. They have attracted great interest due to their high electrical and thermal conductivity, hydrophilicity, high specific surface area and aspect ratio, and chemically active surface, compatible with a wide range of polymers. The use of MXenes as nanofillers in epoxy resins is incipient; nevertheless, the literature indicates a growing interest due to their good chemical compatibility and outstanding properties as composites, which widen the potential applications of epoxy resins. In this review, we report an overview of the recent progress in the development of MXene/epoxy nanocomposites and the contribution of nanofillers to the enhancement of properties. Particularly, their application for protective coatings , electromagnetic-interference shielding, and composites is discussed. Finally, a discussion of the challenges in this topic is presented. As part of the thermosets family, epoxy resins (ERs) are suitable components for advanced engineering applications because they show outstanding mechanical properties and low shrinkage during curing, low residual stresses, and good thermal and chemical resistance ,2. They 2, is probably the most widely used 2D material. Beyond graphene . T. T3T2Cx tability d,e. The reported . The amiRecent work from Xu et al. applied MXene to UHMWPE fibers for electronic textiles applications . Plasma Theoretical studies have also been carried out on ER-MXene composites, either by using the finite-element method or molecular dynamics simulations. Sliozberg et al.  performeThe most relevant results in the mechanical properties of MXene/ER nanocomposites are summarized in With the fast development of technology, there is an increasing interest in polymer composites with high thermal conductivity (TC) and dielectric permittivity for microelectronics, energy storage, and other applications in which a good heat-dissipation performance is desired. The thermal-management capacity of composite materials can also reduce fire risks and system failures of electronic packages. Epoxy resins (ER) are widely used in the electronic fields due to their excellent mechanical properties, good chemical resistance, and high adhesion to substrates. Nevertheless, epoxy resins are characterized by having a very low thermal conductivity. Many attempts have been made to improve their TC by incorporating different inorganic nanoreinforcements, among them graphene/expanded graphite, alumina, and aluminum nitride. Inorganic nanomaterials may enhance both TC and fire safety. In recent years, different studies affirm that TC may be improved by incorporating MXenes to the ER, often reaching better results than other 2D nanofillers ,49,50. MThe addition of small amounts of MXene, between 0.2 and 1.0 wt %, improved the TC up to 141.3% concerning the pure ER . This wo3C2Tx fillers and their aligned distribution in the epoxy matrix. In consequence, depending on the preparation method of the nanocomposite, the distribution of MXene sheets may be improved and increase the anisotropic conductivity. Lin et al. aligned the filler distribution by shear and centrifugal force in stirring by cast-moulding [MXene nanoflakes (multilayer) were introduced into the ER at much higher concentrations, between 5 and 40 wt % of reinforcement . After mmoulding . Due to 3C2 (0.5–3 g) [The influence of MXenes on TC was also studied in carbon-fiber-reinforced ER. As it was mentioned before, CF is usually coated with the MXene nanosheets. However, in this work, a foam was prepared with MXenes (again in multilayer form), cellulose, and carbon fibers by solvent-mixing of the three components and oriented freeze-drying, and then the ER was infiltrated . Therefo0.5–3 g) . TC valu3 redox reaction [Interface thermal resistance between the filler and the ER may limit the TC performance. Therefore, the incorporation of MXene with silver particles was also studied. MXene/Ag powder was prepared by exfoliation, followed by a AgNOreaction . Ag nanoreaction  studied reaction  and usinreaction . Compare3C2Tx nanosheets enhanced the interfacial interaction between the carbonized layer and the ER, preventing the dropping of molten products during burning. It also strengthened the formed foam structure, preventing noncombustible gas release. In consequence, fire performance was improved. Song et al. [Flame retardancy is an interesting feature in a composite to increase fire safety. MXenes have been used to improve flame retardancy in ER. For this purpose, other components are usually added, such as red phosphorous (RP), a classic P-C-N system, zinc hydroxystannate (ZHS), or copper-organophosphate (CuP). MXene sheets were coated by RP through vapor exposition and calcination. RP-MXene was then introduced in the ER, showing excellent results in fire behavior tests . RP is ag et al.  decorateg et al. . The CuPg of a thermoset, but there is a confluence of several factors. First, the nanofiller disrupts the epoxy network; then, it can enhance mobility at the interface and decrease the Tg. Second, the mobility at the interface is strongly dependent on the interfacial interactions between the nanofiller and ER matrix, so Tg may show an increase. Those interactions may be chemical, hydrogen bond, Van der Waals forces, or physical, for example, by interlocking the resin into a rougher surface. Generally, it was reported that the Tg increases as the amount of MXene into the composite increases because interfacial interactions are strong [g [3CN, but composites with higher MXene content showed a decrease in degradation temperature. This decrease was attributed to the dispersion state of Ti3CN into the matrix. For high MXene loads, the amine-based hardener’s preferential adsorption could also affect the epoxy structure after curing.We would like to briefly mention other properties that are included in the field of thermal properties, although they are not usually the target of the functionalization with MXenes. Numerous studies showed changes in the glass transition temperature of the ER when MXene nanofillers were added. The nanofiller may influence the Te strong ,62. Howetrong [g . Anothertrong [g . By conttrong [g . A slighEpoxy resins are components of adhesives, coatings, engineering plastics, and composites. Along with the mechanical and thermal behavior, wear resistance is very important to increase the lifetime of the industrial components. ERs are highly crosslinked and brittle; therefore, their tribological behavior is usually poor in extreme conditions, limiting their use in high-performance devices. Wear resistance and friction may be increased by adding micro or nanofillers . Furthermore, properties such as hardness or impact strength are improved. Two-dimensional nanomaterials enhance the anticorrosive properties by increasing the corrosive ions pathway and providing good tribology. More traditional 2D materials such as boron nitride or graphene are chemically inert and therefore have poorer interaction with the matrix than MXenes, which, in addition, increase the wettability of the ER onto high-energy substrates. MXenes, therefore, are very good candidates to prepare high-performance anti-corrosive/wear coatings.3C2 structure with carbon nanofibers through directional freezing and lyophilization, and then the ER was introduced by vacuum infiltration and cured (3 to 12 wt % MXene content). They observed an increase in hardness and thermal conductivity. Friction coefficient and wear rate decreased 76.3% and 67.3%, respectively, with the highest MXene load, regarding bare ER. The reduction in the wear rate was mainly ascribed to the excellent antifriction feature of Ti3C2 nanosheets. Ti3C2 -OH and -O groups allowed a good interface with ER and may participate in the curing reactions, enhancing the crosslinking at the interface. Under shear force, in high-load composites, the multi-layer Ti3C2 MXene presented was sheared to form a thin sheet, which made the shear strength in the friction process lower and thus had a particular effect on friction reduction. Few MXene sheets were on the CNF surfaces for low-load composites, but the friction coefficient was also reduced because the debris had a better self-lubricating effect. So, increasing the MXene amount does not directly translate to wear behavior. For example, more recently, Li et al. [Several works introduced non-modified MXenes into ER coatings and investigated the tribological properties. Zhang et al.  preparedi et al.  prepared3C2Tx) with APTES, taking advantage of their surface -OH [2N-MXene was mixed (up to 0.5%) with waterborne ER and painted over Al alloys. Functionalization improved the stability of MXene-ER suspensions, and the mechanical properties were enhanced. The storage modulus increased, which brought about resisting plastic deformation and micro-cracking of the H2N-MXene/ER coatings. In addition, the better dispersion of functionalized MXene sheets prevented agglomeration and restacking, reducing stress concentration. The wear rate and friction coefficient decreased by 72.7% and 34% with respect to ER, and the improvement was clearly better when MXene sheets were covalently linked to ER through amino