Although a number of important studies have compared the phenotypes of myeloid cells from acute myeloid leukemia (AML) patients to those of their normal counterparts, there is currently no specific surface phenotype that can universally recognize these cells across AML patients. In this study, we used mass cytometry to characterize the expression of five major embryonic antigens (EAs) in different myeloid cell types from both AML bone marrow (BM) aspirates and normal samples. Our aims were to determine if EAs are abnormally expressed in AML cells and if they can help discriminate AML cells from their normal counterparts. The deregulation of EAs may contribute to AML leukemogenesis and the adoption of a “leukemic stem cell” phenotype. In addition, to understand how EAs may contribute to myeloid cell dysfunction in AML, we evaluated several signaling proteins (SPs) involved in major signal transduction pathways.Background: Embryonic antigens (EA) regulate pluripotency, self-renewal, and differentiation in embryonic stem (ES) cells during their development. In adult somatic cells, EA expression is normally inhibited; however, EAs can be re-expressed by cancer cells and are involved in the deregulation of different signaling pathways (SPs). In the context of AML, data concerning the expression of EAs are scarce and contradictory. Methods: We used mass cytometry to explore the expression of EAs and three SPs in myeloid cells from AML patients and normal bone marrow (NBM). Imaging flow cytometry was used for morphological assessment of cells in association with their OCT3/4 expression status (positive vs. negative). Results: An overall reduction in or absence of EA expression was observed in immature myeloid cells from AML patients compared to their normal counterparts. Stage-specific embryonic antigen-3 (SSEA-3) was consistently expressed at low levels in immature myeloid cells, whereas SSEA-1 was overexpressed in hematopoietic stem cells (HSCs) and myeloblasts from AML with monocytic differentiation (AML M4/M5). Therefore, these markers are valuable for distinguishing between normal and abnormal myeloid cells. These preliminary results show that the exploration of myeloid cell intracellular SPs in the setting of AML is very informative. Deregulation of three important leukemogenic SPs was also observed in myeloid cells from AML. Conclusions: Exploring EAs and SPs in myeloid cells from AML patients by mass cytometry may help identify characteristic phenotypes and facilitate AML follow-up. Embryonic antigens (EAs) maintain pluripotency and self-renewal in embryonic stem (ES) cells during early embryonic development. OCT3/4, SOX2, and Nanog are core transcription factors involved in maintaining ES cell pluripotency and differentiation . OCT3/4 POU5F1B (the gene encoding OCT3/4) is frequently underexpressed in BM nucleated cells [+CD38− versus CD34+CD38+) [POU5F1B in AML patients is associated with unfavorable clinical variables and a poor prognosis [For acute myeloid leukemia (AML), a disease characterized by a differentiation block leading to the accumulation of abnormal immature cells, there are few, contradictory studies pertaining to the expression of EAs. Qinjun et al. showed that OCT4A expression is significantly increased in the bone marrow (BM) nucleated cells of patients with AML , while Jed cells . Additio4+CD38+) . These frognosis ,7. A posIn this study, we used mass cytometry to compare EA expression in different myeloid cell populations from AML cases and normal settings. The main aim of this work was to investigate whether EAs are abnormally expressed in AML cells and if this aberrant expression could be used to optimally discriminate AML cells from their normal counterparts. In addition, we evaluated several important signaling proteins (SPs) such as β-catenin, phospho-p38 (p-p38), STAT3, and p-STAT3, which are involved in different signal transduction pathways regulated by EAs. Identification of deregulated pathways may help discriminate abnormal cells from their normal counterparts, thereby facilitating AML follow-up and advancing the development of new therapeutic approaches.The study design is summarized in the flow chart in 2.NTERA-2 human embryonal carcinoma cells (ACC 527) and three leukemic cell lines  were obtained from the DSMZ–German Collection of Microorganisms and Cell Cultures GmbH . NTERA-2 cells were cultured in Dulbecco’s Modified Eagle’s Medium  with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin solution . The leukemic cells were cultured in RPMI-1640 with 2 mM L-glutamine, 1% penicillin/streptomycin solution, and 10% FCS at 37 °C in 5% CO6 events and were ultimately used for further analysis. The NBM samples used in this study were obtained from healthy BM donors (n = 2) or leftover diagnostic material from non-AML leukemia (n = 2) or multiple myeloma (n = 3) patients who were in long-term complete remission and negative for minimal residual disease. The seven low-quality NBM samples not used in this study came from two healthy BM donors, and from three patients with multiple myeloma in long-term complete remission, one case of follicular lymphoma in long-term complete remission, and a patient with suspicion of idiopathic thrombocytopenic purpura. The healthy donors were aged 22–67 years (median: 51 years), and 45% of patients were females.BM samples from 15 AML patients newly diagnosed at the Hematology and Cell Therapy Department of the University Hospital of Saint Etienne were included in this study. Fourteen samples from normal controls  were also included. However, due to sample low quality (samples with poor viability and a high amount of residues due to frequently clogging during sample acquisition), only 7 NBM and 8 AML files contained more than 0.2 × 10AML cases were diagnosed and classified according to the revised 2016 World Health Organization (WHO) criteria . The chaMass cytometry sample preparation procedures are similar to those used for flow cytometry.g, 5 min). Experiments were performed during the exponential growth phase. Approximately 2–4 × 106 cells were fixed and stained with the viability reagent cisplatin and a panel of 36 antibodies  at the AniRA cytometry facility, SFR BioSciences (UAR3444-US8), Lyon, France.NTERA-2 cells: NTERA-2 cells were harvested by trypsinization  and leukemic cells by centrifugation  was performed to recover the maximum possible number of cells from each sample. A total of 2–4 × 10dy panel , availab+ myeloblasts, CD117+ myeloblasts, and monoblasts, as well as more mature myeloid cells such as immature monocytic cells, monocytes, and mature neutrophils. The representative dot plots for immunophenotypic characterization of immature myeloid cell populations are displayed in Data analysis: First, the FCS files were cleaned with the flowCut algorithm, which automatically removes outlier events due to abnormal flow resulting from obstructions and other technical issues. Cases with few evaluable events and those containing only mature cells were excluded from the study. We observed significant cell loss during the staining process; during cell passage through the mass cytometer; during methanol permeabilization, required for phosphoprotein intracellular labeling; and in some cases when significant cell clumping occurred. Ultimately, 7 FCS files for NBM and 8 FCS files for AML samples  were used in the final step of data analysis, all of which contained approximately 300,000 events per sample. Data analysis was performed with the OMIQ cytometry analysis platform. First, we attempted to use an automated analysis method based on the Uniform Manifold Approximation and Projection (UMAP) for dimensional reduction algorithm. However, this algorithm failed to appropriately discriminate among cell populations, prompting us to use a classical hierarchical classification method. Two possible reasons may underlie this problem. First, we did not perform barcoding before immunostaining, which would have improved data quality by minimizing the variability between samples. Second, the quality of the primary material was sub-optimal with respect to the initial number of cells, hemodilution, and the presence of serum factors favoring cellular agglomeration. The manual sequential gating strategy  allowed + myeloblasts, and immature and more mature monocytic cells in AML M4/M5 cases compared to NBM samples. We also observed an increased percentage of CD34+ myeloblasts in non-M4/M5 AML samples  according to the supplier’s recommendations. Cells were then incubated with a cocktail of the following surface marker antibodies for 15 min at room temperature, protected from light: PerCP-Cy5.5-labelled mouse anti-human CD34 , APC-labelled mouse anti-human CD33 , and V-500-labelled mouse anti-human CD45 . Thereafter, cells were permeabilized with BD Perm/Wash Buffer solution (BD Biosciences) and incubated with AlexaFluor 488-labeled mouse anti-human OCT3/4 antibodies  for 20 