Previously, we reported that pharmacological preconditioning (PPC) leads to a decrease in the amplitude of L-type calcium channel current in the heart. In this study, we examined PPC-associated changes in SOC function. We measured adult cardiomyocyte membrane currents using the whole-cell patch-clamp technique, and we evaluated reactive oxygen species (ROS) production and intracellular Ca2+ levels in cardiomyocytes using fluorescent probes. Diazoxide (Dzx) and thapsigargin (Tg) were used to induce PPC and to deplete internal stores of Ca2+, respectively. Ca2+ store depletion generated inward currents with strong rectification, which were suppressed by the SOC blocker GSK-7975-A. These currents were completely abolished by PPC, an effect that could be countered with 5-hydroxydecanoate , an intracellular mitochondrial energizing solution, or Ni2+ [a blocker of sodium–calcium exchanger (NCX)]. Buffering of ROS and intracellular Ca2+ also prevented PPC effects on SOC currents. Refilling of intracellular stores was largely suppressed by PPC, as determined by measuring intracellular Ca2+ with a fluorescent Ca2+ indicator. These results indicate that influx of Ca2+ through SOCs is inhibited by their ROS and Ca2+-dependent inactivation during PPC and that NCX is a likely source of PPC-inactivating Ca2+. We further showed that NCX associates with Orai1. Down-regulation of SOCs by PPC may play a role in cardioprotection following ischemia–reperfusion.Voltage-dependent Ca Mitochoerfusion .+ channels (mitoKATPs)  . BlockadtoKATPs) . Pharmac opening . In addi opening . PPC redd damage . There h2+ ions are critical mediators in cardiac excitation–contraction coupling, the process that enables the heart chambers to contract and relax. The development of contraction depends on a rise in myoplasmic Ca2+ concentration. During the cardiac action potential, the Ca2+ influx through depolarization-activated Cav1.2 channels in the cell’s plasma membrane triggers the release of Ca2+ from the sarcoplasmic reticulum (SR)  the myocyte, depending on the membrane potential  . Subsequger NCX; , and theniporter . Becauseotential . In the 2+ influx has a dual role in ischemic settings. That is, it has a deleterious role during I/R-induced damage as well as a beneficial role via triggering cardioprotective mechanisms during ischemic preconditioning . As implied by their name, SOCs respond to intraluminal Ca2+ reductions in the endoplasmic reticulum. The stromal interaction molecule (STIM) and Orai proteins have been identified as Ca2+ sensors and store-operated channels, respectively. Their main role is to replenish internal Ca2+ stores (2+ entry (SOCE) has also been described  SOCE is downregulated during PPC, (2) downregulation of SOCE results from SOCs inactivation, and (3) that inactivation involves NCX and mitoKATP channels. Because PPC involves mitoKATP channel opening and increased ROS levels (N-acetylcysteine (NAC). SOCs are inactivated by Ca2+ (2+ concentration ([Ca2+]i) elevation contributes to SOC inactivation, in addition to ROS, we buffered myoplasmic Ca2+ with a Ca2+ chelator. SOC activation has been reported to require actively respiring mitochondria and mitochondrial Ca2+ uptake can be maintained when mitochondria are induced to remain in an energized state , which was injected with sodium heparin .Experiments were conducted with male Wistar rats (300–350 g) employing protocols approved by the Division of Laboratory Animal Units and CINVESTAV-IPN, in compliance with federal law, federal statute, and 2+-free Tyrode’s solution containing 136 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose. Unless otherwise stated, all chemicals were purchased from Sigma–Aldrich . Hearts were recirculated for ∼60 min using Tyrode’s solution supplemented with 70 U/mL type II collagenase  and 0.5 mg/100 mL type XIV protease. Ventricles were minced and shaken two to three times at 2 × g for 7 min in the same solution. Dislodged cells were filtered through a cell strainer  and centrifuged at 72 × g for 2 min. The pellet was re-suspended in Tyrode’s solution and the cardiomyocytes thus harvested were used immediately.Ventricular myocytes were isolated as described previously , with sl4, 1.8 mM MgCl2, 10 mM EGTA, and 15 mM HEPES. The bath solution (pH 7.4) contained 137 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose, 10 μM verapamil, 200 μM ouabain, and 10 mM HEPES. To deplete SR Ca2+ stores, we used the SR Ca2+-ATPase blocker thapsigargin (Tg) at a concentration of 2 μM from a 2-mM stock solution in dimethyl sulfoxide (DMSO). The ROS scavenger NAC was used at a concentration of 2 mM. NCX was blocked with 5 mM Ni2+ ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) 30 μM, Molecular Probes/Thermo Fisher, Waltham, MA, United States, 30 min prior to electrophysiological recordings, as has been done previously . Dzx was added from a 0.1-M stock solution in DMSO to achieve a final DMSO concentration <0.01%. At the concentration used in this study, Dzx is a selective opener of mitoKATP channels . The sel2+]i from single cardiomyocytes as described previously  for 45 min at room temperature. Fura-2 AM was diluted in Tyrode’s solution to a final concentration of ∼5 μM from a stock solution in DMSO containing 9 mM Fura-2 AM and 25% w/v Pluronic F127 . The cells were washed in Tyrode’s solution and attached to laminin-coated coverslips for 20–30 min at room temperature before performing [Ca2+]i measurements. Ratiometric images of Fura-2 AM fluorescence were monitored with an Eclipse TE300 microscope  equipped with a Polychrome V monochromator , allowing high speed changing between 340 and 380-nm excitation wavelengths. Fluorescence emissions were captured though a 510WB80 filter  with an iXon EM + DU885 digital camera . Image acquisition and ratio analysis were performed in Imaging Workbench 6.0 software . [Ca2+]i was estimated as the Fura-2 AM fluorescence ratio (340/380). Ca2+ SR stores were depleted with caffeine (10 mM) and Tg (2 μM). N-Methyl-D-glucamine replaced extracellular Na+ equimolarly (Na+-free solution).We measured i in their microdomains. Ca2+-dependent inactivation of SOCs develops over a period of seconds (2+-dependent inactivation (2+ buffering, consistent with the involvement of Orai1 channels in the generation of SOC currents.Because SOCs are strongly inactivated by Ca2+ , the ext seconds . Among ttivation . In our 2+ produced partial restoration of SOC currents in Tg-treated non-PPC cardiomyocytes suggests that the Ca2+ that inactivates SOCs upon ramp application is likely provided via NCX operating in the reverse mode (2+ influx via NCX is expected to inactivate Orai1 channels in their proximity, providing a functional link between NCX and SOCs. In PPC cardiomyocytes, we found that Tg-generated currents were restored by Ni2+, suggesting that NCX also plays a role in the inactivation of SOCs by PPC. The formation of a complex between Orai1 and NCX in the plasma membrane observed in our co-immunoprecipitation experiments further supports the notion that these proteins may be associated in a cardiac signaling complex promoting inactivation of SOCE during PPC. Because SOC inactivation reduces Ca2+ influx into cardiomyocytes, it could help to attenuate Ca2+ overload and thereby provide protection from I/R damage. In agreement with this possibility, inhibition of NCX has been shown to abolish ischemic tolerance in the heart induced by either the volatile anesthetic sevoflurane or ischemic preconditioning (The present finding showing that NCX blockade with Nirse mode . Ca2+ initioning .2+ buffering capacity by mitochondria during PPC contributes to the promotion of SOC inactivation. Mitochondria are active participants in intracellular Ca2+ signaling and oppose slow SOC inactivation by buffering Ca2+ and, thus, competing with SOC inactivation sites for Ca2+ (2+ through the mitochondrial Ca2+ uniporter and regulate Ca2+ microdomains (2+ influx through SOCs in Jurkat cells is reduced when mitochondrial buffering capacity is compromised by mitochondrial membrane depolarization following Dzx-induced mitoKATP channel opening (Our experiments suggest that a decreased Cafor Ca2+ . In non-odomains . Consist opening  or when  opening , 2000.2+-dependent inactivation of Cav1.2 channels in adult cardiomyocytes (The use of an energizing cocktail solution in the patch pipette delays and attenuates slow SOC inactivation . In PPC myocytes .2+ overload (2+ contents are markedly depleted in intact hearts subjected to I/R (2+ depletion would be expected to activate SOCE.The SOC inhibitor glucosamine  has beenoverload . A potend to I/R . The ens2+-dependent channel inactivation. Additionally, we showed that SOC currents can be restored by maintaining mitochondria in an energized state, which indicates that mitochondria play a key role in these observations, and that the NCX contributes as a source of channel-inactivating Ca2+. Hence, this study suggests that PPC mediated protection against I/R damage may be explained, in part, by decreased SOCE as a result of the largely abrogated influx of Ca2+ through SOCs. Although the present experiments were performed in single cells, it is plausible that PPC would produce similar changes in intact hearts. Future research is needed to test this prediction directly. Given the clinical importance of protecting the heart from I/R damage, a detailed understanding of the cellular mechanisms underlying PPC is relevant to the development of cardioprotective therapies.The main findings of this work are that PPC results in a large inhibition of SOC currents upon depletion of SR, and that this effect is due to ROS and CaThe datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Division of Laboratory Animal Units, CINVESTAV.2+ experiments. EC performed the co-immunoprecipitation experiments. AH assisted with the isolation of cardiomyocytes. JS and MG contributed reagents, material, and acquired financial support for the project. JS analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.JS designed the study. JS, RS, EC, and MG planned the experiments. RS performed the patch-clamp experiments and ROS measurements. EF and MG performed the CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acute myeloid leukemia (AML) is hematopoietic malignancy. This study was designed to develop an individualized prognostic nomogram to predict cancer-specific survival (CSS) and overall survival (OS) of AML.The clinical data of AML patients  diagnosed from 1973 to 2014 were obtained from the Surveillance, Epidemiology, and End Results database. The patients were divided into training cohort  and validation cohort . The prognostic nomograms were designed with clinical variables selected by multivariate Cox regression model in training cohort. The concordance index (C-index), calibration curve, and receiver operating characteristic curve were used to assess the performance of the nomograms.The predictors in nomogram for CSS were AML subtypes, age, sex, region, marital status, and chemotherapy, whereas the predictors for OS were AML subtypes, age, sex, region, race, marital status, and chemotherapy. The C-indexes of the nomograms in internal validation for CSS and OS were 0.712 and 0.703, respectively, whereas the C-indexes in external validation for CSS and OS were 0.712 and 0.705, respectively. The area under the curve of receiver operating characteristic curves for CSS and OS were 0.799  and 0.809 , respectively.The individualized prognostic nomogram could perform relatively accurate prediction of outcome in adult patients with AML. The clinical outcome of AML patients are closely related to immune, molecular, and cytogenetic abnormalities,–5 as well as age at diagnosis, sex, marital status, insurance status, and county-level income.–8 Over the past few decades, diagnosis and treatment in patients with AML has improved, but the overall survival (OS) rate for AML is still low, less than 50%. Therefore, prognostic models need to be established to provide evidence for diagnosis and treatment of AML in clinic.Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignant disease derived from myeloid hematopoietic progenitor cells–12 Recent research shows that nomogram models are built to analyze OS by integrating mutated genes for older patients with AML.The nomogram models have been validated in the prognosis of several malignancies, which can provide good statistical predictions on survival probability.In this study, we tried to design a nomogram model for predicting the survival probability of adult patients with AML, using the Surveillance Epidemiology and End Result (SEER) dataset between 1973 and 2014. The SEER program in the National Cancer Institute's Division of Cancer Control and Population Sciences is the most reliable and comprehensive source of population-based cancer information in the United States, which provides a large dataset for our nomogram models construction. AML subtypes, sex, age at diagnosis, region, race–ethnicities, marital status, and chemotherapy in SEER program were included into the nomogram models analysis. The visual format of the nomogram helps to understand the prognosis of an individual so that their physicians can make a corresponding treatment based on the prognosis.22.1 The clinical data of AML patients diagnosed from 1973 to 2014 were obtained from the SEER database by using the SEER∗Stat program (version 8.3.5). A total of 65,535 records were obtained. In the SEER data, the AML subtypes were classified according to the 3rd edition of the International Classification of Disease Oncology (ICD-O-3) and WHO 2008 definitions.,16 The AML subtypes included in this study are as follows: 9840/3 – acute erythroid leukemia; 9861/3 – AML, NOS; 9865/3 – AML with t , DEK-NUP214; 9866/3 –acute promyelocytic leukemia ) PML/RARA; 9867/3 – acute myelomonocytic leukemia; 9869/3 – AML. inv (3) or t , RPN1-EVI1; 9871/3 – AML with inv (16)(p13.1q22) or t , CBFB-MYH11; 9872/3 – AML with minimal differentiation; 9873/3 – AML without maturation; 9874/3 – AML with maturation; 9891/3 – acute monoblastic and monocytic leukemia; 9895/3 – AML with myelodysplasia-related changes; 9896/3 – AML, t  RUNX1-RUNX1T1; 9897/3 – AML with t , MLLT3-MLL; 9910/3 – acute megakaryoblastic leukemia; 9911/3 – AML (megakaryoblastic) with t , RBM15-MKL1; and 9920/3 – therapy-related myeloid neoplasm. Among the above AML subtypes, ICD-O3 codes 9840/3, 9861/3, 9865/3, 9867/3, 9869/3, 9871/3, 9872/3, 9873/3, 9874/3, 9891/3, 9895/3, 9896/3, 9897/3, 9910/3, 9911/3, and 9920/3 belonged to non-APL AML, whereas 9866/3 belonged to APL. The criteria of region were as follows: East includes Connecticut, Atlanta (Metropolitan), and Rural Georgia; Northern Plains include Detroit (Metropolitan) and Iowa; Pacific Coast includes San Francisco Oakland, Hawaii, Seattle (Puget Sound), San Jose-Monterey, and Los Angeles; and Southwest includes New Mexico and Utah.The SEER program, involving in approximately 26% of the US population, is a publicly available database and primary source of cancer statistics that is supported by the Surveillance Research Program in the National Cancer Institute's Division of Cancer Control and Population Sciences.The following cases were excluded: age at diagnosis <18 years; unknown survival time; unknown marital status; and unknown race/ethnicity. Owing to the small number of patients from Alaska, they might cause bias in survival analysis, so they were also excluded.,18 AML was divided into APL and non-APL. The follow-up time was recorded as the duration of time from the diagnosis to death or the last day of survival information documented in the SEER registry. The variable of “vital status recode” was used to determine the status of survive.The following variables were analyzed: AML subtype, sex, age at diagnosis, region, race/ethnicity, marital status, chemotherapy, cause-specific death, and vital status. It is worth noting that the race/ethnicity of yellow included Chinese, Korean, and Japanese in this study. Additionally, in marital status, married included separated, whereas single included never married or unmarried. According to the prognosis of patients,After exclusion of patients based on the above criteria, 58,882 AML patients were identified for OS analysis. Furthermore, after excluding patients with noncancer-specific death [noncancer-specific survival (CSS)], 42,652 patients were identified as entering CSS analysis. Ultimately, patients were randomly assigned to a training cohort and a validation cohort 1:1 ratio) for OS and CSS analysis  to clarify the independent prognostic value of clinical variables for OS and CSS. Clinically significant variables for OS and CSS, which were selected in multivariate Cox proportional hazard regression models in a backward stepwise manner based on the Akaike information criterion, were assessed for incorporating into the nomogram model. The foreign, rms, hmisc, lattice, survival, formula, and ggplot2 packages in R, version 3.5.1 (http://www.r-project.org/) were applied for nomogram model analysis. Model performance was assessed by internal and external validation, which was performed by discrimination with concordance index (C-index) and calibration curves using 1000 sample bootstrap. Then, all cohorts of patients were given a total score using standard points obtained from the nomogram models, which could predict survival rates of AML patients. The patients were randomly assigned using the Microsoft Excel 2007. The receiver operating characteristic (ROC) curves was used for predictive ability of nomogram in SPSS Statistical Package version 22.0 . A 2-tailed P value <.05 was considered to indicate statistical significance. This study was performed in accordance with the ethical principals of the Declaration of Helsinki for medical research involving human participants.Qualitative variables were categorized prior to modeling based on clinical experience and significance. For continuous variables, the optimal cutoff of age was obtained using X-tile software version 3.6.1 .33.1The clinical characteristics of the patients in the training and validation cohorts for OS and CSS analysis were listed in Table 3.2X-tile software was used to determine the optimal cutoff value of age in total AML patients n = 58,882) after screening, which was applied for univariate and multivariate Cox proportional hazard regression analysis, as well as nomogram model construction. As shown in Figure  after sc3.3Univariate Cox proportional hazard regression analysis for OS and CSS suggested that there were significant differences in survival rates of AML subtypes, age, gender, region, race/ethnicity, marital, and chemotherapy, which could be further included in multivariate Cox regression analysis Table . As show3.4P < .05 of multivariate Cox regression in the CSS, race/ethnicity could not be employed in nomogram. Details of the labels for tick marks and points in nomograms were shown in Table Clinical parameters after multivariate Cox regression selection were channeled into the construction of training cohort nomogram Fig. . However3.5The C-indexes of 1000 sample bootstrap were 0.712 and 0.703 for the CSS and OS predictive nomograms, respectively, which indicated that nomograms for CSS and OS showed relatively precise ability of discrimination. Further calibration curves manifested that the probability of predicted 1, 3, and 5-year CSS and OS in nomograms were well consistent between the predicted outcome and actual observation Fig. .3.6In the external validation cohort, the C-indexes of predictive accuracy for CSS and OS were 0.712 and 0.705, respectively  of ROC curves for CSS and OS were 0.799 [95% confidence interval: 0.792–0.806, Fig. 4,21–23 In this work, we developed 2 nomogram models based on the SEER database to predict CSS and OS for adult patients with AML. Although the predicted and observed probabilities of 1-year OS in the nomograms were not completely consistent, the C-indexes were all higher than 0.7, which achieved considerable prediction accuracy and repeatability when nomograms were applied to training and validation cohorts. Simultaneously, 3 and 5-year CSS and OS in nomograms showed good predictive accuracy. For the nomogram of AUC, the AUC were consistent with the C-index, indicating that the models could provide a good prognostic assessment system in patients with AML.The nomogram model, compared with other predictive models, integrated different clinical variables to offer a more accurate and personalized prognosis assessment system.PML-RAR fusion gene, which was the target site for all-trans retinoic acid. Over the past years, due to the application of all-trans retinoic acid and arsenic trioxide (As2O3), the clinical complete remission (CR) rate and status of the disease-free survival of APL have been significantly improved, and the CR rate has been higher than 90%. However, 3-year OS rate of non-APL AML was still poor, less than 30%. In the present study, the points of non-APL AML in nomograms for CSS and OS were 100 and 94, respectively, indicating that subtype of AML was a strong predictor of prognosis in nomogram models established by the AML data of the SEER program.Here, some variables in the nomogram models were analyzed. Acute promyelocytic leukemia (APL) was generally characterized by the t  chromosomal translocation to generate ,24 The risk ratios of age were more than 1.7 in multivariate Cox regression and the points of age in nomograms for CSS and OS were all more than 60, suggesting that age, especially >74 years old, was a strong predictor of outcome in patients with AML.With the prolonged life expectancy, the incidence of AML was rising in the aging population. Over the past few decades, with the great progress making in the diagnosis and treatment of AML, the outcome of young patients has been greatly improved, but the prognosis of elderly patients (>60 years old), whose long-term OS rate is less than 10%, was still very poor. However, 50% to 60% patients with AML could achieve CR after intensive induction chemotherapy, and the long-term OS rate after chemotherapy could be improved to 15% to 30%.,26 We found that patients without chemotherapy had risk ratios of more than 1.8 and the points in the nomogram were all more than 80, which played an important role in predicting the outcomes of patients.AML is hematopoietic malignancy progressing rapidly, whose natural process is of only a few months.,27 which were consistent with our findings. However, compared with AML subtype, age, and chemotherapy, the points of sex, region, and marital status in nomogram were low, showing that the predictive ability was relatively poor.Studies have shown that sex, region, and marital status were predictors of outcomes in AML patients, relapse, and risk stratification, which may help to improve the reliability and accuracy of the nomogram models. Hence, larger clinical data was needed to validate the accuracy and repeatability of the nomogram models in the future.However, it is worth noting that population-based data of SEER program usually does not include detailed clinical data such as white blood cell,Overall, in this study, the bootstrap-corrected and ROC curve-validated nomogram models could perform comparatively accurate prediction of 1, 3, and 5-year survival probabilities, which were clinically practical and relatively reliable in adult patients with AML. However, an independent external validation data will still be required to validate the nomogram models in the future, making the models more reliable.Conceptualization: Caixia Wang.Data curation: Cunte Chen, Peipei Wang.Formal analysis: Cunte Chen, Peipei Wang.Funding acquisition: Caixia Wang.Methodology: Cunte Chen, Peipei Wang.Supervision: Caixia Wang.Writing – original draft: Cunte Chen, Peipei Wang.Writing – review & editing: Cunte Chen, Peipei Wang, Caixia Wang.