linkage. It must be noted that even when unfunctionalized, the interfacial adhesion between the polar MXene sheets and epoxides is high [Fan et al. functionalized MXene  was obtained, reduced by 79.3% with respect to neat epoxy  and microwave-absorption properties of 2D nanosheets, particularly MXenes, have attracted great interest due to their excellent electrical conductivity, high specific surface area, and chemically active surface. Electrical conductivity (σ) is especially a key factor in enhancing these properties when MXene is used as the filler of the matrix composite. Bearing in mind the low conductivity of epoxy resins, the addition of MXene nanosheets could remediate that handicap, leading to a significant improvement as the MXene loading increases. Recently, MXene/epoxy resin nanocomposites exhibiting good σ values have been reported ,36,52. C3C2 nanosheets were prepared by solvent-homogenized dispersion of the nanosheets with the epoxy resin [3C2 nanosheets-filled epoxy composites due to their microstructure, larger internal boundary layer capacitance, microcapacity network formation, and dielectric properties. The potential use of epoxy-resin-based nanocomposites in the microwave-absorption field can be greatly enhanced by 3D MXene porous structures as absorbing fillers. Zhao et al. fabricated 3D Ti3C2Tx MXene (MX)/carbon foam (CF)/epoxy resin (ER) nanocomposites by adsorption of MXene on a carbon foam surface and freeze-drying, and subsequent vacuum-impregnation of epoxy resin [3C2Tx MXene with magnetic nanoparticles to modify epoxy resin has recently emerged as an interesting strategy to enhance microwave-absorption performances [0.6Zn0.4Fe2O4/Ti3C2Tx (MP) were prepared by the hydrothermal method, and then they were mixed with epoxy resin to be used as a coating on a cement mortar specimen’s surface. The most excellent electromagnetic wave-absorption properties (EMW) were reached when 3 wt % MP was incorporated into epoxy resin, obtaining a reflectivity value 62.9% lower than that of the cement mortar specimen coated with a pure epoxy resin.The fabrication of hybrid absorbers is presented as an ideal alternative to using them as fillers for nanocomposites to improve microwave absorption. Hybrid absorbers offer the advantages of their individual constituents, such as nitrogen-doped graphene , carbon xy resin . The relxy resin . The attormances . Nanocom3C2Tx MXene/epoxy resin, with the removal of partial polar groups on the surface of MXene by thermal annealing, which reached enhanced electrical conductivities and EMI-shielding performance, have been previously reported [3C2Tx MXene with reduced graphene oxide [Nanocomposites of Tireported . Neverthne oxide , carbon ne oxide , naturalne oxide , and silne oxide  by direc3C2Tx content as low as 0.74 vol %, presented a higher value than those of the previously reported polymer nanocomposites with graphene or other nanofillers. Likewise, 3D porous structures of Ti3C2Tx/carbon hybrid foam (MCF), prepared by the sol–gel method, have been published [4 times and 480%, respectively, compared with that of epoxy resin nanocomposites without hybrid foam.Zhao et al. reported the fabrication of epoxy nanocomposites with MXene/reduced graphene oxide (RGO) hybrid aerogels (MGA) obtained by hydrothermal reaction as fillers . Conduct3C2Tx MXene (TCTA) aerogels impregnated with epoxy resin showed excellent anisotropic electrical conductivity and EMI-shielding effectiveness [3C2Tx/polymer matrix nanocomposites. Absorption-shielding effectiveness increased drastically compared to reflection, demonstrating that absorption governed the shielding mechanism due to highly conductive porous networks and the persistence of aligned porous structures. STEM images  used to reinforce epoxy resin nanocomposites have just been reported [Aerogels with aligned porous structures to be used as nanofillers in epoxy resin nanocomposites have been reported with other materials. On the one hand, cellulose nanofibers have broad application in EMI-shielding, energy storage, etc., besides being a green material with good biocompatibility and excellent mechanical properties. Annealed cellulose nanofibers/Titiveness . The annM images c evidencreported . In this2O3 honeycomb as a template via electrostatic adsorption [Recently, another 3D hybrid porous structures/epoxy resin nanocomposite with superior EMI-shielding performance was reported. MXene nanosheets were assembled with honeycomb-structure reduced graphene oxide, first constructed using Alsorption . Next, h− ions through coatings to the metal