min at room temperature in the dark.A total of 2 × 10Data were acquired using an Attune CytPix instrument (Thermo Fisher Scientific). The compensation settings were obtained with CompBeads (BD Biosciences).Statistical analyses were performed in R studio 4.1.2  using Shapiro–Wilk tests, D’Augustino Pearson tests, Wilcoxon tests, and graphical functions. As the number of samples included in the study was low, intergroup comparisons were performed with the Wilcoxon–Mann–Whitney test.1). The staining background was evaluated using the NTERA-2 stained with CD45 and was set at 101. Mean metal intensity (MMI) values between 101 and 102 were considered low-intensity expression, and values above 102 were considered high-intensity expression. As we expected, because NTERA-2 is an undifferentiated cell line, it expressed SSEA-3 but not SSEA-1. In addition, NTERA-2 cells expressed low levels of OCT3/4 and Nanog and high levels of SOX2. With respect to the expression of SPs, NTERA-2 expressed low levels of β-catenin and p-p38 and high levels of STAT3. However, only 36.57% of NTERA-2 cells expressed p-STAT3 at low levels , round cells with scant cytoplasm and immature chromatin patterns. Thus, OCT3/4-positive cells morphologically resemble immature blasts.The Attune CytPix is equipped with a bright-field camera that allowed us to investigate the morphology of the OCT3/4-positive cell populations identified by flow cytometry. The NTERA-2 and leukemic HL60 OCT3/4-positive cells were large  were observed in HSCs, whereas un-phosphorylated STAT3 was detected in more mature cells A,B. β-ca samples C,D.p = 0.017), SOX2-positive , and SSEA-3-positive  HSCs were detected in AML M4/M5 samples compared to NBM. In addition, AML M4/M5 samples contained significantly fewer Nanog-positive CD34+ myeloblasts than normal samples   A. For thp = 0.0025; Nanog MMI: 12.6 ± 1.6 vs. 49.9 ± 30.2, p = 0.0025; SOX2 MMI: 12 ± 1.4 vs. 43.8 ± 17.8, p = 0.0158). In addition, an abnormal increase in SSEA-1 and a reduction in SSEA-3 were observed in AML M4/M5 HSCs compared to their normal counterparts . Unlike the normal myeloblasts, however, CD34+ myeloblasts from AML M4/M5 expressed higher levels of SOX2 than those in NBM .Reduced expression of OCT3/4, Nanog, and SOX-2 was observed in AML M4/M5 cells compared to normal HSCs  as well as increased levels of SSEA-1 . We also observed reduced expression of SSEA-1 and SSEA-3 in AML M4/M5 monoblasts compared to their normal counterparts .Similar to the HSCs, CD34+ myeloblasts from AML M4/M5 samples expressed more SOX2 than their normal counterparts.To conclude, an overall reduction in EA expression was observed in HSCs from AML M4/M5 samples. Moreover, EA expression was absent in non-M4/M5 AML compared to normal HSCs. However, increased SSEA-1 and decreased SSEA-3 expression were observed in HSCs and myeloblasts from AML M4/M5 compared to their normal counterparts. For monoblasts, reduced SSEA-1 and SSEA-3 expression was observed in AML M4/M5 samples compared to normal samples, whereas CD34Next, we evaluated the cytoplasmic expression of two phosphorylated proteins and two transcription factors frequently involved in leukemic cell-associated SPs.+ myeloblasts  and monoblasts   A.p = 0.0158; non-M4/M5 AML vs. NBM: 5.3% vs. 22.2%)  A.+ myeloblasts in AML compared to normal settings, but statistical significance was only attained for non-M4/M5 AML   A.p = 0.0139; non-M4/M5 AML vs. NBM MMI: 14.2 vs. 28.0, p = 0.0217). A reduction in p-p38 intensity was also observed in immature cells from AML compared to normal counterparts but did not achieve statistical significance (p = 0.0479). However, constitutive p-STAT3 expression was more strongly reduced in immature AML cells, including HSCs, from all AML settings compared to their normal counterparts  and non-M4/M5 AML vs. NBM  . Finally, we observed a significant reduction in the percentages of p-STAT3-positive neutrophils in AML M4/M5 compared to NBM   B,C. In a 0.0101) B,C. In cAML represents a heterogeneous group of hematological malignancies characterized by the abnormal proliferation of undifferentiated myeloid progenitors . Various+ myeloblasts, more mature CD117+ myeloblasts, monoblasts, and immature monocytic cells. In addition, we adapted labeling and mass cytometry acquisition protocols to BM samples and established a strategy for data analysis.Although a small number of cases were included, the present study is original and builds upon the few prior studies dedicated to exploring myeloid cell EAs and SPs in AML by mass cytometry. Herein, we designed a 36-antibody panel according to the recommendations of Euroflow , allowinWe observed an overall reduction in EAs in HSCs from AML M4/M5 samples, as well as an absence of EA expression in non-M4/M5 AML samples, compared to normal HSCs. This abnormality might affect cell differentiation. In embryonic cells, quantitative expression of OCT3/4 has been used to define differentiation, dedifferentiation, and self-renewal , whereas+ myeloblasts from AML M4/M5. The pathogenic role of SOX2 in AML is underexplored despite the observation that SOX2 may serve as an oncogene in some cancers [We identified increased SOX2 expression in CD34 cancers .Aberrantly increased expression of SSEA-1, which is a marker for mature cells, along with reductions in the expression of SSEA-3, a marker for immature cells, was observed in HSCs and myeloblasts from AML M4/M5. In addition, reduced expression of SSEA-1 and SSEA-3 was observed in AML M4/M5 monoblasts compared to normal monoblasts. Therefore, the incorporation of these two markers into the panels used for AML M4/M5 follow-up could help distinguish between immature leukemic cells and their normal counterparts.In line with recent studies that used mass cytometry to dissect intratumoral heterogeneity in AML ,18, we oWe observed in AML samples a lower intracytoplasmic β-catenin expression in immature myeloid cells than their normal counterparts from the NBM.Morgan R. G. et al. also noticed an underexpression of the cytoplasmic β-catenin in Wnt-unresponsive AML myeloblasts, and this would be the result of the nuclear localization of β-catenin due a disrupted nuclear-cytoplasmic transport . ImportaTherefore, the abnormal β-catenin expression in AML warrants further investigation.+ myeloblasts from AML M4/M5 but reduced expression levels of p-STAT3, compared to their respective normal counterparts. p-STAT3 was also decreased in immature cells from non-M4/M5 AML samples. The STAT3/5 signaling pathway is induced in leukemic blasts by environmental factors such as granulocyte colony-stimulating factor stimulation or interactions with stromal cells. Narayanan et al. showed that AML patients with low environment-induced STAT3 pathway signaling had inferior outcomes compared to patients with a stronger inducible STAT3 response [In addition, we observed increased protein expression levels of STAT3 in HSCs and CD34response .Reduced p-p38 and p-STAT3 activation was also observed in neutrophils from AML samples compared to normal controls. The p38 MAPK pathway plays critical roles in neutrophil chemotaxis and the regulation of surface receptor expression ; phosphoAs previously reported by Levine et al., the preliminary results of our study show that the exploration of intracellular signaling in AML BM cells is more informative than antigen profile evaluation. The former approach captures the functional state of diseased cells, thus allowing the precise identification and quantification of leukemic clone(s).The possible impact of functional signaling analysis in AML, as well as the prognostic impact of abnormal EA, β-catenin, p-p38, and p-STAT3 expression, needs to be examined in future studies. One limitation of our study is its small cohort size; follow-up studies with larger patient cohorts are required to further validate our findings.A major advantage of mass cytometry is the ability to measure more than 40 cellular parameters from specimens in which sample volume is limited. However, there are also disadvantages. First, cell loss during the staining process, estimated at ∼30%–50% of the initial cell number , can occOur work provides a framework for using mass cytometry to characterize EAs and monitor cell signaling phenotypes in myeloid cells from AML BM samples. Functional signaling analysis is more informative than antigen profile evaluation in AML due to extensive intra- and inter-patient immunophenotypic heterogeneity. Therefore, this method may also improve genetic diagnostic approaches to risk stratification.Mass cytometry is an extremely useful tool to explore antigens with low-intensity expression, such as EAs, and to measure multiple intracellular markers such as phosphorylated proteins. However, further optimization of the technology, labeling protocols, and data acquisition approaches for BM samples is required to improve cell recovery and increase detection of rare events such as leukemic stem cells.