Mesenchymal stromal cells (MSCs) are an essential element of the bone marrow (BM) microenvironment, playing a crucial function in regulating hematopoietic stem cell proliferation and differentiation. Recent findings have outlined a putative role for MSCs in hematological malignancy development. So far, conflicting results have been collected concerning MSC abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In particular, a considerable amount of evidence has been accumulated strongly supporting a permissive role of MSCs in malignancy evolution to MDS, while a potentially causative or promoting function performed by MSCs in AML has not yet been fully clarified. Here, we compared MSCs isolated from healthy, MDS, and AML subjects to investigate MSC alterations and to emphasize putative common and/or diverse features.We isolated and expanded MSCs from AML patients (AML-MSCs) and MDS patients (MDS-MSCs), and we analyzed and compared their phenotypic and functional properties with respect to each other and versus healthy donor-derived MSCs (HD-MSCs).We found that stable MSC cultures could be easily established from HD and MDS mononuclear BM-derived cells, while a substantial fraction (25%) of AML patients failed to yield MSCs. Nevertheless, isolated MDS-MSCs and AML-MSCs, as well as HD-MSCs, contained the basic features of MSCs. Indeed, they displayed similar surface marker expression and efficient capacity to differentiate versus osteogenic and adipogenic lineage in vitro. We also proved that MDS-MSCs and AML-MSCs, analyzed by fluorescence in-situ hybridization, did not harbor leukemic cell cytogenetic abnormalities. Moreover, MDS-MSCs and AML-MSCs were similar in terms of ability to sustain AML cell viability and immune-regulatory capacity. However, we were also able to detect some differences between AML-MSCs and MDS-MSCs. Indeed, we found that the frequency of rescued MSCs was lower in the AML group than in the HD and MDS groups, suggesting that a reduced number of MSC precursors could inhabit AML BM. Instead, MDS-MSCs showed the lowest proliferative capacity, reflecting some intrinsic and particular defect.Overall, our results elucidated that MDS-MSCs and AML-MSCs did not show macroscopic and/or tumor-related defects, but both displayed functional features potentially contributing to favor a leukemia-protective milieu. Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are two biologically and genetically heterogeneous groups of clonal myeloid neoplastic disorders characterized by hematopoietic stem cell (HSC) dysregulation and ineffective hematopoiesis , 2. In pBM samples were obtained from 32 AML patients  and 26 MDS patients . In addition, BM from 12 healthy donors (HDs)  was used as the control  , and plated at an initial seeding density of 1.6 × 105 cells/cm2. After 2–3 days, the nonadherent cell fraction was removed by rinsing cells with phosphate-buffered saline solution (PBS), and monolayers of adherent cells were cultured until they reached 70–80% confluence. Cells were then detached by trypsin solution  , reseeded at a density of 3.5 × 103 cells/cm2, and used for experiments within passages 3–5. Cell growth was analyzed by direct cell counts at each passage.BM-derived MSCs were isolated from BM aspirates of HDs (HD-MSCs) or patients affected by acute myeloid leukemia (AML-MSCs) or myelodysplastic syndrome (MDS-MSCs) at diagnosis, and were expanded ex vivo as previously described . Briefly5 cells were incubated, in the presence of the antibodies listed, in PBS/0.5% FBS at room temperature with light protection for 15 min. Cells were rinsed in PBS and analyzed by flow cytometry . A minimum of 10,000 events was collected in list mode on FACSDiva software.For immunophenotype studies, dual-color immunofluorescence was performed using the following panel of phycoerythrin (PE)-conjugated or fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies: anti-human CD13, anti-human CD19, anti-human CD34, anti-human HLA-DR, anti-human CD44, anti-human CD45, anti-human CD73 (Becton Dickinson), anti-human CD14, anti-human CD29, anti-human CD105 (Biolegend), and anti-human CD90 (Chemicon). The cell autofluorescence level was used as the negative control. For cell-surface staining, 1 × 103 cells/cm2 and grown in osteogenic differentiation medium (Lonza) containing l-glutamine, mesenchymal cell growth serum MCGS, dexamethasone, ascorbate, β-glycerophosphate, and pen/strep. The medium was replaced every 3–4 days. Cell cultures were stopped at day 21 for histological staining and total RNA extraction. Calcium deposition was determined using Alizarin red staining as previously described [4 cells/cm2 on a Lab-Tek II coverglass chamber  and grown for 3 days in adipogenic induction medium (Lonza) containing additional h-insulin, l-glutamine, MCGS, dexamethasone, indomethacin, 3-isobuty-1-methyl-xanthine, and pen/strep followed by 3 days in adipogenic maintenance medium containing h-insulin, l-glutamine, MCGS, and pen/strep. Both steps were repeated up to day 18 when cell cultures were stopped for histological staining and total RNA extraction. Fat droplets within adipogenic differentiated cells were identified using the Oil Red O staining method as previously described [w/v) Oil Red O solution (Sigma Aldrich) with gentle shaking at room temperature for 15 min. Images were collected with an Axiovert 40 CFL microscope (Carl Zeiss Microscopy).To induce osteogenic differentiation, MSCs were seeded at 3.1 × 10escribed . Brieflyescribed . Brieflyt) values for differentiation specific genes , Runt-related transcription factor (RUNX) 2) and an endogenous reference gene ) were determined automatically using the 7900 ABI PRISM system (Applied Biosystems). Relative quantification was calculated using the ΔCt comparative method [t value of the endogenous reference gene from the Ct value of the specific gene. PPARγ or RUNX2 cDNA levels in undifferentiated cells were taken as 1. In some experiments, cDNA synthetized as already described from a Universal RNA (Agilent genomics) was used as the reference value and taken as 1. All reactions were performed in duplicate. Primer probes for PPARγ, Hs01115513_m1, RUNX2, Hs00231692_m1, GAPDH, and Hs00266705_g1 were purchased from Applied Biosystems.Total RNA was isolated using an RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions and quantified by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). For cDNA synthesis, 1 μg of denatured total RNA was reverse transcribed using an Improm II kit (Promega) and random hexamers (Promega) in a 20 μl final volume according to the manufacturer’s instruction. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the ABI-PRISM 7900 Sequence Detection System (Applied Biosystems). The qRT-PCR reactions were performed using a 96-well Optical Reaction Plate. For each PCR run, 1 μl of cDNA product was mixed with 2× Platinum Super mix (Thermo Fisher Scientific) in a total volume of 25 μl. The thermal cycling conditions consisted of an initial stage of 50 °C for 2 min, and 95 °C for 10 min, 40 cycles of melting at 95 °C for 15 s, and annealing and elongation at 60 °C for 1 min. Threshold cycle . Fluorescent in-situ hybridization (FISH) was carried out on fixed cells according to the manufacturer’s instructions with appropriate specific probes. The following commercially avaialble probes were used for MSC analysis: EGR1 FISH Probe Kit, LSI MLL Dual Colour, Break Apart Rearrangements Probe and LSI PML/RARα Dual Colour, and Dual Fusion Translocation Probe Kit (Vysis); and CBFβ/MYH11 Translocation, Dual Fusion Probe, and RB1 Deletion Probe (Cytocell). Images were analyzed using a fluorescence microscope NIKON E1000 equipped with FITC/TRITC/AQUA/DAPI filter sets and the Genikon imaging system software (Nikon Instruments). At least 200 nuclei were counted for each sample.2 and after 24 h AML cells were seeded with transwells on MSC layers (1:10). After 4 days of cocultures, AML cells were harvested and analyzed by flow cytometry. In apoptosis experiments, after 4 days of cocultures, AML cells were harvested and labeled with annexin-V/propidium iodide (PI) . Briefly, cells were washed in PBS and then incubated with Annexin-V-FLUOS/PI incubation buffer with light protection at room temperature for 15 min and analyzed by flow cytometry as already described.MSCs were seeded at the density of 20,000 cells/cmIn proliferation experiments, before seeding, AML cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (BioLegend). Briefly, cells were washed twice in PBS and incubated with CFSE (5 μM) with light protection at room temperature for 4 min. Cells were then washed twice in cold medium  and analyzed by flow cytometry as already described.+/CD4+/CD25+ cells were gated on CD4+ cells.MSCs derived from HDs, AML patients, or MDS patients were cocultured for 7 days in autologous RPMI with allogeneic peripheral blood mononuclear cells (PBMCs) (ratio 10:1). After 7 days, PBMCs were harvested and stained using the intracellular staining kit FOXP-3/Transcription Factor Buffer Set (eBioscience/Thermo Fisher Scientific) including the monoclonal antibodies (mAbs) PE-conjugated anti-human FOXP3 , APC-H7-conjugated CD3 , FITC-conjugated CD4 , and APC-conjugated CD25 . For each sample, isotype-matched irrelevant mAb staining was used as the control. At least 10,000 events were analyzed by flow cytometry as already described. FoxP3t test or one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test for multiple comparison. All analyses were performed using GraphPad Prism software (version 6.0). Differences were considered statistically significant at p < 0.05.Data are presented as mean ± SEM of at least three independent determinations. Statistical differences between groups were determined by Student’s N = 26) and AML-MSCs (N = 32) at diagnosis from treatment-free patients to rule out potential bias due to chemotherapy effects. As a control, MSCs were isolated and expanded from healthy subjects . MSCs were successfully obtained from all samples of HDs and from almost all the MDSs , while only 75% (24 out of 32) of the AML samples were able to generate MSCs (Table P < 0.01), while the MDS-MSC frequency had an intermediate value  . This pattern was particularly exacerbated in the MDS-MSCs  Fig. . This suML) Fig. . In ordeSCs Fig. . These dMSCs isolated as already described were expanded and analyzed at P3 or P4 to ascertain typical biological properties according to the minimal criteria to define “bona fide” MSCs derived from BM . The immNext, we examined the differentiation capacity of MSCs isolated from MDS and AML patients, in comparison with MSCs isolated from HDs. Thus, MSCs were induced to differentiate versus the osteogenic or adipogenic lineage, as described in Methods, and analyzed after 3 weeks by differentiation-specific histological staining. As shown in Fig. N = 3 MDS, N = 3 AML), we found that neither MDS-MSCs nor AML-MSCs presented the same chromosomal alterations, typical of myeloid malignancies, as those detected in MNCs at diagnosis , which are known to suppress immunity also in hematological malignancies [+/CD25+/FoxP3+ cells). We demonstrated that MDS-MSCs and AML-MSCs efficiently induced Tregs with no significant differences between them or versus HD-MSCs (Fig. In AML, and especially in MDS, immune dysregulation participates in the establishment of a leukemic permissive milieu. We wondered whether there were differences in the immune-regulatory functions of HD-MSCs, MDS-MSCs, and AML-MSCs. In particular, we investigated the ability of MDS and AML patient-derived MSCs to induce CD4gnancies . We therIn the last few years, the possible contribution of BM-MSCs to the pathogenetic/pathophysiologic process of MDS and AML has gained increasing interest. However, so far, understanding of the MSC role in supporting leukemia in vivo has been hindered by a low level of characterization and conflicting results. In particular, it has not been completely clarified whether AML-MSCs and MDS-MSCs share common features contributing to a disease-permissive microenvironment and preferentially supporting neoplastic cells.In this study, we characterized and compared MSCs derived from MDS and AML patients with MSCs derived from HDs as a reference control. We showed that both MDS-derived and AML-derived MSCs met the MSC criteria proposed by the International Society for Cellular Therapy  and are The main abnormality we detected in MDS-MSCs was a marked decrease in their proliferation potential. The growth pattern of MSCs isolated from MDS patients has been questioned, with some authors showing a reduced expansion potential –32, and It is noteworthy that, as expected, MDS samples in our study showed a higher median age than that of AML and HD samples. This could be relevant in differentiation assay results. Indeed, it was reported that MSC osteogenic and adipogenic potential decreased during aging –40. Howe. FISH data in MDS in the literature confirmed that cytogenetic aberrations in MDS-MSCs, if present, differed from chromosomal markers in altered hematopoietic cells [Some authors suggested that genetic alterations in MSCs might represent a specific mechanism of leukemogenesis. Indeed, they showed that MDS and AML patients, with genetic abnormalities in their in-vitro expanded MSCs, had a worse overall and disease-free survival than the normal karyotype . Howeveric cells , 33, 42.MSCs have a unique immune-regulatory and immunosuppressive ability. Since aberrant immune responses have been associated with the pathophysiology of AML, and especially of MDS, we decided to test the immune-modulatory properties of MDS-MSCs and AML-MSCs. In particular, we decided to investigate the ability of MDS and AML patient-derived MSCs to induce Tregs. Indeed, Tregs have been recognized as essential contributors in microenvironment immunomodulation and ultimately in helping leukemic cells to evade immune surveillance , 44. In It has been demonstrated that MDS-MSCs are defective in hematopoiesis supporting functions , 49, 50,Our data demonstrate that MDS-MSCs and AML-MSCs share common features such as phenotype, differentiation capacity, absence of leukemia-specific genetic abnormalities, ability to sustain AML cell viability, and immune-regulatory capacity in vitro. However, AML-MSCs were more difficult to isolate from BM, while MDS-MSCs showed a lower proliferation potential. Overall, MDS-MSCs and AML-MSCs did not present macroscopic defects and/or abnormalities directly related to leukemia, but both displayed functional differences that, translated in vivo, could potentially help to turn the BM microenvironment from hostile to supportive for leukemic cells.