substrate. To overcome these problems, additives for acquiring high-efficiency corrosion protection are required.Among organic coatings, epoxy resins (ER) are the most common approach for the corrosion protection of metallic structures . Epoxy c3C2Tx has been the representative and most investigated MXene as an additive in epoxy coatings. This paper reviews different MXene/epoxy resin coatings to protect metal substrates from corrosion.Two-dimensional nanosheets have great potential to improve the barrier performance of epoxy coatings. Graphene , hBN 7979, and t3C2Tx MXene, with F, O, and OH groups, has excellent adsorption stability for the epoxy group (and the benzene structures of DGEBA-based epoxy). Yan et al. [3C2Tx in waterborne epoxy resin using a liquid-phase blending method. The presence of Ti3C2Tx at 0.5 and 1.0 wt % content inhibited the penetration of an electrolyte and considerably improved the corrosive resistance of epoxy coatings, which was attributed to the intrinsic properties of Ti3C2Tx and its strong barrier effect. However, the largest aggregates were detected with increasing content. The optimal addition of Ti3C2Tx in the epoxy coating was 1.0 wt %.Based on the theoretical calculations, it has recently been reported  that Ti3n et al.  disperse3C2Tx, with 3-aminopropylthiethoxysilane (APTES), exhibiting good dispersibility and compatibility in the epoxy coating. Incorporating as low as a 0.5 wt % content of NH2-Ti3C2Tx sheets was enough to exhibit excellent anti-corrosion performance on an aluminum alloy substrate. This greatly improved performance was mainly attributed to the adhesion strength between the coating and metal substrate by the interaction between amino of APTES with hydroxyl groups on metal. Regarding water uptake, a significant contribution of amino-functionalized Ti3C2Tx sheets was observed. Impedance spectroscopy (EIS) in a 3.5 wt % NaCl aqueous solution was used to consider corrosion protection. The impedance modulus at low frequency (|Z|0.01Hz) from Bode-impedance plots is commonly used as a semi-quantitative indicator for assessing anticorrosion performance. Usually, the higher |Z|0.01Hz represents better corrosion protection. For neat epoxy coating, the |Z|0.01Hz value was 2.34 × 108 Ω cm2 24 h after initial immersion. After 4 weeks of immersion, the value decreased by three orders of magnitude, implying rapid coating destruction. When unfunctionalized MXene was incorporated (at 0.25 and 0.5 wt % content), significantly higher values after 4 weeks of immersion were reached compared to neat epoxy (about one order of magnitude higher). Meanwhile, the |Z|0.01Hz values for amino-Ti3C2Tx/epoxy were remarkably higher, especially at 0.5 wt % content (from 3.09 × 109 Ω cm 24 h and 1.02 × 107 Ω cm2 after 4 weeks), which was two orders of magnitude higher than neat epoxy coating, indicating excellent barrier properties. More recently, Li et al. [3C2Tx MXene, with notable anticorrosion improvement from the better dispersibility and chemical bonding in the ER. The positive effect of epoxy functionalization on corrosion resistance was confirmed by EIS measurements using a 3.5 wt % NaCl solution as an immersion medium. The highest impedance was achieved for the 0.5 wt % MXene coating. In addition, it was further believed that GPS-Ti3C2Tx could repair the defects and compact the epoxy coating. In addition, the authors suggested that compared with amino-functionalized MXene (APTES-Ti3C2Tx), the GPS-Ti3C2Tx nanosheet should be more suitable for practical application in epoxy coatings. Given the similarity between MXene and graphene, it makes sense to compare the behavior of both. Pourhashem et al. [The dispersion degree of the MXene nanosheets significantly affects the anti-corrosion performance of coatings. In addition, similar to graphene, MXene may have the opposite effect due to the formation of electrodes by the highly conductive network in the coating matrix. Because of this, investigations have been focused on finding a method that could improve the dispersibility of MXene in the epoxy coating and inhibit galvanic corrosion. Yan et al.  synthesii et al.  focused m et al.  found th3C2Tx at low loading, but a change in GPS-Ti3C2Tx content from 0.3 to 0.7 wt % caused aggregation, weakening the barrier protection. A Nyquist diagram with a larger capacitive arc diameter corresponded to a better anticorrosion property. A significant reduction in capacitive semicircle diameter occurred for all coatings during immersion. This phenomenon is universal in EIS measurement. For neat ER, the