In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP3R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP3 sensitivity. We hypothesized the IP3R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP3R1 regulation by IP3, cytosolic, and luminal Ca2+ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca2+ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP3 ligand sensitivity. Single-channel IP3R1 studies revealed that the conductance of IP3R1-WT and -D2594K channels is similar. However, IP3R1-D2594K channels exhibit higher IP3 sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP3R1-D2594K showed a bell-shape cytosolic Ca2+-dependency, but D2594K had greater activity at each tested cytosolic free Ca2+ concentration. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels’ pore cytosolic exit affects the channel’s gating behavior thereby explaining the enhanced ligand-channel’s sensitivity.Inositol 1,4,5-trisphosphate receptor (IPThe online version contains supplementary material available at 10.1007/s00424-023-02796-x. The reproducibility of this response was examined by performing agonist dose–response curves by duplicate in three separate experiments. ATP stimuli greater than 1 µM evoked a significantly larger Ca2+ release transients when the mutant IP3R1 was expressed.To evaluate the functional impact of IP2+ release events are altered by the mutated IP3R1, Ca2+ puffs were imaged in intact HEK cells expressing IP3R1-WT or IP3R1-D2594K. IP3R-mediated Ca2+ puffs are analogous to RyR-mediated Ca2+ sparks in striated muscle. Compared to the global Ca2+ transients, the attributes of Ca2+ puffs reflect closely the molecular IP3R functional properties, such as unitary conductance and single-channel gating (opening/closing) kinetics. Ca2+ puffs were promoted by addition of 20 nM of extracellular ATP. Our dose–response titration experiments ranging from 10 to 100 nM indicated that 20 nM ATP consistently promoted Ca2+ puffs and rarely induced global intracellular Ca2+ transients. Note these local Ca2+ signals are not the result of extracellular Ca2+ entry because the extracellular solution contained 0 mM Ca2+. Ca2+ puffs were measured during 2-min periods that were preceded and followed by control periods (no extracellular ATP present). Figure 2+ puffs recorded in cells expressing IP3R1-WT or IP3R1-D2594K channels. Confocal images obtained under these conditions indicate that HEK cells expressing IP3R1-D2594K channels exhibit substantially higher Ca2+ puff activity elicited by ATP than those expressing IP3R1-WT. Subcellular areas with high Ca2+ puff activity were identified and selected as regions of interests (ROIs). In these selected regions, x-time images were generated with 10-µm kymograph lines to define Ca2+ puffs’ properties, which are better visualized by their fluorescence intensity profile plotted under each x-time image. From this type of fluorescent traces, Ca2+ puff kinetic attributes  were measured. Frequency of occurrence was directly quantified from selected ROIs in the x–y linescan images. Figure 3R1-D2594K-expressing cells showed more than 2.5-fold higher Ca2+ puffs frequency , while the mutant puffs’ kinetics exhibited significantly longer decay (2.28 ± 0.09 vs 0.35 ± 0.01 s), longer half duration (2.39 ± 0.09 vs 0.68 ± 0.02 s), longer time to peak (1.43 ± 0.04 vs 0.57 ± 0.02 s), and longer rise time (0.76 ± 0.03 vs 0.35 ± 0.01 s) than their counterpart from WT. To evaluate the contribution of HEK293 cells endogenous IP3Rs to Ca2+ release events, IP3R1-D2594K and IP3R1-WT cells were not induced with tetracycline, and linescan confocal images were recorded during application of external ATP. Under these conditions, infrequent or no intracellular Ca2+ signals were recorded in either cell type, indicating that the activity of endogenous IP3R was not detectable. Overall, these cellular results . However, the mutant IP3R1 is far more active (compared to WT) at 5 µM IP3. Figure 3]. These data were well fitted by a Hill equation with significantly different EC50 values (660 nM for WT and 406 nM for mutant). In addition, IP3 activated the mutant IP3R to a significantly higher Po level. The maximal Po reached at 10 µM IP3 was 3.6-fold higher for the IP3R1-D2594K compared to the WT channel. Thus, the IP3R1-D2594K mutation increased the sensitivity and efficacy of IP3 activation.To gain insight about the mechanism for the enhanced response to ATP by cells expressing IP3R1-D2594 residue was replaced by alanine, the concentration-dependent response to IP3 was reduced, while replacing it by a lysine or arginine, the response was substantially increased  at − 40 mV of membrane potential, which favored cytosol-to-lumen Ca2+ flux. Under these conditions, the luminal Ca2+ dependency was essentially eliminated, as indicated by the straight lines fitted to the data points. In contrast, Fig. 2+ flux, the luminal Ca2+ dependency of both types of channels becomes bell-shaped, resembling the cytosolic Ca2+ dependency. This bell-shaped behavior indicates the predominance of activation over inhibition at low luminal [Ca2+], and the predominant inhibitory effect as luminal [Ca2+] is increased. However, the activity of IP3R1-D2594K (open circles) as a function of luminal [Ca2+] was more than threefold greater than that of the WT channel (filled circles) and peaked at about one order of magnitude lower luminal [Ca2+] . These dramatic effects observed across most of luminal Ca2+ levels tested were unlikely due to the mutant conducting a larger flux because as shown in Fig. 3R1 unitary conductance.The mechanism of luminal Caresearch , 48–50; 3R and RyR share many structural and functional similarities. In RyR2, the negatively charged residue E4872 electrostatically interacts with the positively charged residue R4874 in the neighboring subunit to stabilize the closed state. When open, E4872 moves away from R4874, but closer to the negatively charged residue E4878. These residues (E4872 and E4878) are thought to be involved in electrostatic interaction with luminal Ca2+  and membrane potential on the duration of IP3R1-WT opening and closing events. Single IP3R1-WT channel activity obtained at three different luminal [Ca2+] at + 40 mV . The duration of IP3R1 opening and closing events varied with luminal [Ca2+]. We analyzed mean open time (MOT) and mean closed time (MCT) separately because lumen-to-cytosol Ca2+ flux carried by an open channel may act on cytosolic Ca2+ regulatory sites and extend mean open time . Interestingly, single IP3R1 MOT displayed a bell-shaped dependency on EDF. As indicated by the vertical arrows, MOT decreased as the ER [Ca2+] is reduced. Arrows represent the expected EDFs at room temperature, for an ER that is full of Ca2+ (solid arrow), a depleted ER (dash arrow), and an extremely depleted ER (square dot arrow). Note that the activation phase lies within the physiological range of electrochemical driving forces for Ca2+. The MOT behavior is reminiscent of the IP3R1’s bell-shaped cytosolic Ca2+ sensitivity  also exhibited a profound dependency on EDF as illustrated in Fig. 2+ binding site (in the channel itself and/or in an accessory protein), our analysis indicates that the IP3R1 dependence on luminal [Ca2+] is strongly influenced by the Ca2+ ions passing through the IP3R1. Unfortunately, the same type of kinetic analysis could not be performed on IP3R-D2594K channels because multiple (not single) mutant channels were typically present. However, based on our cytosolic and luminal [Ca2+] studies, we believe mutant channel dwell times would likely exhibit similar dependencies on EDF.As mentioned, the IPme . Intuitsee Fig.  and stro3R1-D2594 residue critically contributes to ligand interactions  of multiple IP3R1 binding sites. Thus, the IP3R1-D2594 residue is located in an ideal position to influence gating kinetics by establishing specific interactions within the pore to stabilize and prevent hyperactivity.In summary, a ligand gated channel’s gate interacts  with ligand binding sites. Our data show the intra-pore near-the-gate IPSupplementary file1 (PDF 578 KB)Below is the link to the electronic supplementary material.


Kras and Trp53, also with trans-differentiation of pancreatic acini. However, we do not know when, in the course of cancer progression, physiological functions are lost by mutant acinar cells, nor can we assess their capacity for the production of pancreatic juice components. Here, we investigated whether two mutations—KrasG12D and Trp53R172H—present simultaneously in acinar cells of KPC mice (model of oncogenesis) influence cytosolic Ca2+ signals. Since Ca2+ signals control the cellular handling of digestive hydrolases, any changes that affect intracellular signaling events and cell bioenergetics might have an impact on the physiology of the pancreas. Our results showed that physiological doses of acetylcholine evoked less regular Ca2+ oscillations in KPC acinar cells compared to the control, whereas responses to supramaximal concentrations were markedly reduced. Menadione elicited Ca2+ signals of different frequencies in KPC cells compared to control cells. Finally, Ca2+ extrusion rates were significantly inhibited in KPC cells, likely due to the lower basal respiration and ATP production. Cumulatively, these findings suggest that driver mutations affect the signaling capacity of pancreatic acinar cells even before the changes in the epithelial cell morphology become apparent.Glandular pancreatic epithelia of the acinar or ductal phenotype may seem terminally differentiated, but they are characterized by remarkable cell plasticity. Stress-induced trans-differentiation of these cells has been implicated in the mechanisms of carcinogenesis. Current consensus links pancreatic ductal adenocarcinoma with onco-transformation of ductal epithelia, but under the presence of driver mutations in   Studies on the phenotypic transition and its role in pancreatic carcinogenesis revealed a considerable capacity of pancreatic acinar cells (PACs) to undergo morphogenetic changes and, as a result, acquire a duct cell-like phenotype. The consensus has