B. monnieri extract (BME) is an abundant source of bioactive compounds, including saponins and flavonoids known to produce vasodilation. However, it is unclear which components are the more effective vasodilators. The aim of this research was to investigate the vasorelaxant effects and mechanisms of action of saponins and flavonoids on rat isolated mesenteric arteries using the organ bath technique. The vasorelaxant mechanisms, including endothelial nitric oxide synthase (eNOS) pathway and calcium flux were examined. Saponins (bacoside A and bacopaside I), and flavonoids (luteolin and apigenin) at 0.1–100 µM caused vasorelaxation in a concentration-dependent manner. Luteolin and apigenin produced vasorelaxation in endothelial intact vessels with more efficacy (Emax 99.4 ± 0.7 and 95.3 ± 2.6%) and potency (EC50 4.35 ± 1.31 and 8.93 ± 3.33 µM) than bacoside A and bacopaside I . Pretreatment of endothelial intact rings, with L-NAME (100 µM); an eNOS inhibitor, or removal of the endothelium reduced the relaxant effects of all compounds. In K+-depolarised vessels suspended in Ca2+-free solution, these active compounds inhibited CaCl2-induced contraction in endothelial denuded arterial rings. Moreover, the active compounds attenuated transient contractions induced by 10 µM phenylephrine in Ca2+-free medium containing EGTA (1 mM). Thus, relaxant effects occurred in both endothelial intact and denuded vessels which signify actions through both endothelium and vascular smooth muscle cells. In conclusion, the flavonoids have about twice the potency of saponins as vasodilators. However, in the BME, there is ~20 × the amount of vaso-reactive saponins and thus are more effective. Bacopa monnieri (L.) Wettst. or Brahmi, is an Ayurvedic medicine traditionally used as a memory enhancer. Along with memory improvement, it is known to promote mental health, as a neurotonic and cardiotonic agent. B. monnieri extract (BME) clearly has a cognitive enhancing potential and neuroprotective effects . Th. Th50 4.However, higher potency of aglycone flavonoids compared to saponin glycosides may be due to sugar moieties interfering with the molecule interacting with the binding sites responsible for the vasorelaxation as suggested by previously, i.e., lipophilic groups in the ring skeleton of flavonoids increased their vasorelaxant activity . This pr50 and reduced Emax which imitated the effect of endothelial denudation, suggesting the relaxation was mainly medicated by NO. This accords with observations made by Jin et al. that a cyclooxygenase (COX) inhibitor did not affect the relaxation induced by apigenin  HEPES, 10; KH aerated ,49,50,51B. monnieri active compounds (0.1–100 µM), including luteolin, apigenin, bacoside A or bacopaside I were added cumulatively.Following stabilization, endothelial intact rings of mesenteric arteries were pre-contracted with 10 µM PE. After the contraction had become constant, the B. monnieri active compounds  induced vasorelaxation, endothelial denuded arteries were used. The data of effect of active compounds were presented as %relaxation.Successful endothelial denudation was confirmed by the absence of relaxation upon addition of 10 µM ACh. For investigation of the role of endothelium in 0.1–100 µM B. monnieri active compounds  induced vasorelaxation were evaluated in endothelial intact ring pre-treated with NG-nitro-L-arginine methyl ester , an inhibitor of eNOS, for 30 min prior to 10 µM PE exposure.The role of the endothelial relaxing factor, NO, in 2+-free Krebs’ solution (containing (mM): ethylene glycol-bis (ß-aminoethyl ether)-N,N,N,N tetra acetic acid (EGTA), 0.01; NaCl, 122; KCl, 5; HEPES, 10; KH2PO4, 0.5; NaH2PO4, 0.5; MgCl2, 1 and glucose, 11 (pH 7.3)) for 30 min followed by replacing with Ca2+-free Krebs’ solution containing 80 mM K+ for 10 min which depolarizes the vascular smooth muscle cells, thus opening VOCCs. Various concentrations of CaCl2 were then added (0.01–10 mM) in a logarithmic progression. After obtaining the maximum response, the baths were washed out and replenished with Ca2+-free Krebs’ solution for 30 min. The Ca2+-free 80 mM K+ solution was then re-applied following pre-incubation for 10 min with either: 10 µM active compounds or 1 µM nicardipine (antagonist of VOCCs). Concentration-response curves to cumulative addition of CaCl2 were then repeated and compared with maximum contraction evoked by previous control CaCl2 challenges.Endothelial denuded mesenteric arteries were equilibrated in Ca2+ loading of the SR Ca2+ stores, endothelial denuded mesenteric arteries were exposed to 80 mM K+ solution for 5 min, and then washed out with Ca2+-free Krebs’ solution containing 1 mM EGTA for 10 min. The arterial rings were then challenged with 10 µM PE  which release Ca2+ from the SR thereby eliciting a transient contraction [+ solution for 5 min, and washed out with Ca2+-free Krebs’ solution containing 1 mM EGTA and 10 µM active compounds for 10 min. The arterial rings were again challenged with 10 µM PE. The PE-induced contractions were compared in the presence or absence of active compounds.To stimulate initial Catraction . The sam50 and Emax were compared using unpaired Student’s t test. Values are expressed as mean ± SEM. A p-value < 0.05 was considered significant. ‘n’ is the number of vascular rings used, each ring originating from a different animal.Statistical analyses used GraphPad Prism version 5.00 for Windows, . Data from each concentration-effect curve was analysed using non-repeated two-way ANOVA. Curve fitting in the figures was generated by the same software using non-linear regression. ECB. monnieri active components, including both saponins and flavonoids, produced vasodilatory effects on rat isolated mesenteric arteries partially via endothelial dependent release of vasodilators and also by direct effects on vascular smooth muscle cells via blockade of Ca2+ influx and its release from SR. This study for the first time reports the comparative vasodilatory effects of saponins and flavonoids found in B. monnieri extract. However, B. monnieri extract, flavonoids i.e., luteolin and apigenin would be more potent vasodilators but saponins have a greater effect because of their greater contents. Accordingly, the clinical benefits on enhanced blood flow and cognitive function may arise from a combination of flavonoids and particularly the saponins.This study demonstrated that


In vivo, the sonophore QC1-pHLIP could detect malignancies at higher contrast than its fluorescent analog ICG-pHLIP, which was developed for fluorescence-guided surgical applications. PHLIP-type optoacoustic imaging agents in clinical settings are attractive due to their ability to target breast cancer and a wide variety of other malignant growths for diagnostic purposes. Intuitively, these agents could also be used for visualization during surgery.Breast cancer is the most common type of malignant growth in women. Early detection of breast cancer, as well as the identification of possible metastatic spread poses a significant challenge because of the structural and genetic heterogeneity that occurs during the progression of the disease. Currently, mammographies, biopsies and MRI scans are the standard of care techniques used for breast cancer diagnosis, all of which have their individual shortfalls, especially when it comes to discriminating tumors and benign growths. With this in mind, we have developed a non-invasive optoacoustic imaging strategy that targets the acidic environment of breast cancer. A pH low insertion peptide (pHLIP) was conjugated to the dark quencher QC1, yielding a non-fluorescent sonophore with high extinction coefficient in the near infrared that increases signal as a function of increasing amounts of membrane insertion. In an orthotopic murine breast cancer model, pHLIP-targeted optoacoustic imaging allowed us to differentiate between healthy and breast cancer tissues with high signal/noise ratios.  While there have been significant developments in diagnosis and treatment of malignant tumors, treatment of many cancers remains a challenge due to difficulties in diagnosing and identifying them without advanced, and infrastructure-heavy equipment. This is particularly relevant in breast cancer, the most common diagnosed cancer in women, affecting almost 270,000 new individuals yearly in the US alone4. For breast cancer, early detection of primary tumors significantly improves overall survival2. The survival differences are most pronounced between breast cancer patients that were diagnosed with either local versus non-local tumors. Women with localized tumors, which represent 60% of the total breast cancer population, have a >99% 5-year survival rate. The survival rate drops to 70–85% for regional and to 25% for distant metastases. Intuitively, diagnosing breast cancer early represents major benefits in terms of increased survival and also decreased number of treatments. For increasing the number of patients who are diagnosed when their disease is still local, non-invasive and early screening technologies are critical.Malignant growths originate from alterations in the genome, including changes in epigenetic regulation, point mutations, gene deletions, duplications and chromosomal rearrangements6. Despite improvements in mammography techniques, breast magnetic resonance imaging (MRI) is complementary for screening asymptomatic women at increased risk of breast cancer7. This is partly due to the limited sensitivity of mammograms, which declines markedly with increasing breast density and leads to false-negative results9. Women with high breast densities are often screened with ultrasound (US)10, but detected abnormalities are often found to be not cancer12. The combination of these screening tests (mammography/ultrasound and breast MRI) is effective, but it is avoided in practice for an average risk patient due to higher cost14. Optoacoustic imaging on the other hand has evolved into a clinically translatable platform which can provide limited functional and molecular information15. The impact of optoacoustic imaging has been shown through a number of clinical studies for various breast screening of patients20. It has the primary advantage of imaging at depths of up to 7 cm with submillimeter resolution, high contrast to noise ratio, real time acquisition, portability and does not require ionizing radiation23. In the near future, optoacoustic imaging systems could be the cost-effective alternative for first point of care testing.Regular systematic screening for breast cancer is therefore key in increasing the overall survival rate24. In breast imaging, tumor heterogeneity limits the general use of cell surface biomarkers for breast cancer imaging26. On the other hand, one general finding which is largely independent from an individual tumor cell’s expression profile is that the pH at the surface of cancer cells is lower than bulk extracellular pH27. The surface acidity of cancer cells is a general physical characteristic that is independent of tumor perfusion and found across an entire tumor30. Therefore, a low pH is a key characteristic in an alternative targeted imaging approach. We hypothesized that a low pH optoacoustic tracer would enable us to non-invasively image breast cancers.So far, optoacoustic imaging often relied on contrast free methods, but to enable a molecularly specific readout, sonophores could be necessary31. Their mechanism of action is based on the protonation of aspartic and glutamic