smallest semicircles’ diameter in the Nyquist plots during the immersion stage are commonly observed along with the worst anticorrosion property. Unmodified Ti3C2Tx coatings only exhibited a slight improvement in Nyquist plots compared with neat ER. For the GPS-Ti3C2Tx coatings, the semicircle diameter increased when the amount was increased from 0.3 to 0.5 wt % but decreased significantly when the content was increased because aggregates were formed (TEM images showed excellent dispersion and uniform GPS-Tie formed .3C2Tx) on graphene oxide (GO) nanosheets without functionalization and incorporating them into an epoxy zinc-rich resin [3C2Tx, and GO-Ti3C2Tx zinc-rich epoxy coatings) remained within the scope of cathodic protection at the initial stage of immersion. However, coatings with Ti3C2Tx presented cathodic protection for a more extended period of immersion, especially for GO-Ti3C2 coatings, because they were able to provide electrical contact between zinc particles and the steel substrate, ensuring that sacrificial properties were not suppressed, which brought about a longer service life of the epoxy coating.MXene functionalization is not always necessary. Taking advantage of the traditional cathodic protection of zinc, heterostructures were designed by grafting MXene -3-methylimidazolium bromide, an ionic liquid (IL), for waterborne epoxy coatings, to ensure superior oxidative stability of MXene in air. The IL simultaneously served as a high-efficiency corrosion inhibitor to prevent metals from corrosion and as a dispersant of Ti3C2Tx MXene into the epoxy coating. The electrochemical results confirmed an increase in the impedance modulus of the coating by 1–2 orders of magnitude higher relative to neat epoxy, containing small amounts of MXene. The superior anticorrosion performance of the composite coatings was mainly ascribed to the great increase in highly dispersed IL-MXene nanosheets and the extra property caused by the formation of IL passive films.Zhao et al.  prepared3C2Tx/epoxy coating over a nitriding layer was used to protect aluminum alloy [3C2Tx/epoxy coating kept the ability to repair the defects of the nitriding layer. Additionally, the impedance at low frequencies increased by two orders of magnitude after 4 weeks of immersion in a 3.5 wt % NaCl solution, confirming a significant advantage over the neat epoxy. The authors assigned this excellent performance to the barrier network formed by Ti3C2Tx and the shield of the nitriding layer.An inorganic–organic multilayer protection system consisting of Tium alloy . It is w3C2Tx heterostructures with promising anticorrosion abilities using a (2D) layered double hydroxide (LDH) nanoclay based on Mg2+ and Al3+ assembled on surface MXene (MXene@LDH). Combining LDH antifriction performance as lubrication additives and, as recently reported, its active corrosion protection [3C2Tx MXene flakes. The MXene@LDH heterostructures exhibited excellent dispersibility and compatibility with the epoxy resin matrix. Comparing EIS measurements, larger capacitive arcs in Nyquist plots were observed for all fabricated coatings, LDH, MXene, and MXene@LDH epoxy composites, compared with neat epoxy. All formulations gradually deteriorated as the immersion time increased, but the MXene@LDH system displayed the largest radius in Nyquist curves for the different immersion times evaluated.Meng Cai et al.  designedotection , the assc (coating resistance) and Rct (charge-transfer resistance) are related to the resistance of the penetration of electrolytes, and theoretically, the higher value of Rc and Rct means good corrosion protection, i.e., a larger Rc represents better resistance of the coating. Thus, the Rc and Rct values of all the coatings display a decreasing tendency with immersion time. Higher Rc and Rct values compared to neat epoxy are commonly reported with the addition of MXene into the epoxy coating. However, important differences were observed depending on the type of functionalization used [Electrical equivalent circuit models a provideion used ,83,89.3C2Tx hybrid as an additive for epoxy coating. Excellent anticorrosion behaviors under both atmospheric pressure and a simulated deep-sea environment were detected. A small amount of SF-Ti3C2Tx was sufficient to show an impedance value of 1.31 × 108 Ω cm2, which is four orders of magnitude greater than pure ER. As a result of the MXene modification with SF fibers, the surface became rough, helping to improve the adhesion between Ti3C2TX and the epoxy resin. The addition of 0.5 wt % SF-Ti3C2TX was beneficial to