been established that PC arises equally often from the acinar and ductal epithelia.y in PC6 .7 rarely alerts patients who may simply ignore symptoms of a “clinically silent” disease in the pancreas. However, the pathophysiological mechanisms that drive the transition of normal epithelial cells to the premalignant stages of pancreatic carcinogenesis are not well understood.The Kras-driven malignant transformation not only perpetuates pancreatic carcinogenesis but also induces clonal epithelial loss of function , thus re2+-rich fluid.2+ serve as a physiological signal for enzyme release from acinar epithelial cells. However, when these signals are dysregulated10 and escalate into global and sustained Ca2+ elevations, digestive enzymes become activated prematurely in situ in PACs,10 causing necrosis and the release of damage-associated molecular patterns (DAMPs) from injured and dead cells.11 Surprisingly, the role of pathophysiological Ca2+ signals is much less evident in the development of PC, compared to other pancreatic pathologies also characterized by the inflammatory background, such as acute pancreatitis (AP).In normal pancreatic epithelia, the secretion of digestive hydrolases by acinar cells occurs via exocytosis concurrent with the secretion of Ca2+ signals in PACs isolated from KPC  mice, a genetic animal model of pancreatic carcinogenesis.12 Owing to the presence of mutations in single alleles of the Kras and Trp53 genes under the control of the Pdx1-Cre construct, KPC mice recapitulate a multistep  model of PC development  that in normal PACs evoke cytosolic Ca2+ oscillation. In another set of experiments, we applied menadione (Men) that elicits a more irregular pattern of intracellular Ca2+ elevations in PACs: oscillations, calcium transients, formation of the cytosolic Ca2+ plateau, or a combination of all of the above. A detailed numerical analysis of the area under response curves, as well as a direct comparison of the proportions of different types of cytosolic Ca2+ signal patterns, shows that, on average, Ca2+ elevations in KPC cells were more prominent and prolonged compared to controls . The application of a Ca2+ extrusion protocol revealed a significantly slower Ca2+ efflux from the stores in KPC PACs compared to control-Cre cells, but the expression levels of the plasma membrane Ca2+ ATPase (PMCA) remained unchanged. Finally, the analysis of the cellular energy metabolism showed decreased mitochondrial respiration in KPC cells compared to normal PACs. Collectively, these findings suggest an impaired Ca2+ handling in premalignant secretory epithelium expressing KrasG12D and Trp53R172H, even when no morphological changes are yet evident in PACs.This has prompted us to study cytosolic Caelopment . Using Km HEPES, phenol red free, were purchased from Agilent, USA; horse anti-mouse IgG Antibody (H + L), peroxidase (#PI-2000), was purchased from Vector, USA; Matrigel, growth factor reduced, phenol red-free, was purchased from Corning, USA; NucView 488 & RedDot 2 Apoptosis and Necrosis Kit was purchased from Biotium, USA; TAE buffer was purchased from Bio-Rad, USA; and Thapsiargin was purchased from Cayman, USA.2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 3,12-Bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-dioic acid (EGTA), 3-(morpholin-4-yl)propane-1-sulfonic acid (MOPS), acetylcholine, anti-vinculin mouse monoclonal antibody (#V9131), bicinchoninic acid (BCA), calcium chloride, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), collagenase type IV, dimethyl sulfoxide (DMSO), eosin with phloxine, ethanol, hematoxylin, Immobilon Western Chemiluminescent HRP Substrate, magnesium chloride, menadione, oligomycin, potassium chloride, radioimmunoprecipitation assay buffer (RIPA), rotenone with antimycin A, sodium chloride, Tris-buffered saline (TBS), and Tween 20 were purchased from Sigma-Aldrich/Merck, USA; Agarose, DNA Perfect Leader, and SimplySafe loading dye were purchased from EURx, Poland; Amersham Hybond 0.2 PVDF blotting membranes were purchased from Cytiva, UK; anti-calcium pump pan PMCA ATPase mouse monoclonal antibody (#ab2825) was purchased from Abcam, UK; CellTiter-Glo 3D Cell Viability Assay was purchased from Promega, USA; cOmplete mini, EDTA-free protease inhibitor cocktail, and KAPA mouse genotyping mix were purchased from Roche Diagnostics GmbH, Germany; distilled water, Genomic Mini isolation kit, and Tris buffer were purchased from A&A Biotechnology, Poland; Fluo-4 AM, Hoechst   33258, NuPAGE 4%-12% Bis-Tris Gels, and Nunc 96 Well White/Clear Bottom Plates, TC Surface were purchased from Thermo Fisher Scientific, USA; glucose, glutamine, pyruvate, Seahorse XF Cell Mito Stress Test Kit, and XF DMEM Medium pH 7.4, 5 m 5 mm tail tip samples were collected from young animals after weaning. DNA was extracted using a Genomic Mini isolation kit, eluted in Tris buffer, and stored for a long term at −20°C.The animal genotype was confirmed by polymerase chain reaction (PCR) using DNA extracted from the tail tips and specific primers under the conditions described in Kras and Pdx1-Cre; 3%: Trp53, respectively), 1% or 3% agarose gels were made by dissolving agarose in TAE buffer. SimplySafe loading dye was then added to allow DNA detection in agarose gels. Casted gels were positioned in a Sub-Cell GT Horizontal Electrophoresis System , covered with TAE buffer, loaded with 10 μL of DNA or 3 μL of DNA Perfect Leader, and the samples were run at 90 V for 30-60 min. The samples were visualized using a ChemiDoc XRS + Imaging System , and the genotypes were determined based on product sizes given in For the separation of PCR products 12 mouse breeding pair (mixed background) was transferred from Cardiff University to the Jagiellonian University institutional animal units in 2018. The animals were kept in a 12 h light/dark regimen, in individually ventilated cages (up to 5 mice) with aspen wood bedding material and environmental enrichment, and free access to food (standard rodent chow diet) and water. All procedures involving animals were performed in accordance with the ARRIVA guidelines,13 and ultrasonographic tumor detection/palpation was carried out according to license No. 113/2020, issued by the II Local Ethics Committee for Animal Experimentation . Up to 9-month-old tumor-free KPC mice , or age/sex-matched control animals , were humanely killed by cervical dislocation14 (for cytosolic Ca2+ measurements) or CO2 inhalation (other experiments). The pancreatic tissue was removed for further experimental procedures.The KPC (LSL-Kras16In our mouse model of pancreatic carcinogenesis (KPC mice), the presence of pancreatic abnormalities  was examined by abdominal palpation on a weekly basis. In certain cases, KPC mice were also monitored by ultrasound imaging (USG) using a Vevo 2100 high-frequency, high-resolution digital imaging platform , according to the previously described protocols.18 inspected under a light microscope DMi8 , photographed, and analyzed.Pancreatic tissues were fixed in formalin , embedded in paraffin, and cut into 4 μm sections. The sections were then stained using our standard hematoxylin and eosin (HE) staining protocol,m) NaCl, 140; KCl, 4.7; HEPES, 10.0; MgCl2, 1.0, and glucose, 10.0 was supplemented with 1.0 m m Ca2+19 (in Ca2+ extrusion protocol: 10.0 m m Ca2+14). PACs were isolated as described previously14 from the pancreata of control-Cre mice, or tumor-free pancreatic tissues of KPC mice, and loaded with a Ca2+-sensitive fluorescent indicator Fluo-4 AM . After loading, PACs were transferred to a flow chamber, and the experiments were performed in continuous perfusion with NaHEPES-based solutions. The cells were visualized using an LSM-880 AiryScan AxioVert confocal microscope equipped with an EC Plan-Neofluar 40×/1.30 Oil DIC M27 objective . Excitation was set to 488 nm laser light and emission to 593-630 nm. A series of images were recorded at 256 × 256 pixel resolution; two consecutive frames were averaged, and the time resolution was 1 image per 2 s. Fluorescence signals were plotted as F/F0, where F0 was an averaged signal from the first 10 baseline images, normalized as previously described.20Unless otherwise stated, extracellular buffer NaHEPES containing  for 10 min in the absence of extracellular Ca2+. Next, again in Ca2+-free extracellular buffer, 10 μm Tg was applied for 200 s, and then the extracellular Ca2+ concentration was increased to 10 m m, which induced Ca2+ influx to the cytosol. Once a cytosolic plateau was reached, extracellular Ca2+ was reduced to 0 in the presence of 2 m m EGTA. This abrupt removal of extracellular Ca2+ unmasked Ca2+ extrusion across the plasma membrane. This phase of the response was further analyzed and compared between PACs isolated from control-Cre and KPC mice. For every recorded Ca2+ trace, the normalized fluorescence that corresponds to half the decrease between the maximum and minimum F/F0 values was calculated as follows: F1/2 = Fmin + (Fmax − Fmin)/2, according to the previously described methods.21 The time values corresponding to Fmax (tmax), and F1/2 (t1/2) were calculated from the linear fit to the extrusion phase. Finally, t1/2 was calculated as the difference between t(F1/2) and tmax.For the CaKPC and control-Cre mouse pancreatic tissue samples were homogenized using a TissueLyser , in RIPA buffer supplemented with a protein inhibitor cocktail. The protein concentration was assessed by the BCA assay. 