acid residues, which induces a conformational change of pHLIP’s secondary structure and allows the peptide to insert itself into cell membranes. Indocyanine green (ICG) is a well-established fluorescent dye in scientific literature and its use in humans is known for a variety of fluorescence and optoacoustic imaging applications38. A pHLIP peptide conjugated with indocyanine green (ICG) has recently been reported to differentiate normal from neoplastic tissue in various animal tumor models42. To date, however, only one study was carried out using a fluorescent pHLIP for optoacoustic imaging43. One of the most striking advantages of ICG-pHLIP is that upon binding to cells, fluorescence is enhanced by 20–25 times, which increases the tumor to background ratio35. While, intuitively, this enhancement represents a significant advantage for fluorescence imaging, it is a disadvantage for optoacoustic imaging, a technology that is based on the generation of acoustic signals which rely on the energy transfer to heat (non-radiative decay). An imaging agent which does not disseminate its absorbed energy through photons could therefore be more advantageous to consider for optoacoustic applications.The water soluble pH low insertion peptides (pHLIPs) are a class of membrane-binding peptides which target acidity at the surface of cancer cells46. Dark quenchers have gained some attention and holds promise to generate higher optoacoustic signals than their more commonly used fluorescent counterparts since they are strong absorbers and undergo non-radiative decay48. The dark quencher IRDye QC1 is structurally similar to ICG  Does multi-spectral optoacoustic tomography (MSOT) with QC1-pHLIP allow non-invasive delineation of breast cancer in a murine orthotopic breast cancer model? (ii) How does the sonophore QC1-pHLIP compare as an optoacoustic tool to fluorescent ICG-pHLIP? We believe that molecularly targeted sonophores can overcome several hurdles currently hampering the development and broad clinical use of the MSOT. We believe that optimizing and validating QC1 pHLIP for cancer detection is of high value for detecting both localized and non-localized breast tumors. Such an optoacoustic imaging strategy could reduce the frequencies of biopsies, provide accurate monitoring of breast tumors and increase the precision of surgical interventions.left)) and ICG ) belong to the cyanine dye family. The IRDye QC1 is a subclass of heptamethine type, containing a heterocyclic ring in the center giving rise to a more photostable dye than its parent cyanine family dyes. Full chemical characterization of QC1-pHLIP is shown in Fig. R of var3 pHLIP was found at 17.9 min (95.3% AcN), whereas QC1-pHLIP was found at 17.0 min (99.2% AcN). The retention time of ICG-pHLIP was 28.0 min (97.7% AcN). At the same concentration, the appearance of QC1-pHLIP is dark teal, whereas ICG-pHLIP appears green. The extinction coefficient of QC1-pHLIP at 810 nm is 41,000 cm−1 M−1 and it is similar to the extinction coefficient of ICG-pHLIP at 810 nm which is 42 000 cm−1 M−1, shown in Fig. left) and QC1-pHLIP  are shown and directly compared with the shape of its UV/Vis absorption spectra  is a synthetic peptide derived from the C-helix of bacteriorhodopsin which contains 27 amino acids with the following sequence: (D-Ala)-(D-Cys)-(D-Asp)-(D-Asp)-(D-Gln)-(D-Asn)-(D-Pro)-(D-Trp)-(D-Arg)-(D-Ala)-(D-Tyr)-(D-Leu)(D-Asp)-(D-Leu)-(D-Leu)-(D-Phe)-(D-Pro)-(D-Thr)-(D-Asp)-(D-Thr)-(D-Leu)-(D-Leu)-(D-Leu)-(D-Asp)-(D-Leu)-(D-Leu)-(D-Trp). To provide the first optoacoustic dark quencher pHLIP, the focus was on obtaining QC1-pHLIP as an acid specific probe, capable of sensing and targeting acidity at the surface of cancer cells in tumors. The scheme of QC1-pHLIP and its mode of action is shown in Fig. LIP Fig. , right atra Fig. . ICG-pHLtra Fig. . A class50. While evaluating the acoustic nature of the pHLIP imaging agents in vitro, the fluorescent behavior of ICG-pHLIP was also assessed. ICG-pHLIP was incubated with increasing numbers of cells of the murine breast cancer cell line 4T1  in phosphate buffered saline (PBS) media containing 10% glucose for 7 min at 37 °C). Increasing the concentrations of 4T1 cells while keeping the pHLIPs concentration constant at 5 µM, yielded dramatically increased fluorescence . Fluorescence signal difference starts to plateau after that  and 3b), and it was therefore critical to assess the behavior of both the fluorescent ICG-pHLIP and dark quencher QC1-pHLIP in an optoacoustic in vitro setting. Using the same experimental conditions carried out in the fluorescence imaging setting, the optoacoustic flow phantom device was used. Plotting the optoacoustic imaging intensities (a.u.) at various wavelengths  and spectrally unmixed CLS score for ICG-pHLIP  and the statistical significance was found between cell-free media and 0.1 × 106 cells (****p = 0.0001) and 1 × 106 cells .Fluorescent dyes such as ICG are known to quench at higher concentrationsnce Fig. . The bighat Fig.  with onltro Fig. . For ICGLIP Fig. , showed LIP Fig. , n = 3 aex vivo organs  were lined up on top of a clear plastic membrane coated with colorless ultrasound gel  and ICG pHLIP (right) were obtained. Ex vivo tumors of QC1-pHLIP injected mice showed higher optoacoustic signals, with QC1-pHLIP showing about 33-fold  signal enhancement between the injected and non-injected group tumors (**p = 0.003), shown in Fig. 53.For comparing the sonophore QC1-pHLIP to its fluorescent counterpart ICG-pHLIP, an orthotopic murine breast cancer model was set up. Imaging for both ICG-pHLIP and QC1-pHLIP  was performed 24 h post i.v. injection. The mice were sacrificed and transcardial perfusion was carried out with saline before major organs of interest  were resected. The high optical density of blood represents a major component of the endogenous optoacoustic signal and thus, excised organs were imaged with and without prior perfusion Fig. . Optoacogel Fig. . Optoacogel Fig.  and muscgel Fig.  before aex vivo studies were notable, with QC1-pHLIP clearly outperforming ICG-pHLIP in optoacoustic sensitivity .Using an imaging agent that does not emit energy in the form of photons (as fluorophores do) could provide higher signal intensities for optoacoustic imaging applications. QC1, a dark quencher, dissipates its absorbed energy mostly in the form of non-radiative decay. A large portion of energy is emitted 60. Based on this and our data, we believe that pHLIP-based optoacoustic imaging could enter the clinic as well.The synthesis of the QC1-pHLIP was achieved in one step. The sulfhydryl of single cysteine residue of pHLIP reacts with QC1 maleimide to QC1-pHLIP in high yields (70%). It is important to note that under HPLC neutral buffer conditions where acetonitrile is the mobile phase, two close peaks corresponding to two interchangeable conformations of QC1-pHLIP were observed Fig. . Attachiright) and Fig. in vitro studies , QC1 pHLIP shows a higher signal to noise ratio of 32-fold compared to 2-fold with ICG pHLIP. In vivo, the signal enhancement for QC1 pHLIP is 100% and 35% for ICG pHLIP. As expected, the presence of endogenous contrast agents at greater depths in the presence of skin account for the dissimilarity between ex vivo and in vivo results.ICG-pHLIP has a higher tendency to aggregate and molecularly stack as compared to QC1-pHLIP, which becomes evident at higher concentrations Fig. . QC1-pHLies Fig. . The ex in vivo studies corroborate this hypothesis. This suggests that further investigations into the use of dark quenchers as sonophores are not only necessary, but could yield highly significant results. In theory, pHLIP could be conjugated to any other dark quencher. With the current trend pointing towards optoacoustic imaging taking on more and more complex imaging tasks, we believe that studying and understanding the photophysical and biological properties of small molecule sonophores will enable us to create new, better and more sensitive imaging probes for optoacoustic imaging. The systematic chemical studies of these sonophores will potentially increase the likelihood of molecular optoacoustic imaging to become a standard of care technology.Intuitively, it would be premature to assume that sonophores generally make better optoacoustic imaging agents than fluorophores. However, because dark quenchers efficiently absorb energy and do not emit photons, a higher fraction of the absorbed energy could potentially be dissipated as heat, which can make them more potent optoacoustic imaging agents. Our In conclusion, emerging concepts such as pHLIP could provide the discoveries and platforms for new and alternative sonophores in which we can leverage unparalleled capabilities of optoacoustic technologies for cancer detection in patients. Our ability to reverse engineer fluorescent ICG-pHLIP into a dark quencher QC1-pHLIP suggests that routine non-invasive imaging of breast cancer could be achieved with optoacoustically labeled pHLIP sonophore.In vacuo refers to the use of a Büchi Rotavapor R-300 rotary evaporator using the vacuum pump V-710. All reactions were magnetically stirred, and room temperature refers to 20–25 °C. High performance liquid chromatography (HPLC) purification and analysis was performed on a Shimadzu UFLC system equipped with a DGU-20A degasser, SPD-M20A UV detector, a LC-20AB pump system, and a CBM-20A communication BUS module. HPLC solvents . HPLC purification was performed on an Atlantis dC18 reversed-phase 5 µm silica, 4.6 mm × 250 mm column. Purification was performed using 10–60% CH3CN method  at a flow rate of 1 mL min−1. Liquid chromatography-mass spectrometry (LC-MS) using electrospray ionization (ESI) was recorded using a Waters instrument with SQD detector for mass identification. A lyophilizer  was used for freeze drying. A 100-US high pressure  syringe pump was used to control the flow rate and facilitate delivery of solutions and cells inside the phantom flow system. An automated cell counter  was used for counting the number of cells. Planar (2-D) fluorescence imaging was carried out using the IVIS Spectrum . In vitro fluorescence confocal microscopy was carried out using a Leica TCS SP8. Fluorescence imaging was also carried out using the Odyssey Classic fluorescence scanner (Li-COR).IRDye QC1 maleimide was purchased from LI-COR . Variant 3 (Var3) pHLIP with the amino acid sequence shown in Fig. +), m/z calculated for [C202H281ClN38O57S5K2]3+ 1476.60 found 1475.91 [M + 2 K + H]3+. HPLC, tR = 17.0 min (99.2% AcN), λmax (PBS/DMSO) 810/800 nm.Var3 pHLIP  was dissolved under nitrogen in DMSO/PBS mixture (300 µL: 700 µL) followed by the addition of IRDyeQC1 maleimide . The reaction mixture was stirred for 24 h at room temperature. The mixture was lyophilized overnight and the dried powder slowly re-suspended in acetonitrile (700 µL). The mixture was purified on a HPLC. The purified product was lyophilized overnight to obtain 360 µg (70% yield) of a teal powder. LC-ESI-MS  with path lengths of 0.231 cm and 0.300 cm for 75 µL and 100 µL, respectively. UV/Vis absorbance and fluorescence spectra were measured on SpectraMax® M5 Multi- Mode Microplate Reader. Samples were measured together with a corresponding reference solvent contained in a matched well and volume. Measurements were recorded in triplicates at 25 °C. The absorbance scan was performed with an integration time of 0.5 seconds and range from 350 nm to 1000 nm in 5 nm resolution. Spectra and linear calibrations were plotted using