achieve better corrosion resistance, even after immersion for 60 days at atmospheric pressure, and exceptional adhesion strength. Moreover, the |Z|0.01Hz still maintained ~108 Ω⋅cm2 after immersion for 240 h under severe hydrostatic pressure, offering ineffective failure prevention in harsh deep-sea environments.The work of Chen et al.  used silPhosphorylated chitosan (p-CS)  and biod3C2Tx MXene nanosheets modified with APTES and cerium cations (Ce3+) as an eco-friendly corrosion inhibitor to fabricate novel self-healing epoxy nanocomposite coatings. MXene-Ce3+ was introduced in the saline solution and into the epoxy resin and showed important improvement of the total impedance relative to epoxy, attributed to the release of Ce3+ cations into the corrosive medium, creating cerium-based components (cerium hydroxides/oxides), which act as a physical barrier for electrolyte diffusion, prolonging the durability of the coating. Another novel functionalization involving embedded carbon dots (CD) on the surface of MXene has recently been published [3C2TX/epoxy composite compared with a randomly dispersed CD-Ti3C2TX/epoxy composite coating. The authors reported more than four orders of improvement in the impedance modulus because CD can easily be absorbed on the steel surface, providing an additional passivation effect. In addition, the parallel arrangement of the CD-Ti3C2Tx nanosheet can inhibit corrosion by eliminating connections of MXene/MXene and MXene/metal in the vertical direction, perpendicular to the diffused direction of corrosive species. An extraordinary Rc was reached, four and two orders of magnitude higher than that of neat ER and random CD-Ti3C2Tx coatings, respectively. In this work, the aligned CD-Ti3C2Tx epoxy coating showed the highest anti-corrosion performance reported, even compared to other forms of 2D nanosheet composite coatings such as graphene and BN-based epoxy composite coatings.Novel strategies have also been reported. For example, Haddadi et al.  synthesiublished . A (CD)-In summary, functionalized-MXene provides a promising method for designing nanocomposite coatings for high-performance and long-term metal protection. Nevertheless, despite the publications reported to date, it still remains a challenge to design a simple and effective process to fabricate MXene-based epoxy coatings with high anti-corrosion performances. More efficient protection strategies for MXene nanosheets are still to be explored. The most relevant results in the anticorrosive properties of MXene/ER nanocomposites are summarized in 3C2Tx MXenes, curcumin (cur)—which has multifunctional therapeutic properties—and epoxy-capped and phenylboronic-acid-decorated hyperbranched polyether (EHBPE-PBA), which was obtained through an epoxy/thiol curing chemistry. The resulting biomedical patch presented photo/thermal-induced self-healing ability and notable antibacterial activity owing to the high photothermal-conversion efficiency of Ti3C2Tx MXenes under near-infrared radiation at 980 nm. Furthermore, hybrid patches evidenced good surface wettability and water absorption thanks to the hydrophilicity of MXene nanosheets and the PEG-based polymer backbones forming EHBPE-PBA.The extraordinary physical and chemical properties of MXenes previously reviewed have attracted wide interest in the areas of mechanical engineering, optics, energy, and electronics. Currently, these materials are broadening their applications in the biomedical field. This is mainly due to their large surface area and strong absorbance in the near-infrared region. Furthermore, they present a highly efficient photothermal effect and more powerful antibacterial activity than other two-dimensional nanoparticles, such as graphene. Zou et al. reported the fabrication of multifunctional MXene/hyperbranched epoxy biomedical patches . The hyb3C2Tx MXene loaded with 2-methylimidazole zinc salt (ZIF-8) nanocontainer@benzotriazole (BTA) multifunctional composite filler incorporated were also reported [MXene nanosheets can absorb light and microwaves; hence, the thermally induced self-healing behavior of the coatings incorporating MXene was investigated. An epoxy coating, a reversible crosslinking network based on the Diels–Alder reaction, with dispersed MXene flakes, was fabricated . MXene nreported . The coa3C2Tx MXene, resulting in a doping level as low as 30 ppm, the most effective loading compared to those previously reported. The strong interaction between the functional groups and unsaturated bonds on the MXene surface and epoxy matrix in the interface region generated