15 μg protein samples were loaded into the NuPAGE 4%-12% Bis-Tris precast gels and resolved  using SDS/PAGE electrophoresis in MOPS buffer. The overnight transfer (25 V) in a Mini Trans-Blot Cell  was performed onto the 0.2 μm PVDF blotting membranes. Then the membranes were blocked in 5% non-fat dry milk in TBS supplemented with 0.05% Tween-20 (TBS-T). The membranes were incubated  with primary antibodies: anti-calcium pump pan PMCA ATPase mouse monoclonal antibody (1:1000) or anti-vinculin mouse monoclonal antibody (1:5000), and then were washed with TBS-T . Next, the membranes were incubated  with the secondary antibody: horse anti-mouse IgG Antibody (H + L), peroxidase (1:5000), and then were washed with TBS-T . Protein detection was performed in a ChemiDoc Imaging System  using Immobilon Western Chemiluminescent HRP Substrate. The ImageLab software was used for the densitometric analysis.m Men  in extracellular NaHEPES buffer, containing 1 m m Ca2+ or Ca2+-free. One hour before the incubation end, the cells were stained with the NucView 488 & RedDot 2 Apoptosis and Necrosis Kit, which reports caspase-3/7 activity with a green fluorescence signal and the presence of dead cells with far-red fluorescence signal, according to the manufacturer’s instructions. PACs were transferred to a glass-bottom chamber and imaged using a DMI8 fluorescence microscope equipped with an HC PL APO 40x/1.30 OIL objective and a DFC7000GT camera . The following parameters were applied for NucView 488 imaging: excitation 490 nm, 5% illumination power, an FITC emission filter; and for RedDot 2 imaging: excitation 660 nm, 9% illumination power, and a CY5 emission filter. A total of 15 random images were collected, and live, apoptotic, and necrotic cells were counted, and their numbers were averaged and presented as percentages of the total ± SEM. (2) For the ATP-based cell viability measurements, a CellTiter-Glo 3D Cell Viability Assay was used to quantify ATP levels in acinar cells freshly isolated from KPC and control-Cre mice, following the manufacturer’s protocol. Briefly, equal volumes of extracellular NaHEPES buffer containing PACs and CellTiter-Glo 3D Reagent were transferred to a 96 Well White/Clear Bottom Plate, shaken for 5 min, and left for 25 min at RT to stabilize the samples. The luminescence signals were recorded using an Infinite M200 Plate Reader , and the results were normalized post-assay to the protein levels assessed by the BCA assay run in a reference plate, using the same cell aliquot as for the assay.(1) For cell death assessments, PACs were stimulated for 2 h at RT with 60 μ23 with some modifications. Briefly, 8-well cell culture plates were thin-coated with Matrigel 12 h before the measurements. PACs were isolated on ice, transferred to the plates, and allowed to attach. Next, extracellular NaHEPES buffer was replaced with XF DMEM pH 7.4 medium, supplemented with 10 m m glucose, 1 m m pyruvate, and 2 m m glutamine. The cells were incubated for 15-30 min at 37°C in a CO2-free I5110 dry oven , and then a mitochondrial stress test was performed using a Seahorse Bioscience XF HS Mini Analyzer . The cells were injected with sequential fluxes of:23 1 µg/mL oligomycin applied to inhibit ATP synthase and cause a rapid hyperpolarization of the mitochondrial membranes; 0.3 µm FCCP, an uncoupling agent of oxidative phosphorylation, which restores proton transport across the mitochondrial inner membrane; and 2 µg/mL mix of antimycin A and rotenone, an inhibitor of complexes I and III, and thus a blocker of mitochondrial phosphorylation. The results were normalized post-assay to the cell numbers calculated by counting cell nuclei stained with Hoechst 33258 and analyzed using the QuPath software.24 Basal respiration, ATP production, maximal respiration, proton leak, spare respiratory capacity, non-mitochondrial oxygen consumption, and coupling efficiency were calculated with normalized oxygen consumption rate (OCR) changes.Metabolic functions of acinar cells isolated from KPC and control mice were assessed using a Seahorse XF Cell Mito Stress Test Kit, according to the previously described protocols,2+ traces), box and whisker plots  showing individual data points together with a median and/or a mean, 10 × 10 dot plots (response abundancies), or bar charts (cell death assay) showing a mean ± SEM. For statistical analysis, appropriate parametric or nonparametric tests were applied using the GraphPad Prism 8.0.1 software (detailed description in the figure legends), and the significance threshold was set at P < .05.Quantitative results were presented as averaged/representative linear plots (CaKras (G12D) and Trp53 (R172H), under the control of the Pdx1-Cre system, which promotes the development of PC. Control cells were obtained from Cre mice expressing the Pdx1-Cre construct, but in these animals, both alleles of Kras and Trp53 genes were free from cancer-inducing point mutations. KPC and control-Cre PACs were isolated by collagenase digestion of the mouse pancreata, and they were essentially identical in terms of their morphological features. In order to characterize the capacity of KPC or control-Cre PACs to maintain physiological Ca2+ signals evoked by ACh, we first applied low 50 n m concentrations of the stimulant for 600 s and recorded Ca2+ responses induced in control-Cre  in the oscillatory responses recorded in KPC PACs compared to control-Cre acinar cells (m Ca2+) for 200 s, PACs were treated with a supramaximal concentration of 10 μm ACh in order to trigger large responses. Global Ca2+ releases recorded in PACs isolated from control-Cre mice represented a typical single Ca2+ response characterized by a rapid Ca2+-release phase followed by a steady decline in cytosolic Ca2+ levels (P < .001) areas under the responses recorded in KPC vs control-Cre PACs  that carry driver mutations in trol-Cre  and KPC trol-Cre . While cmulation , in KPC esponses . As evidmplitude  and B. Decreased , but theecreased  and B. Aar cells , with ex+ levels . Similar+ levels , but in Cre PACs , with me2+ signals in KPC and Cre acinar cells. Historically used as a cost-effective substitute for vitamin K in fortified foods, Men evokes cytoplasmic Ca2+ elevations that take the form of oscillatory responses, Ca2+ transients, or Ca2+ plateaus . Owing to the presence of double bonds in its naphthoquinone ring, Men can initiate the production of reactive oxygen species in PACs. Cell stimulation with 60 μm Men elicited cytosolic Ca2+ responses in control-Cre , a single spike followed by the plateau (13%), and the plateau alone (25%). The corresponding values in control-Cre cells were much lower: 22% of the overall plateau-type responses, that is, 10% of transients on top of the plateau, 4% of a single spike followed by the plateau, and the plateau alone in 8% of total responses recorded in control-Cre cells 21 (2+-free extracellular buffer supplemented with thapsigargin (Tg), a potent inhibitor of the endomembrane Ca2+ pump, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), to empty intracellular Ca2+ stores. The extracellular Ca2+ concentration was then changed to 10 m m, which caused an influx of Ca2+ into the cytosol —this was evidenced by the increase in cytosolic Ca2+ until a prolonged plateau was formed in both control-Cre and KPC cells  sodium-calcium exchanger (NCX) activity,25 almost the entire Ca2+ extrusion is dependent on the plasma membrane Ca2+ ATPase (PMCA) in these cells.21 When extracellular Ca2+ was removed, KPC cells not only maintained a Ca2+ plateau longer than control-Cre cells but were also characterized by slower rates of Ca2+ extrusion than control PACs. The analysis of cytosolic Ca2+ clearance revealed a statistically significant difference (****P < .0001) in the PMCA-driven extrusion between KPC and control-Cre cells, here described as the time required for Ca2+ concentration to decrease by half the maximum amplitude (t1/2). The t1/2 values were much higher in KPC cells compared to control-Cre PACs: 47.91 vs 33.91 [s], respectively (2+ extrusion across the plasma membrane was slower than in control-Cre cells and this effect was not due to decreased expression of PMCA (In order to compare the speed of Cations)21 . We firsPC cells . Abrupt PC cells . The conectively . Importaectively  and D. T of PMCA .2+ extrusion rates found in KPC acinar cells compared to control-Cre PACs, we investigated mitochondrial respiration in these cells (P < .05) in KPC cells compared to normal PACs (6 cells]), spare respiratory capacity (30598.66 vs 28917.73 [pmol/min/106 cells]), non-mitochondrial oxygen consumption (6009.10 vs 4867.59 [pmol/min/106 cells]), proton leak (10 845.15 vs 11 641.88 [pmol/min/106 cells]), and coupling efficiency (66 vs 57 [%]) were not markedly altered in KPC cells  area under the response curve to 50 n m ACh, but a significantly lower (***P < .001) area under the transient-type responses to supramaximal 10 μm doses of ACh in PACs isolated from KPC mice compared to control-Cre cells , wherein the mutant KRAS allele was deleted owing to the homologous recombination.26 Colorectal cancer cells were found to have SOCE repressed, and that protected these mutant cells from the apoptotic stimuli and cell death.26 Active removal of Ca2+ from the cytosol to the extracellular environment across the biological membranes is essential for maintaining low resting cytosolic Ca2+ levels.27 Processes of Ca2+ extrusion by Ca2+ pumps require constitutive hydrolysis of ATP, but this is the metabolic “price to pay” in order to prevent accumulation (overload) of Ca2+ in the cytosol and cell death.28 Notably, sustained elevations in cytosolic Ca2+ reduce ATP synthesis.29 Since KPC acinar cells were characterized by significantly lower rates of Ca2+ extrusion (****P < .0001) than control-Cre PACs , and so was the production of ATP (*P < .05) in PACs isolated from KPC mice  in mitochondrial respiration (G12D. This, in turn, decreases their capacity for fine-tuning the compartmental Ca2+ concentration, resulting in a less regular pattern of the cytosolic Ca2+ signals than in control-Cre acinar cells (Secretory acinar epithelia may undergo a phenotypic transformation into duct-like cellular structures, which, under the condition of cell stress and the presence of driver mutations in genes that encode Kras and Trp53 proteins, contribute toward pancreatic carcinogenesis . Here we signals -4, the c signals , and mit signals . First, al cells  and B. Sre cells . Althougre cells , the prore cells  and B. Wre cells  and B, tsolution . James eCre PACs , and PMCCre PACs  and D, wCre PACs . ProductCre PACs  and founKPC mice . Of notecapacity , the spacapacity , were noxtrusion  and B. Axtrusion  and C, axtrusion , it is ppiration , but alsar cells  and 4.


Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca2+ channels critical for EC coupling in close proximity, the L-type Ca2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca2+-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches.The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the CaThe online version contains supplementary material available at 10.1007/s00395-023-00984-5. In adulhiPSC-CM , 49, 50.the cell , 57. In coupling . So far,coupling , 45, 59,coupling , 19, celcoupling , 46, 51.2+ handling and contraction [2+ signalling [Recent research has focussed intensively on the investigation of the effect of topographical and physical cues on the maturation of hiPSC-CM during culture. Cardiomyocytes grown on micropatterned substrates enhanced cell alignment and improved Catraction , 40, 54.gnalling .Apart from environmental parameters, the question arises which cellular mechanisms may be involved in tubulogenesis. Few proteins have come into the focus of attention as potential key players in the process of t-tubule and dyad formation, specifically caveolin-3, nexilin and junctophilin. While overexpression of caveolin-3 prevented the loss of tubular network in cardiomyocytes from pressure overload-induced failing mouse hearts , loss of2+ homeostasis and fibre organisation of skeletal muscle cells [Here, we focus on the potential role of the bridging integrator-1 protein (BIN1) in t-tubule biogenesis , 14, 17.le cells , 52, butle cells , 55.2+ handling at the level of EC coupling. Our data demonstrate that a cardiomyocyte-specific microarchitecture comprising a t-tubular-like membrane network is required for efficient EC coupling in hiPSC-CM and, thus, for the development of robust and mature cardiomyocytes. Our findings open new avenues for future hiPSC-cardiomyocytes development and applications as cardiac cell grafts.To induce further maturation of hiPSC-CM, we merged two approaches and combined 3D reshaping together with BIN1 expression to trigger structural remodelling and maturation. We tested the hypothesis that reshaping hiPSC-CM in pre-designed cuboid 3D micro-scaffolds and overexpression of BIN1 lead to tubulogenesis and improve CaHuman iPS cell lines were kindly provided by Dr. Lukas Cyganek, Stem Cell Unit Göttingen, University Medical Center Göttingen (UMG). Wild-type iPSC line UMGi014-C clone 14 (isWT1.14) was generated from dermal fibroblasts using the integration-free Sendai virus and described previously . Two oth2+ imaging, cellular electrophysiology, quantitative PCR, Western blotting and statistical analysis are provided in the Supplemental file section.Detailed methods for cardiomyocyte preparation from hiPSC and culture, 3D micro-scaffold production, SEM imaging, AAV6-mediated transduction, immunocytochemistry, PLA, live Cap = 0.005) and protein (p = 0.017) levels in hiPSC-CM transduced with BIN1 compared to control, which only showed low levels of BIN1 protein , RYR2, SERCA2 (ATP2A2) and Na+/Ca2+ exchanger (NCX). The mRNA levels of these targets remained unaffected by BIN1 overexpression.To test our hypothesis whether 3D reshaping induced structural remodelling, single hiPSC-CM were plated on a planar surface and in 3D micro-scaffolds with hexagonal and cuboid shapes. To recapitulate the rectangular structure of adult cardiomyocytes, 3D-cuboid scaffolds were designed accordingly to provide a long axis and spatial restrictions to further grow in height as described before . Hexagonn p = 0.07 levels In comparison to rod-shaped adult ventricular cardiomyocytes, which present parallel-aligned and organised myofibrils, 2D-cultured hiPSC-CM are rather flat with no defined long axis formation. To assess the shape of hiPSC-CM and myofibril structures in 3D micro-scaffolds, the organisation of the sarcomeric α-actinin and actin was investigated. As shown in Fig. p < 0.05; Fig. In the next step, we investigated the synergistic potency of 3D reshaping and BIN1 overexpression on triggering the generation of membrane invaginations forming t-tubules, which are critical for proper EC coupling. Immunolabeling of BIN1 confirmed the expression of BIN1 in transduced hiPSC-CM presenting strong sarcolemmal invaginations indicative of the development of an early but still unstructured tubular network  were measured using the indicated voltage clamp protocols. In each cell, the current–voltage (IV) relationship, the voltage dependence of activation and the voltage dependence of inactivation were examined with independent voltage protocols. From the IV curve, the current amplitudes were measured to assess the peak Ca2+ current amplitudes in dependence of the clamped membrane potential. Total membrane current was then normalised to cell capacitance to obtain the LTCC current density per cell, a measure to compare the different values of different cells and cell sizes. Figure CaL recorded from hiPSC-CM of the different experimental groups. On average, the peak ICaL amplitudes at different potentials were not significantly different in the experimental groups. This finding was further confirmed by measuring and comparing the voltage-dependent activation and inactivation states of ICaL. From the steady-state activation and inactivation curves  of current decay measured at + 10 mV was compared among the different experimental groups  and spatial dynamics  = 3.401, p = 0.036) of the Ca2+ sparks.Since Io-4 Fig. A. Intereo-4 Fig. B and spao-4 Fig. C. In addo-4 Fig. D. Two-wa2+ transients in confocal line-scan imaging using the Ca2+-sensitive fluorescent indicator fluo-4. Representative line-scan images and line profiles of each experimental group are summarised in Fig. 2+transient kinetics revealed that 3D reshaping significantly reduces time-to-peak  = 8.77, p < 0.001, Fig. 2+ transients at the level of Ca2+ release and reuptake in the second hiPSC-CM line . Interestingly, cuboid BIN1-overexpressing hiPSC-CM revealed the best synchronised Ca2+ transients compared to any other conditions whereas remarkable delays of Ca2+ release were observed in the corresponding control groups  and current-stimulated Ca2+ transients were measured simultaneously with the following stimulation protocol: INa was inactivated by a voltage ramp from the holding potential (VH) of − 80 mV to − 40 mV, then Ca2+ release was triggered by two separate voltage steps to − 25 mV and + 10 mV  during prolonged caffeine application  indicative of a tight association of the SR and the sarcolemma in BIN1-overexpressing hiPSC-CM. In contrast, NCX staining revealed a dotted distribution pattern over the entire sarcolemma .Finally, dependence of Cants Fig. C, D. At -CM Fig. D, E. Thiion Fig. F. The exA comparison of the presented data is summarised in Table Despite their cardiogenic properties, hiPSC-CM differ from adult cardiomyocytes in many structural and functional details, which are generally summarised and connoted as immature features. The term immaturity in this context is inspired and derived from the immature characteristics of prenatal or neonatal cardiomyocytes. Despite distinct differences in the development, differentiation and properties of native or stem cell-derived cardiomyocytes, the concept of immaturity conveys the idea that, in compliance with native cells, further maturation of hiPSC-CM towards an adult phenotype is possible. Since the immaturity of hiPSC-CM limits their application for clinical purposes, current research focuses intensively on the development of efficient maturation strategies to achieve an adult-like phenotype that may be best suited for a cell-based therapy of the failing heart.2+ handling and, thus, the basis of EC coupling in hiPSC-CM. In line with previous work [In this study, we focussed on the combined effects of changes in cell shape towards more adult morphology and molecular enhancement of BIN1 expression on Caous work , our datous work , the addous work , piezo cous work , 60 and/ous work  to elici2+ channel was reduced to less than 40 nm in 3D-reshaped BIN1-overexpressing hiPSC-CM, reminiscent of the narrow dyadic cleft of adult, i.e. mature cardiomyocytes. This close vicinity is indispensable for the functional interaction of LTCCs and RyR2s via rapid diffusion of Ca2+ ions, thus ensuring efficient EC coupling.In addition to morphological remodelling by changing cell shape, we also overexpressed BIN1 in the hiPSC-CM. Previous reports suggested a possible role of BIN1 in the generation of membrane tubular networks , 41, 52.2+ clock mechanisms may provide a plausible explanation. At the molecular level, hiPSC-CM expressed all essential Ca2+ handling proteins contributing to Ca2+ influx, release and removal [2+ release from the SR is key to the Ca2+ clock mechanism [2+ currents and both spontaneous and Ca2+-triggered Ca2+ release events, respectively.One hallmark of immaturity in hiPSC-CM is their spontaneous contractile activity. Although varied mechanisms underlying this autorhythmicity have been hotly debated, the interplay between so-called membrane and Ca removal  to a simechanism , 34, 53.2+ current revealed no difference in its properties and kinetics among the different groups of hiPSC-CM, but Ca2+ signalling differed at the functional level pointing to a more mature Ca2+ handling in the cytoplasm. One indicator for this in structurally remodelled hiPSC-CM was the reduction in spontaneous Ca2+ release events from the SR. While non-patterned control cells revealed a high frequency of Ca2+ sparks and related spontaneous Ca2+ release events at resting conditions, their occurrence was significantly decreased in cuboid BIN1-overexpressing hiPSC-CM. Detailed analysis of spontaneous and Ca2+-triggered Ca2+ release events revealed faster Ca2+ transients in 3D-remodelled cells irrespective of the shape both at the level of Ca2+ release from the SR (TTP) and Ca2+ reuptake via SERCA2 and extrusion via the NCX (FDHM), which is in accordance with previous studies of either BIN1-overexpressing or 3D-reshaped iPSC-CM [Electrophysiological evaluation of the LTCC-dependent Ca iPSC-CM , 46.2+ transients [2+ handling and specifically in desynchronised Ca2+ transients in the affected cardiomyocytes with the consequence of severely reduced EC coupling and decreased contractile force [2+ handling in cardiomyocytes, and therefore represent a major goal to achieve in hiPSC-CM [2+ transient, which is initiated by diffusion-dependent activation of the RyR2s via Ca2+ influx through the LTCCs, revealed strong desynchronization at the onset of the Ca2+ transients. This pattern can be best explained by inward propagation of the released Ca2+ from the peripheral cell membrane towards the cell centre rather than true CICR. In contrast, cuboid BIN1-expressing hiPSC-CM demonstrated a spatio-temporally synchronised onset of Ca2+ release from the SR. Due to the many BIN1-induced membrane invaginations, LTCCs are also located deep in the centre of the cell. As a consequence, sarcolemmal depolarization and LTCC-mediated Ca2+ influx lead to local CICR via junctional RyR2s simultaneously throughout the entire cell and not only at the cell periphery.In dysfunctional cardiomyocytes originating from different models of cardiac disease , 13, losansients . Furtherle force , 57. HenhiPSC-CM . In non-2+ channels and Ca2+ influx currents, Ca2+ release from the SR was significantly greater in these cardiomyocytes due to shorter Ca2+ diffusion distances and, thus, a more effective EC coupling gain. This increased gain is an important measure for amplification of the cytosolic Ca2+ signal through RyR2-mediated Ca2+ release from the SR and, therefore, confirms that structural remodelling leads to robust EC coupling, indicative of important functional maturation processes at the level of single hiPSC-CM.This improvement of junctional CICR was further corroborated by the significantly enhanced EC coupling gain in cuboid 3D-reshaped, BIN1-overexpressing hiPSC-CM. Our data demonstrate that despite similar levels of expression of the Ca2+ handling machinery suggests that BIN1 may also represent a promising target for the treatment of heart failure. Potential therapeutic effects of BIN1 gene therapy have already been tested at the preclinical level with preliminary success [In summary, our data provide strong evidence that structural remodelling of hiPSC-CM at the level of cell morphology and membrane organisation leads to optimization of the functional interaction between LTCCs and RyR2s, the key players of EC coupling in cardiomyocytes. In this context, the role of BIN1 as important steering wheel for membrane invaginations and consequently for the development of a more mature Ca success , 63. WitHere, we have addressed different levels of structural remodelling in the context of the rather complex development of fully mature cardiomyocytes from pluripotent stem cells that is in addition accompanied by some technical limitations. Our experimental model served primarily the identification of specific cues and features that are needed to drive hiPSC-CM to further maturation. In this context, one significant success in the maturation process is already a reduction in variability, as seen from our data. In the presented form, the tool of reshaping is not suited for in vivo experimentation, but importantly, the gain of knowledge that both longitudinal shape and t-tubule formation are required for functional maturation, will have to be considered for the preparation of cell grafts. In future studies, the impact of the composition of the extracellular matrix (ECM) and its stiffness  as well Supplementary file1 (DOCX 765 KB)Below is the link to the electronic supplementary material.