Prism 7 .Absorption and fluorescence spectra were measured in a 96-well plate  for imaging. After imaging, the flow rate was increased 2× and the phantom was washed with at least 10 mL PBS.A custom-made flow-mediated optoacoustic set up was prepared for optoacoustic characterizations of the agents. A clear, cylindrical hollow membrane tubing was placed at the center of a cylindrical mold . The soft tissue mimicking phantom was freshly prepared by adding 15% v/v intralipid® 20%, I.V. fat emulsion (to provide the scattering), and 0.01 mM Direct Red 81 (for absorption) to a pre-warmed solution of 5% v/v agarose Type 1 (solid in <37 °C) in Milli Q water (18.2 ΩM cm at 25 °C). Compared to the previously described methodA pre-clinical multi-spectral optoacoustic tomography (MSOT) device  was used for imaging. It is equipped with an array of 256 detector elements which are cylindrically focused, having a central ultrasound frequency of 5 MHz and up to 270° coverage. The phantoms were aligned so that the illumination ring coincides with the detection plane, i.e. the curved transducer array being centered around the phantom. Data acquisition was performed in the wavelength range 680–900 nm in 10 nm steps, using 10 averages frames per wavelength, which equates to 1 s acquisition time per wavelength per section. The optical excitation originates from a Q-switched Nd:YAG laser with a pulse duration of 10 ns and a repetition of 10 Hz. Light is homogenously delivered to the phantom using a fiber split into 10 output arms. The fiber bundle and the transducer array are stationary, and the sample holder moves along the z-direction allowing longitudinal acquisition of different imaging planes using a moving stage. MSOT measurements were performed in a temperature controlled water bath at 34 °C. All of the variable parameters during the measurement were kept constant, i.e. optoacoustic gain, laser power, focus depth, frame averaging, frame rate and high/low pass filters. For optimal acoustic coupling, we waited at least 5 min before initiating the scan, so that the phantom equilibrates to the temperature of the water bath before measurement. To compare the optoacoustic signal of ICG-pHLIP and QC1-pHLIP, serial dilutions at 2 µM, 5 µM, 10 µM, 15 µM and 20 µM in DMSO were prepared. Solutions were delivered to the center of the imaging phantom device using a syringe pump. In between measurements, the flow device was washed with PBS (10 mL).2) and deoxyhemoglobin (Hb) reference spectra are included in the software package. Orthotopic tumors were implanted in the same position and distance from the surface of the skin and from animal to animal. Hence, all of the animals are affected by light scattering to the same extent. In addition to the relative known location (mm) of the animal placed on the mice bed, the location of tumor was identified based on the presence of deoxyhemoglobin. Quantitative information was obtained by defining the ROI manually within a 2D slice MSOT image. For spectrally unmixed injected pHLIPs, the mean pixel intensities per cross-section in the volume of interest (VOI) was first plotted as classical least square (CLS) score vs. position (mm) to assess the signal strength in the tumor. Decreasing signal strength over the distance away from the tumor, creating a parabolic shape is in agreement with the analysis. The maximum “mean signal per cross-section” was used as a quantitative indicator of probe binding in the tumor. Overall optoacoustic intensities (a.u.) were reported at all imaging wavelengths (680–900 nm). For all cases, unless otherwise stated, the (CLS) method for spectral deconvolution was used and reported as CLS scores. The following reference spectra were used: hemoglobin (HbO2), deoxyhemoglobin (Hb) and either ICG-pHLIP or QC1-pHLIP. In order to compare the direct intensities of all the images between ICG-pHLIP and QC1-pHLIP, the optoacoustic spectra (2 µM) were normalized to their maximum optoacoustic (810 nm) wavelength. The overall optoacoustic intensities  are then overlaid with either multi-spectrally unmixed ICG-pHLIP (green) or QC1 (cyan) pHLIP channel.Spatial reconstruction and multi-spectral processing (MSP) of the data was performed using the ViewMSOT software suite  and a backprojection algorithm at 200 mm (100 µm) resolution. The normalized optoacoustic reference spectra . At 70% confluence, the RPMI media was removed, washed with PBS, trypsinized and suspended in RPMI media. The cells were counted (1:2 dilution) and the appropriate volumes transferred into separate falcon tubes . The cells were spun down into pellets (1200 rpm for 5 min) and carefully re-suspended into the appropriate volume of saline. The solution containing the pHLIP imaging agents (35 µL) was added to the cell suspension (35 µL). The cells were mixed thoroughly by pipetting up and down. After 10 min incubation time, optoacoustic and fluorescent imaging was carried out.A solution of cell free media containing saline, 20% DMSO and either ICG-pHLIP or QC1-pHLIP (10 µM) was prepared. A second preparation contained murine breast cancer 4T1 cells, which had been grown and maintained in Roswell Park Memorial Institute (RPMI) media (pH 7.4) and 10% fetal bovine serum (FBS) with penicillin/streptomycin under standard cell culture conditions (5% COnu (6–8 weeks old) were used in the study. The mice were split into three groups of four mice, namely for the ICG-pHLIP injected group, the QC1-pHLIP injected group and a non-injected control group. All animal procedures, other than tail vein injections, were performed with the animals under general 2% isoflurane inhalation anesthesia. In order to test the ability of ICG-pHLIP and QC1-pHLIP to target tumor tissue, orthotopic allografts were created using the mouse breast cancer cell line 4T1. The 4T1 cells were injected into the 4th mammary fat pad just inferior to the nipple of a female mouse (1 × 106 cells in 30 µL 1:1 RPMI medium and Matrigel). The tumors were allowed to grow for 6–7 days  before the ICG-pHLIP or QC1-pHLIP formulation were injected. The mice were equally split into groups of three cohorts (n = 4): (i) An ICG-pHLIP injected group; (ii) a QC1-pHLIP injected group; (iii) and a non-injected group. Tumor-bearing mice (n = 4) were imaged with the MSOT prior to injection. The experiments were randomized, and 200 µL of 6 mM kg−1 of ICG-pHLIP or QC1-pHLIP formulation were injected into mice via tail vein. The mice were re-imaged at 24 h post injection. Both pHLIP agents were dissolved in 10% v/v glucose in phosphate buffer saline (PBS). For MSOT imaging timepoints, the experiments were conducted on the murine breast cancer model (n = 3) that were injected with 200 µL of 120 µM QC1-pHLIP in PBS. Tumor optoacoustic signals were monitored non-invasively using the MSOT imaging system after 30 min, 4 h, 8 h, 12 h and 24 h post intravenous injection. Animals injected with ICG-pHLIP were imaged at 30 min, 4 h, 8 h, 12 h and 24 h on an IVIS Spectrum  system for planar fluorescence imaging. Perfused and non perfused mice studies shown in Fig. All animal experiments were done in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Memorial Sloan Kettering Cancer Center (MSKCC) and followed the National Institutes of Health guidelines for animal welfare. Healthy Hsd:athymic female mice NudeFoxn1After 24 h post intravenous injection and imaging, muscle and tumors were harvested and fixed in 4% paraformaldehyde  in PBS overnight at 4 °C, thoroughly rinsed with PBS, then kept in 70% ethanol. Tissues were embedded in paraffin and 5 mm thick sections were sliced (10 µm) from the paraffin block. The sections were either imaged with confocal microscope (Leica TCS SP8) or stained with hematoxylin and eosin (H&E) and scanned with a Mirax digital slide scanner  for histological analysis.Supplementary Info- optoacoustic imaging of QC1 pHLIP


The PFD treatment promoted an increase in both peak contraction and kinetics of shortening and relaxation. Moreover, the amplitude and kinetics of Ca2+ transients were enhanced as well. Excitation–contraction coupling (ECC) was also investigated, under whole-cell patch-clamp conditions. In keeping with a previous report, PFD increased twofold the density of Ca2+ current (ICa). Notably, a similar increase in the magnitude of Ca2+ transients was also observed. Thus, the gain of ECC was unaltered. Likewise, PFD did not alter the peak amplitude of caffeine-induced Ca2+ release, indicating stimulation of Ca2+-induced–Ca2+-release (CICR) at constant sarcoplasmic reticulum Ca2+ load. A phase-plane analysis indicated that PFD promotes myofilament Ca2+ desensitization, which is being compensated by higher levels of Ca2+ to promote contraction. Interestingly, although the expression of the Na+/Ca2+ exchanger (NCX) was unaffected, the decay of Ca2+ signal in the presence of caffeine was 50% slower in PFD-treated cells (compared with controls), suggesting that PFD downregulates the activity of the exchanger. PFD also inhibited the production of reactive oxygen species, under both, basal conditions and the presence of oxidative insults . Conversely, the production of nitric oxide was either increased  or remained unchanged (in ventricular myocytes). Protein levels of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) were also investigated. eNOS values did not exhibit significant changes. By contrast, a dual regulation was observed for nNOS, which consisted of inhibition and stimulation, in ventricular and atrial myocytes, respectively. In the latter cells, therefore, an up-regulation of nNOS was sufficient to stimulate the synthesis of NO. These findings improve our knowledge of molecular mechanisms of PFD action and may also help in explaining the corresponding cardioprotective effects.Pirfenidone (PFD) is used to treat human pulmonary fibrosis. Its administration to animals with distinct forms of cardiovascular disease results in striking improvement in cardiac performance. Here, its functional impact on cardiac myocytes was investigated. Cells were kept 1–2 days under either control culture conditions or the presence of PFD (1 mM). Subsequently, they were subjected to electrical stimulation to assess the levels of contractility and intracellular Ca An action potential (AP) activates voltage-gated Ca2+ channels of the plasma membrane (CaV1.2), allowing entry of extracellular Ca2+ (ICa) that activates ryanodine receptors (RyR2) and a consequent release of Ca2+ from the sarcoplasmic reticulum (SR). The resulting increase in cytosolic Ca2+ (Ca2+ transient) activates the contractile machinery, and relaxation occurs as the levels of Ca2+ decay thanks to the activity of both the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and the Na+/Ca2+ exchanger  is of pivotal relevance for cardiac contractility. ECC relies, in turn, on a critical phenomenon known as CaA small synthetic compound termed pirfenidone  is used to treat human pulmonary fibrosis. Many studies have reported that PFD also improves cardiac performance in animal models of: atrial fibrillation AF, , myocardV1.2, by a TGF-β1 independent mechanism  is thought to be involved . Neverthechanism .V1.2 and CICR. PFD also elicited a marked decrease in NCX activity and promoted changes in the synthesis rate of cytosolic free radicals [reactive oxygen species (ROS) and nitric oxide (NO)]. Interestingly, the compound also regulated the expression of neuronal NO synthase (nNOS), but not of that corresponding to NCX or eNOS. These findings may