a high potential barrier under the action of an external field. Consequently, the energy was not enough to overcome the potential barrier, and most of the carriers were effectively trapped at the interface, suppressing the surface charge accumulation of the insulator.MXene is a 2D nanomaterial with an extensive ion channel, allowing moderate charge adsorption capacity and a large specific surface area that is chemically very active. MXene/epoxy nanocomposites have been fabricated to eliminate the surface charge accumulation on insulators . Epoxy rEpoxy resins are thermosets with interesting physicochemical properties for numerous engineering applications. They are also an ideal matrix of fiber-reinforced polymer composites. Considerable efforts have been made to improve their properties by adding nanofillers to their formulations. The selection of nanofillers has been marked not only by the intrinsic functional properties afforded to the material but also by their ease of synthesis, availability, and compatibility with the matrix. One of the most recent and most promising functional materials is MXenes, which can be obtained from MAX phases. MXenes are thermal and electrical conductors and show optimal mechanical performance. They are also hydrophilic with high-energy surfaces that are compatible with a wide range of polymers and may be functionalized/decorated with inorganic and organic compounds.In epoxy resins, MXenes have been added with four main objectives: (i) to improve the mechanical performance of epoxy resins and fiber-reinforced epoxy composites, (ii) to increase the thermal and electrical conductivity of the resin, (iii) to improve the tribological and anti-corrosive behavior of epoxy resin coatings, and (iv) to improve radiation absorption for EMI shielding.Despite the novelty of MXenes, considerable notable achievements have been attained on those objectives. From the point of view of mechanical properties, the introduction of MXenes into the epoxy resin improves the mechanical properties: the modulus, strength, and toughness. Nevertheless, a significant drawback of MXenes is the aggregation and re-stacking of the nanosheets for relatively high nanofiller loads, which limits their performance. The step of dispersion into the resin is, in fact, a fundamental stage to achieve good properties in the composite. In addition, MXenes must be manipulated with care and inert atmospheres at low temperatures to avoid oxidation. Recent efforts showed that the chemical surface groups of the delaminated MXenes can be functionalized with a variety of compounds and polymers to increase their compatibility with the resin, increase their dispersion, and protect MXenes from oxidation, with promising results. MXenes have also shown their usefulness in improving the behavior of fiber-reinforced epoxy composites. Taking advantage of their chemistry, MXenes have been used to modify the surface of activated carbon fibers, improving the fiber–matrix interfacial interaction. Remarkable increments in ILSS and IFSS have been reported. It should be noted that, although MXenes hold up well compared to other materials with similar structures, such as graphene, they provide additional interesting functionalities.The improvement of the electrical and thermal conductivity of the epoxy resins by MXenes has been established. Notable electrical conductivities have been reported with low MXene loads, making their epoxy composites interesting for EMI shielding, anti-static coatings, and electronics and energy-related applications. The thermal conductivity of epoxy resins is highly increased by MXene addition. Especially in combination with Ag nanoparticles for reducing interface thermal resistance among the nanofiller and the epoxy matrix, MXene nanocomposites showed high conductivity and low thermal expansion coefficients. This effect widens the use of epoxy resins as thermal interface materials. The application of MXenes in epoxies for fire safety is now being explored. Preliminary works showed synergies when MXenes are combined with classic flame-retardant materials in the epoxy resin.Epoxy resins are intrinsically brittle, so their tribological behavior is poor, a major drawback for their use as adhesives or protective coatings. MXenes have advantages with respect to traditional anticorrosive and antifriction nanofillers because their hydrophilicity enhances the interaction with the matrix and increases the epoxy resin’s wettability onto high-energy substrates. High-performance coatings with low friction and wear and/or anticorrosive