Given their predominance in atria versus ventricles, SK channels are considered a promising atrial-selective pharmacological target against atrial fibrillation (AF), the most common cardiac arrhythmia. However, the precise contribution of SK current (ISK) to atrial arrhythmogenesis is poorly understood, and may potentially involve different mechanisms that depend on species, heart rates, and degree of AF-induced atrial remodeling. Both reduced and enhanced ISK have been linked to AF. Similarly, both SK channel up- and downregulation have been reported in chronic AF (cAF) versus normal sinus rhythm (nSR) patient samples. Here, we use our multiscale modeling framework to obtain mechanistic insights into the contribution of ISK in human atrial cardiomyocyte electrophysiology. We simulate several protocols to quantify how ISK modulation affects the regulation of AP duration (APD), Ca2+ transient, refractoriness, and occurrence of alternans and delayed afterdepolarizations (DADs). Our simulations show that ISK activation shortens the APD and atrial effective refractory period, limits Ca2+ cycling, and slightly increases the propensity for alternans in both nSR and cAF conditions. We also show that increasing ISK counteracts DAD development by enhancing the repolarization force that opposes the Ca2+-dependent depolarization. Taken together, our results suggest that increasing ISK in human atrial cardiomyocytes could promote reentry while protecting against triggered activity. Depending on the leading arrhythmogenic mechanism, ISK inhibition may thus be a beneficial or detrimental anti-AF strategy.By sensing changes in intracellular CaNEW & NOTEWORTHY Using our established framework for human atrial myocyte simulations, we investigated the role of the small-conductance Ca2+-activated K+ current (ISK) in the regulation of cell function and the development of Ca2+-driven arrhythmias. We found that ISK inhibition, a promising atrial-selective pharmacological strategy against atrial fibrillation, counteracts the reentry-promoting abbreviation of atrial refractoriness, but renders human atrial myocytes more vulnerable to delayed afterdepolarizations, thus potentially increasing the propensity for ectopic (triggered) activity. It iAF (cAF) , 7. To aAF (cAF) –11. For proposed , 13, as nd IKur) , 11.2+-activated K+ (SK) channels are emerging as a promising atrial-selective pharmacological target against AF (ISK) shortens atrial (but not ventricular) APD under physiological conditions. ISK is modulated by Ca2+  (2+ cycling and sarcoplasmic reticulum (SR) Ca2+ release to transmembrane potential (Em) dynamics (ISK-voltage relationships have been observed in many studies in both native and cloned channels from various species (2+ and Mg2+) cations  but also to shorten the ERP, which may facilitate the formation of reentry circuits (proarrhythmic). Moreover, an altered dynamic APD regulation at fast rates may influence the induction of alternans   and can dynamics . Despite species –25, and  cations –29. Recet manner , 31. Thelternans , possiblISK on the electrophysiology of human atrial myocytes and their vulnerability to Ca2+-driven arrhythmias using mathematical modeling and simulation, which have proven useful tools for gaining mechanistic insight into AF mechanisms and developing novel antiarrhythmic pharmacological approaches  was modified by reducing the time constant of inactivation to reproduce experimental data at physiological temperature  compared with the baseline nSR condition. Our default cAF model does not account for any AF-associated SK channel remodeling. However, we performed a separate set of simulations in which both SK channel conductance and Ca2+ sensitivity were altered as seen in AF  on AP and Ca2+ transient (CaT) properties at different pacing rates  was estimated at steady state for both nSR and cAF. To investigate how GSK changes affect atrial refractoriness, we determined the ERP simulating an S1–S2 premature stimulation protocol. First, for each condition studied, we identified the diastolic threshold of excitation (DTE). The S1 stimulus  was applied at a basic cycle length (BCL) until steady state. To determine the ERP, we then applied the premature S2 stimulus (same duration and amplitude as S1) at progressively smaller S1–S2 intervals by decrements of 5 ms. As previously described , we identified the maximum pacing thresholds required for the development of voltage  and Ca2+-  driven alternans. The occurrence of DADs was assessed by stimulating the models at a fast rate for 10 s in the presence of 1 µM isoproterenol, and then pausing the stimulation. By progressively increasing the stimulation frequency (1-ms decrements in BCL), we identified the maximum pacing threshold required for the development of DADs .We studied the role of GSK modulation on spontaneous Ca2+ release events (SCRs) and consequent DADs at different pacing rates . Each simulation consisted of a quiescent period of 4 s, a pacing period of 28 s to achieve a steady state and a final quiescent period of 5 s for observation of oscillations in Em and global [Ca2+]i. Whenever present, we quantified the amplitude of the voltage oscillation (ΔEm) by measuring the difference between the first peak and diastolic minimum during the final quiescent period. Then, we identified the corresponding SCR as the nearest [Ca2+]i peak preceding the voltage oscillation and determined its amplitude (ΔCa) by calculating the difference between the peak and diastolic minimum during the final quiescent period. We used cutoff values of 300 nM and 10 mV for oscillations in [Ca2+]i and Em, respectively.We used the group of 10 3-D models to assess the effect of GFollowing an established approach , we builDATA AVAILABILITY).Simulations of the updated Grandi et al. model were performed with a standard desktop using MATLAB , v. R2022a. Simulations of the Zhang et al. 3-D model  were performed with a computing cluster with Intel Xeon CPU E5-2690 v4 at 2.60 GHz 28 CPUs (56 threads) + 132 GB. Data analysis was performed with MATLAB using a standard desktop. Model codes are freely available on the authors’ website  on steady-state AP and CaT properties at varying stimulation frequencies , the cAF model displays shorter AP and ERP, and a more negative (polarized) resting Em (RMP) compared with nSR. Despite the depressed cytosolic CaT, ISK peak in cAF is comparable to that in nSR when the conductance does not undergo arrhythmia-driven remodeling , which rise both well above the Kd used to model Ca2+ activation of ISK  during the AP (not shown). The described differences between nSR and cAF AP and CaT features are maintained in the presence of GSK perturbations. Our simulations show that increasing GSK shortens the atrial AP and ERP, hyperpolarizes RMP, and reduces CaTamp in both nSR and cAF myocytes . We applied multivariable linear regression to quantify the sensitivity of APD, CaTamp, and ERP (assessed in each model variant at 1-Hz pacing) to changes in the perturbed model parameters  and background Cl− currents. CaTamp is less sensitive to K+ currents and primarily affected by changes in maximal conductance of L-type Ca2+ channels (GCaL) and transport rates of Na+/Ca2+ exchanger (vNCX) and Na+/K+ ATPase (vNKA), which are also positively correlated with APD changes, along with GNaL. Taken together, our results suggest that increased ISK mainly causes APD and ERP shortening. These alterations have the potential to promote arrhythmogenesis as they increase the susceptibility to reentry, which can initiate arrhythmia within the atrial tissue and contribute to its persistence over time . In agreement with the results shown in SK for voltage- and Ca2+-driven alternans, respectively), indicating that propensity for alternans is enhanced in disease. Although GSK changes minimally alter BCL thresholds in nSR , increasing GSK moderately elevates the BCL thresholds in cAF . We then determined the BCL thresholds for voltage-driven alternans in each model variant of our nSR and cAF populations and performed multivariable linear regression  indicates that the contribution of ISK to the development of alternans is limited compared with that of other channels or transporters. Our results show that the parameters that are more influential for the pacing threshold for alternans in both nSR and cAF are GCaL and the maximal rate of SR Ca2+ release through RyRs . Increasing these parameters would reduce the pacing threshold, thereby protecting against alternans.When pacing the cAF model at fast rates, our simulations revealed the development of alternans B, anotheF models A and B. gression  to quantgression C. The poISK in the development of DADs, a Ca2+-driven phenomenon that can cause triggered activity, thereby contributing to AF initiation , confirming the disease-associated increase in DAD propensity. Furthermore, increasing GSK reduces the BCL thresholds in both nSR  and cAF , thereby protecting against arrhythmias. As described for alternans, we determined the BCL thresholds for DADs in each model in our populations and performed linear regression . Our analysis also highlights that the sensitivity to changes in GSK is limited if compared with changes in other parameters that are directly involved in the regulation of Ca2+ cycling, such as GCaL, vNCX, vNKA, and vRyR,rel. We next separated our nSR and cAF populations in two subgroups of model variants based on the development of DADs at 2-Hz pacing . Using this cutoff, we identified 