be essential to explaining how PFD exerts its cardioprotective effects.Here, we have investigated whether PFD regulates the function of cardiac myocytes. Our present results show that, indeed, in both atrial and ventricular myocytes, PFD improves the magnitude and kinetics of contractility. This can be explained by a concomitant stimulus on both CaInstitutional Animal Care and Use Committee , complies with the Mexican Official Norm NOM-062-ZOO-1999 and is in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .Atrial and ventricular myocytes were obtained as described elsewhere . The pro2 incubator, kept at 37°C and maintained in the presence of standard culture medium [control (CN)] or culture medium supplemented with PFD (1 mM). Unless otherwise specified, experiments were performed 1–2 days after of have initiated the PFD treatment. This protocol was chosen based on previous determinations of the maximal effect of PFD on CaV1.2 . The heart was then quickly removed by thoracotomy, cannulated (via aorta) and mounted into a homemade Langendorff system. A warm (37°C) digestion buffer containing a mixture of collagenase type II and protease was retrogradely perfused, and the tissue (either ventricles or right atrium) was excised as soon as became softened. Subsequently, samples were carefully triturated, centrifuged and the resulting myocytes were plated into laminin-coated coverslips, which in turn were lying into 35 mm Petri dishes. The cells were transferred to a COn CaV1.2 .2+ transients and ICa were investigated simultaneously using a combination of the voltage-clamp technique and epifluorescence  and exhibited an electrical resistance of approximately 4 MΩ. The cell capacitance (Cm) was estimated from linear capacitative currents, which were acquired under both cell-attached and whole-cell conditions. Leak currents were eliminated on-line, using a p/N leak subtraction procedure. For each cell, the peak value of ICa was normalized by Cm and plotted as a function of Vm. The resulting I–V curves were fitted according to the following Boltzmann equation:CaGmax represents the maximal Ca2+ conductance, V1/2 is the potential for half-maximal activation of Gmax, k is a slope factor, and Vrev is the apparent reversal potential of ICa.where 2+ were assessed using Fluo-4. The dye was excited using a 100 W mercury arc lamp (at 470–490 nm), and the emitted fluorescence was collected at 515–550 nm using a photomultiplier tube. The magnitude of Ca2+ transients was estimated as the maximum fluorescence value during test pulses (F), divided by the basal fluorescence observed before the pulse (Fb), according to: ΔF/F = (F – Fb)/Fb. The gain of EC coupling (ECC Gain) was calculated by normalizing maximal ΔF/F values to its respective peak value of ICa. The increase in Ca2+ signal induced by caffeine (30 mM) was used to estimate the amount of releasable SR Ca2+ content. Caffeine was applied via a local, rapid perfusion system (Warner Instruments Corporation), first in the absence and then in the presence of extracellular Na+ (see Recording solutions). For each cell, the NCX function was examined at the holding potential  by analyzing Ca2+ signals remaining at the end of caffeine applications (25 s).Levels of free cytosolic Ca2+ transients and mechanical properties were recorded simultaneously in intact (non-patch clamped) myocytes, with the aid of an IonOptix system , and changes in myocyte length during shortening and re-lengthening were measured at both ends of the cell. In ventricular myocytes, the average sarcomere length was also investigated. Briefly, an area of well-defined striations was selected, and then the average periodicity of Z-line densities was determined . The sampling frequencies for recordings of contractions and intracellular Ca2+ were 0.25 and 1.0 kHz, respectively.Ca2DCF DA and DAF-FM DA; 2+-insensitive point), and its fluorescence was used to normalize signals corresponding to CM-H2DCF DA and DAF-FM DA . Both sensors were excited at 470–490 nm and examined at 515–550 nm.Production of ROS and NO was estimated using fluorescence sensors for these molecules , anti-eNOS , anti-nNOS , and anti-β-tubulin . Secondary: goat anti-mouse IgG .Protein levels of NCX, nNOS and eNOS, were investigated by western-blot analysis . The cel2 2, MgCl2 0.5, NaH2PO4 0.3, lactic acid 1.1, and pyruvic acid 3 (pH = 7.4).The Tyrode’s solution contained (in mM): NaCl 130, HEPES 25, Glucose 22, KCl 5.4, CaCl2+ transients and ICa were recorded in the presence of the following solutions. External (in mM): methanesulfonic acid 145, TEA-OH 115, HEPES 10, CaCl2 5, MgSO4 2, 4-aminopyridine 5, 9-anthracene carboxilic acid 1, glucose 10 and 2-3-butanedione monoxime 2. Internal (in mM): Cs-Asp 100, CsCl 10, HEPES 10, Cs-EGTA 1, CaCl2 0.2, glucose 10, TEA-HCl 20, ATP-Mg 5, GTP-tris 0.05 and Fluo-4 K5 0.15.In atrial myocytes, CaCa and Ca2+ transients were recorded in the presence of slightly different solutions . External (in mM): methanesulfonic acid 125, TEA-OH 115, HEPES 10, CaCl2 10, MgSO4 8, 4-aminopyridine 5, 9-anthracene carboxilic acid 1, 2-aminoethyl diphenylborinate 0.01, Creatine 25, glucose 10 and 2-3-butanedione monoxime 2. Internal (in mM): Cs-Asp 160, CsCl 10, HEPES 10, Cs-EGTA 6, CaCl2 1, glucose 10, ATP-Mg 5, TEA-HCl 25, MgCl2 0.5, GTP-tris 0.05 and Fluo-4 K5 0.05.In ventricular myocytes, I2+ responses triggered by caffeine (30 mM), which was applied using the above-cited external solutions (supplemented or not with 140 mM Na+. EGTA (0.5 mM) was added as well, to chelate any contaminating Ca2+. When required, TEA was omitted to balance the osmolarity.The NCX activity was investigated by comparing intracellular CaIn all patch-clamp solutions, the pH was adjusted to 7.3.t-test, and ANOVA was used for multiple statistical comparisons.All data were analyzed using the following software packages: SigmaPlot , IonWizard , and pCLAMP . Experimental results are shown as means ± SEM from the indicated number of experiments (n). Two groups were compared by Student’s All experiments were performed at room temperature (∼24°C).Ca is larger in PFD-treated myocytes than in controls  and PFD-treated (n = 16) myocytes: −9.3 ± 2.0 and −20.9 ± 2.7 pA/pF (p < 0.003), and 0.34 ± 0.08 and 0.57 ± 0.06 ΔF/F (p < 0.05). Some examples of these determinations are show in Figures Our previous work has shown that the density of Icontrols . Thus, wCa and Ca2+ transients was investigated , in accord with our previous work ; and 184 ± 24 and 297 ± 25 nS/pF . No significant differences were found in the voltage-dependent parameters of GCa (mid-point and slope factor), between control and PFD-treated myocytes.In subsequent experiments, the voltage-dependence of I Figures . Fittingous work . SpecifiCa that is not redundant for ECC. In other words, the extra ICa elicits the same degree of Ca2+ release as its respective control. The activity of SERCA was examined next, as illustrated in Figure 2+ transient was fitted to a simple exponential equation. The resulting average values of τ are shown in Figures The gain of ECC was also investigated, and it was found identical in control and PFD-treated myocytes Figures . This re2+, caffeine was applied under both absence and presence of extracellular Na+ , between control and PFD-treated myocytes . To quantify this observation, for each cell, F0 was normalized to its corresponding peak caffeine response (FPeak). The resulting average values were: 0.59 ± 0.04 and 0.78 ± 0.04 (p = 0.001), for control (n = 30) and PFD-treated cells (n = 31). This analysis suggests that PFD inhibits Na+-independent Ca2+ extrusion systems.Two different concentrations of extracellular Na Figures , 0 Na+. + to decreasing [Ca2+] was next estimated, from FNa/F0 ratios (1.0 means no contribution). Surprisingly, in PFD-treated cells, the NCX activity was not detected , whereas in control myocytes the exchanger contributed by 45% . The NCX function was also more directly compared between control and PFD-treated cells , for control (n = 21) and PFD-tread cells (n = 29). These data suggest that PFD inhibits the NCX activity by 30%.The contribution of NaThen we wonder if the effects observed in ECC . With regard to ventricular myocytes, PFD did not alter the amplitude of the Ca2+ transient but increased the average values of systolic Ca2+ . Additionally, PFD increased both maximum rate of Ca2+ output  and lag time between electrical stimulus and initiation of Ca2+ transient decay . As a consequence of these changes, the PFD treatment resulted in an increased integral of the initial phase of the Ca2+ transient .We also build phase-plane diagrams, to analyze myofilament Cay Figure . The cory Figure  but alsoy Figure . Althougy Figure , an incry Figure . This iny Figure  and suggtization , which i2+ transients were not investigated, the finding that PKI does not interfere with the magnitude or kinetics of cell shortening  phosphorylates key elements of ECC, and thus we investigated whether this protein might be involved in the stimulus of contractility. Ventricular myocytes were exposed 30 min to PKI (a cell-permeable PKA inhibitor), which failed in reverting PFD effects , revealing a 15–30% inhibition of ROS production . As predicted from results shown in Figure PFD vs. CN). Additionally, in both CN  and PFD-treated cells  the oxidative insults caused a significant increase in ROS production. The production of ROS, however, proved to be identical in the following three experimental conditions: CN, PFD + ACA, and PDF + H2O2  or hydrogen peroxide  is sufficient to increase the synthesis of NO . Conversely, a reduction in these levels  is not sufficient for changing the production of NO .Pirfenidone also did not alter the expression of eNOS . Our results demonstrate, for the first time, that PFD upregulates not only CaV1.2 but also CICR and ECC. Remarkably, PFD also: enhances the degree and kinetics of contractility, inhibits Ca2+ extrusion via NCX (and other secondary pathways), and lowers oxidative stress.Cardiac myocytes are of paramount relevance for blood pumping, and its function is compromised in a number of cardiac conditions. On the other hand, many studies show that PFD-treated animals are less prone to develop cardiac dysfunction . Howeverin vivo condition.Below we discuss molecular mechanisms that may underlie our findings, along with the corresponding implications for the in vivo conditions. Unfortunately, however, deciphering the underlying mechanisms is complicated because they may comprise several interrelated variables and processes —such as resting membrane potential, the fraction of inactivated channels, the affinity of myofilaments for Ca2+, and cytosolic ionic composition . Here we have combined these approaches and obtained evidence suggesting that, by enhancing ICa and SR Ca2+ release, PFD stimulates the contractility of both the sarcomere and cardiomyocytes. We cannot discard, however, a potential contribution of other mechanisms.Investigating contractility under physiological-like situations  is essential because the corresponding results are more likely to be significant for position . On the V1.2 channels . Evidence exists indicating that in rat atrial myocytes this happens, even under basal conditions. This is because Ca2+ transients elicited by ICa cannot recruit RyRs located in central regions of the cell, due to the absence of t-tubules. Besides, the mitochondria act as a buffer of Ca2+ that complicates free diffusion of the ion, thereby preventing the activation