properties have been prepared. For tribological performance, the best results are obtained when nanoparticles are introduced on the surface of MXenes. Those nanoparticles increase the roughness and favor mechanical interlocking with the matrix, help to reduce the friction coefficient, and may also protect MXenes from oxidation. Two-dimensional nanofillers have also been used to improve corrosion protection because of their barrier properties, which create tortuosity paths to delay the corrosion when microcracks or defects appear in the coating. MXenes show more important advantages: forming brick-wall structures that block the diffusion into the resin. A good dispersion of the MXene sheets is again fundamental to achieving optimal behavior. Recently, interesting results have been found with heterostructures created with MXenes and graphene or LDH nanoclays or by adding other components, reducing the contact between the MXene layers. Functionalized MXene introduction on epoxy coating formulations is undoubtedly a promising route to obtain high-performance anticorrosive coatings.3C2Tx MXene/epoxy nanocomposites by blending may result in a simple and easily applicable method. Nevertheless, one of the drawbacks is the high MXene loading required to reach an effective conductive network. Synergistic effects in improving EMI-shielding and microwave-absorption performances have been provided by combining MXenes and other additives, such as magnetic nanoparticles, reduced graphene oxide, and silver nanowires. Fabrication of ordered structures with aligned conductive networks is an interesting strategy to improve conductivity at low filler loadings. We have identified recent works in which 3D hybrid conductive networks have been used as fillers of epoxy resin matrix exhibiting outstanding EMI-shielding properties. We consider this strategy a doubtlessly valuable methodology; nevertheless, it is not yet a significantly explored field. The freeze-drying fabrication process allows achieving porous conductive networks with low MXene content while maintaining a 3D structure. Despite this, MXenes are easy to oxidize, and EMI-shielding composite foam materials have poor air stability; therefore, the development of improved alternate fabrication methods continues to be a challenge.MXenes have acquired consideration in electromagnetic-interference-shielding and microwave-absorption fields for use as fillers, owing to their outstanding electrical conductivity, chemically active surface, and high specific surface area. The fabrication of Ti3C2. Consequently, it is necessary to expand studies to other members of the extensive MXene family, not only carbides but also nitrides and carbonitrides. Moreover, except for a few papers, authors do not systematically analyze the influence of the size of the MXene sheets on the overall performance. The MXene dimensions may be a consequence of the delamination and dispersion procedure used, which may give rise to non-comparable results. A lack of standardization is also common in many nanomaterials and delays the research advances. At the current moment, anticorrosive coatings and EMI shielding appear to be applications with the most significant potential, but this may change if the availability of MXenes substantially increases. It is interesting to also note that, even the antibacterial and antifouling properties of MXenes begin to be established, there is a deficiency of studies related to this important feature on their epoxy nanocomposites with perspectives in environmental applications. This exhaustive review summarizes the previously reported properties exhibited by Ti3C2Tx MXene/epoxy resin nanocomposites. We are looking forward to providing insight into future trends and challenges for developing materials with enhanced properties for next-generation applications.In summary, MXenes have demonstrated that they may enhance a range of properties of great interest in epoxy resins. However, new challenges are still present that require addressing. The synthesis of MXenes has to be improved to reduce costs and favor their application. The oxidation of MXenes is an undesired possibility that has to be minimized by controlling the storage and application conditions. Functionalization will mitigate this drawback and help avoid the restacking of nanosheets. Many studies showed the potential of MXenes, the influence of the MXene’s chemical and physical structure on the composite performance remains unexplored. Indeed, most of the research on MXenes/epoxy nanocomposites has been performed with Ti