of this “reserve” of RyRs ((i) PFD inhibits the Ca2+ uptake by Na+-independent slow systems, which likely include mitochondrial transport; and (ii) PFD-treated cells must present higher [Ca2+]i in microdomains of the cell periphery, due to exacerbated ICa.Our previous work demonstrated that PDF increases the number of functional Cachannels . This fiifferent , and thu of RyRs . The resCa to trigger Ca2+ release, in the face of an intact SR Ca2+ load. β-adrenergic stimulation reverses this “defective EC coupling,” which likely involves a change of the microarchitecture of the dyad  in these conditions, membrane depolarization is more expedite than in an AP, and (ii) the recording solution does not contain Ca2+-binding biomolecules (as opposed to the cytosol). Thus, in patch-clamp experiments, PFD probably improved the DHPR/RyR functional interaction more efficiently (see above), because of both enhanced activation kinetics of ICa and optimized diffusion of the ion. These differences could also account for the observation that in intact ventricular myocytes PFD did not alter the rate of Ca2+ output, but instead increased the latency for reaching the peak of the Ca2+ transient as well as the corresponding integral  PFD accelerates Ca2+-dependent inactivation of CaV1.2. As can be seen in Supplementary Figure Ca inactivation. Thus, while the influx of Ca2+ via CaV1.2 is higher during the first ∼10 ms, a subsequent accelerated kinetics of inactivation imposes a drastic reduction of Ca2+ influx  PFD increases the absolute level of SR Ca2+ uptake, in proportion to its effects on Ca2+ transients. The observation that large and small Ca2+ transients return both to basal values with similar time constants (τ) means that the activity of SERCA is unchanged . Accordingly, if one displays A100 values as a function of the peak Ca2+ transient amplitude, it becomes clear that PFD increases the velocity of Ca2+ uptake as a consequence of enhancing the magnitude of Ca2+ transients , inflammation  and oxidative stress  are involved. To date, however, the molecular targets responsible for these changes remain unknown (Pirfenidone has been the subject of numerous studies regarding long-term  unknown . Free ra unknown . NeverthThe present study was designed to characterize long-lasting and persistent modifications of cardiomyocyte function (as opposed to rapid and readily reversible). Hence, cells were exposed to PFD for a minimum of 4 h  and experiments were performed 15–50 min after finishing the treatment . Therefore, the changes we have observed persist long after PFD removal. Moreover, at least for the case of ROS and NO production, it took a minimum of 8 h of PFD treatment to detect a significant effect , and this, in turn, influenced free radicals production and cell function.atrium), whereas the stimulation of NO generation requires 24 h to become evident . Moreover, these findings are also in line with results of Kar and collaborators, showing that prolonged exposure of HL-1 atrial myocytes to H2O2 results in low expression levels of nNOS — do not reproduce the PFD effect on ICa  on evidence derived from hepatocytes . Howeverd CaV1.2 . Additiod CaV1.2 . Moreoved CaV1.2  could beIn vivo, PFD decreases the susceptibility to develop arrhythmias (AF and ventricular tachycardia), and these effects are thought to depend on a concomitant inhibition of fibrosis  ventricular developed pressure in mdx mice  left ventricular ejection fraction in rats subjected to myocardial infarction (Stimulation of CICR, ECC, and contractility, on the other hand, may be of critical relevance to explaining why PFD improves: (farction .The inhibition of ROS production by PFD could be relevant in more general terms, because these free radicals are involved in several cardiac conditions, including arrhythmias, myocardial infarct, hypertrophy, alcoholic cardiomyopathy, and heart failure (for reviews see In vivo, the underlying mechanisms are likely numerous and interrelated. Here, its effects on isolated myocytes were investigated, limiting influences from additional tissues and cells. Our results show for the first time that PFD stimulates the function of cardiac myocytes. Other in vivo effects may be regarded as “auxiliary” because the compound acts on essential components of the cardiac muscle-pump. In particular, it stimulates CICR, ECC, and contractility. Moreover, it also decreases ROS production, suggesting that its effects on myocytes are cytoprotective.Pirfenidone has been long acknowledged to be of therapeutic potential for a number of heart symptoms. DO-G performed experiments and analyzed data. AM-V and GA performed experiments, analyzed data, and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Changes in [Ca2+]i was determined using fluorescence microscopy. Cell viability was evaluated by the neutral red method, and cell cytotoxicity by the MTT method. The [Ca2+]i gradually increased when cells were exposed to 0.1 mM gentamicin for 10, 20, and 30 min. The presence of extracellular Ca2+ was found to be necessary to stimulate the increase in [Ca2+]i induced by gentamicin, since this stimulus disappeared by using 1.8 mM EGTA (a Ca2+ chelator). Morphological changes were observed with scanning electron microscopy in epithelial cells exposed to the antibiotic. Furthermore, with the MTT method, a decrease in metabolic activity induced by gentamicin was observed, which indicates a cytotoxic effect. In conclusion, gentamicin was able to alter [Ca2+]i, change the morphology of MDCK-C11 cells, and promote cytotoxicity.It is well known that the aminoglycoside antibiotic gentamicin is capable of causing damage to kidney cells. Given the known involvement of Ca Gentamicin is an aminoglycoside antibiotic that has a broad spectrum of action against gram-negative bacterial infections and it is more recommended in severe cases . Since t2+ release from intracellular stores  and cellular cytotoxicity, using MDCK-C11 cells, a clone that has several properties that resemble those of intercalated cells of the distal nephron.Given the known involvement of Ca3. Cells were grown at 37°C (pH 7.4) in a humidified 95% air and 5% CO2 incubator . These cells were used from passages 75 to 90.A subtype (clone) of MDCK cells denominated C11 (MDCK-C11), obtained from Dr. H. Oberleithner , was cultured in MEM , supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, 10 mg/mL streptomycin, and 2 g/L NaHCO2+]i cell cultures were monitored for FLUO-4 fluorescent dye intensity emission. The excitation spectrum for this fluorescent marker is 480 nm, whereas the fluorescence was measured at 520 nm every 1 second. The data are reported in arbitrary units of fluorescence (AUF). The resulting changes in fluorescence intensity during the experiments are reported as percentages relative to the fluorescence intensity at baseline . Cells grown to confluence on glass coverslips were incubated for 20 min with 10 µM FLUO-4 . After this, the glass coverslips were rinsed and placed into a thermoregulated chamber (37°C) assembled on an inverted epifluorescense microscope . Areas of interest containing MDCKC11 cells were identified at 20× magnification using the software AxioVision 4.8.1. Following a stabilization period with the control solution, the cell monolayer was exposed for 10, 20, and 30 min to a control solution containing 0.1 mM gentamicin sulfate  with a recording period of about 1 min. Effects of the CaSR agonist 0.3 mM Gd3+, the calcium chelating agent 1.8 mM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; Amresco, USA), and 50 mM BAPTA (Santa Cruz Biotechnology) were evaluated under the same conditions compared to the effects of gentamicin.Qualitative changes in i over time as observed in the representative recording shown in 3+, a CaSR agonist, the cytosolic ionized calcium increased to 107.9±19.6% after 10 min, 111.9±9.3% after 20 min, and 116.1±8.0% after 30 min. These values were significantly lower than those observed with gentamicin, as shown in 2+]i observed might be an influx of extracellular Ca2+ or activation of intracellular signaling pathways that promote release of calcium from the endoplasmic reticulum. In the absence of extracellular calcium (from the addition of 1.8 mM EGTA to medium containing gentamicin), the [Ca2+]i was significantly lower than control values at all exposure times .Incubation of MDCK-C11 cells monolayers with 0.1 mM gentamicin sulfate determined a significant increase of i) that was higher than that obtained with gadolinium. This finding indicated that gentamicin would have a more pronounced effect on the activation of CaSR. Moreover, in the presence of BAPTA, there was no change in the [Ca2+]i level, even in the presence of gentamicin, because this drug is able to chelate all cytoplasmic Ca2+. An increase in the [Ca2+]i level greater than 30% may be responsible for triggering the toxic effect of this aminoglycoside on MDCK C11 cells. This effect does not depend on the endocytic pathway, but occurs by stimulation of Ca2+ per Ca2+, because in the absence of extracellular Ca2+ (EGTA), the [Ca2+]i level remained at the basal level.Like gadolinium, neomycin, and tobramycin, gentamicin is also known as an agonist of CaSR ,19. StimIn this study, we also evaluated the viability of MDCK-C11 cells exposed to gentamicin. Cells exposed to the antibiotic gentamicin showed no change in the uptake of neutral red dye, indicating that the endocytosis function was not impaired by the antibiotic. With regard to the evaluation of the cytotoxicity caused by gentamicin, MDCK-C11 cells showed a significant decrease in metabolic activity with 48-h exposure, which indicates metabolic stress.2+]i level. A high concentration of Ca2+ released from the endoplasmic reticulum promotes a consequent high Ca2+ uptake by mitochondria. In these organelles, high Ca2+ levels activate apoptosis pathways , cells failed to remain adhered, losing their adhesion to the extracellular matrix (ECM), possibly by modulation of the receptors that enable cell adhesion. Among several molecules related to ECM, the transmembrane glycoproteins integrins can be emphasized, which have α and β domains and are membrane receptors. These biomolecules interconnect the internal cell cytoskeleton with the ECM . Among tin vitro studies. However, this dose of gentamicin was shown to alter metabolism and morphology of MDCK-C11 cells. Gibbons et al. (The dose of gentamicin used in the present study (0.1 mM gentamicin sulfate) was smaller than that used in other s et al.  showed ts et al. .2+]i levels in MDCK C11 cells, which is dependent on the presence of Ca2+ in the extracellular medium. The gentamicin-induced increase in [Ca2+]i levels was significantly higher compared to the increase caused by a CaSR agonist, gadolinium. Gentamicin altered metabolic activity and morphology of cells that resemble those of intercalated cells of the distal nephron. Intercalated cells play an important role in the acid-base balance of the organism, contributing to pH homeostasis by reabsorbing bicarbonate and secreting the hydrogen ion in accordance with the body's needs. The findings of the present study demonstrate, for the first time, that the toxic effect of gentamicin on intercalated cells can lead to changes in the metabolism of these cells. These changes promote cell death, which could contribute to the progression of renal failure.In conclusion, our results demonstrated that gentamicin was capable of promoting a significant increase in [Ca