218 and 570 model variants exhibiting DADs within the nSR and cAF populations, respectively (inset). As previously described . Interestingly, the BCL threshold for SCR is not affected by GSK modulation (right), suggesting altered coupling between Em and [Ca2+]i with increased GSK. To confirm this hypothesis, we analyzed both supra- and subthreshold Em and [Ca2+]i oscillations obtained after stimulating the cells at a pacing frequency of 3 Hz (SK significantly reduces the amplitude of Em oscillations (ΔEm). The amplitude of the [Ca2+]i oscillations (ΔCa) is not different between 50% and 100% GSK, but is significantly reduced with 200% GSK. To assess changes in Em-Ca2+ coupling, we calculated the ΔEm/ΔCa ratio and identified a significant reduction with an increase in GSK. Notably, we obtained similar results when investigating different pacing rates between 2 and 5 Hz . Taken together, these results indicate that the protective effect of GSK activation against the development of DADs is due to a strengthening of the repolarizing force that counteracts the Ca2+-dependent depolarization.To get mechanistic insight into the subcellular processes involved in the development of triggered activity, we simulated our recently developed 3-D model of the human atrial myocyte with spatially detailed Ca cycling , 51. We dulation A, right, of 3 Hz B and C. ISK modulation on the regulation of APD, CaT, and ERP, as well as the occurrence of arrhythmogenic alternans and DADs. Our findings indicate that increased GSK promotes APD and ERP shortening, while slightly increasing the propensity for alternans, thereby primarily promoting the development of an arrhythmogenic reentrant substrate. Our simulations also demonstrated that enhanced ISK limits Ca2+ cycling and counteract the occurrence of DADs, thus exerting a protective effect against triggered activity. This protective effect is due to an enhanced repolarizing force  when GSK is increased. Overall, these results , as suggested by our functional experiments . In our cAF simulations, we also assumed unchanged Ca2+-dependent activation of ISK compared with nSR. Experiments in atrial cardiomyocytes from patients have shown an increased affinity for intracellular Ca2+ in cAF versus nSR . As discussed earlier, this is because SK channel function is controlled by the levels of Ca2+ in cleft and subsarcolemmal compartments that, during the AP, rise well above the Kd values used here. Our data revealed minor changes in the propensity for alternans and DADs, with the former being facilitated and the latter being counteracted by decreased Kd,SK . The limited impact of increasing Ca2+ affinity is confirmed with the 3-D model of Zhang et al. . Although BCL threshold for SCRs is not affected, decreasing Kd,SK lowers the BCL threshold for DAD in two (out of 10) atrial myocyte models at nominal GSK   . Based o4571385) , 74. It uring HF , 67, whehythmias . Similarntricles . Bonilla of EADs . On the ntricles . These oISK in female versus male animals  signaling. These pathways are important regulators of atrial myocyte function , but will also depolarize the surrounding tissue and reduce the sink when cells are coupled, thus enhancing source-sink mismatch  and Cafunction , and canfunction . Since bfunction , 49, fur tissues . Moreoveactivity . Similarmismatch .2+ can block SK channels at IC50 of ∼20 µM . Lastly, our atrial myocyte models were built assuming homogeneous distribution of SK channels through the plasmalemma , as a larger fraction of channels “senses” the Ca2+-dependent effect. Further studies assessing how this spatial proximity influences local and global Ca2+ signaling and Em dynamics are clearly warranted.It has been shown that Caf ∼20 µM , which cmyocytes , 86. Whechannels . We founISK inhibition may be suitable for the treatment of patients with cAF. This strategy, however, may cause an increase in vulnerability for cellular DADs, thus potentially promoting atrial ectopy. Nevertheless, if the increased ectopy caused by SK channel inhibition cannot induce reentry, which is opposed by the ERP-prolonging effect, the net outcome will be primarily antiarrhythmic. Overall, these observations are similar to those made for APD-prolonging class III agents. The use of these drugs that target the hERG K+ channel (responsible for the delayed rectifier K+ current IKr) is constrained by the potential risk of inducing life-threatening ventricular arrhythmias through EADs  used in this study and all simulated data presented here are freely available for download at the following links: 10.6084/m9.figshare.23982561.v1https://doi.org/10.6084/m9.figshare.23982561.v1.Supplemental Figs. S1, S2, S3, S4, S5, S6, and S7: This work was funded by National Institutes of Health Grants T32HL086350 (to N.T.H.), R01HL131517 (to E.G. and D.D.), and 1OT2OD026580-01, R01HL141214, and P01HL141084 (to E.G.); and R00HL138160 (to S.M.); American Heart Association Predoctoral Fellowship 20PRE35120465 (to X.Z.) and Postdoctoral Fellowship 20POST35120462 (to H.N.); German Research Foundation (DFG) Grant Do 769/4-1 (to D.D.); European Union  Grant 965286 (to D.D.); The Netherlands Organization for Scientific Research (NWO/ZonMW Vidi) Grant 09150171910029 (to J.H.); Norwegian Center for Research-Based Innovation Precision Health Center for Optimized Cardiac Care (ProCardio) Grant 309762 (to M.M.M.); and Burroughs Wellcome Fund-Doris Duke Charitable Foundation “COVID-19 Fund to Retain Clinical Scientists” award (to S.M.).No conflicts of interest, financial or otherwise, are declared by the authors.E.G. and S.M. conceived and designed research; N.T.H. and X.Z. performed experiments; N.T.H. and X.Z. analyzed data; H.N., M.M.M., J.H., D.D., E.G., and S.M. interpreted results of experiments; N.T.H. and S.M. drafted manuscript; X.Z., H.N., M.M.M., J.H., D.D., and E.G. edited and revised manuscript; N.T.H., X.Z., H.N., M.M.M., J.H., D.D., E.G., and S.M. approved final version of manuscript.


However, the literature presents conflicting data regarding the effects of AET on Ca2+ transit and cardiac function in rats with heart failure resulting from aortic stenosis (AoS). This study aimed to evaluate the impact of AET on Ca2+ handling and cardiac function in rats with heart failure due to AoS. Wistar rats were distributed into two groups: control  and aortic stenosis . After 18 weeks, the groups were redistributed into: non-exposed to exercise training  and trained  for 10 weeks. Treadmill exercise training was performed with a velocity equivalent to the lactate threshold. The cardiac function was analyzed by echocardiogram, isolated papillary muscles, and isolated cardiomyocytes. During assays of isolated papillary muscles and isolated cardiomyocytes, the Ca2+ concentrations were evaluated. The expression of regulatory proteins for diastolic Ca2+ was assessed via Western Blot. AET attenuated the diastolic dysfunction and improved the systolic function. AoS-ET animals presented an enhanced response to post-rest contraction and SERCA2a and L-type Ca2+ channel blockage compared to the AoS. Furthermore, AET was able to improve aspects of the mechanical function and the responsiveness of the myofilaments to the Ca2+ of the AoS-ET animals. AoS animals presented an alteration in the protein expression of SERCA2a and NCX, and AET restored SERCA2a and NCX levels near normal values. Therefore, AET increased SERCA2a activity and myofilament responsiveness to Ca2+ and improved the cellular Ca2+ influx mechanism, attenuating cardiac dysfunction at cellular, tissue, and chamber levels in animals with AoS and heart failure.Aerobic exercise training (AET) has been used to manage heart disease. AET may totally or partially restore the activity and/or expression of proteins that regulate calcium (Ca The practice of exercise training plays an important role in: 1) improvement of the physical fitness of practitioners, which is essential to optimizing the performance of daily activities  improvem; (2) disLower cardiorespiratory fitness and worse functional capacity and quality of life are common features of heart failure ,12,13. A2+) handling, one of the main factors in positive cardiac adaptation LT); and the ratios of lactate concentrations at lactate threshold and at exhaustion by respective velocities at each moment ([LAC](LT)/Vel(LT), and [LAC](Ex)/Vel(Ex)).The AET prescription was based on the analysis of lactate curves, with the daily training speed equivalent to the lactate threshold (LT). This point was determined through the graphic plotting of lactate concentrations versus the running speed of the stages. The moment when there was a change in the linearity of the curve as a function of the increase in speed, established by visual inspection, was considered the LT . The varAET was started 18 weeks after surgery and involved a rat-specific treadmill running program  five days a week for 10 consecutive weeks ,32,33. T®, Sespo Indústria e Comércio Ltd.a- Divisão Vetbrands, Jacareí, São Paulo, Brazil) and xylazine . The following variables evaluated cardiac structure: LA normalized to the aortic diameter (LA/Ao), left ventricle diastolic diameter (LVDD), left ventricular systolic diameter (LVSD), posterior wall diastolic thickness (PWDT), interventricular septum diastolic thickness (ISDT), and relative wall thickness (RWT). The following parameters assessed ventricular function: heart rate (HR), midwall fraction shortening (FS), ejection fraction (EF), posterior wall systolic velocity (PWSV), early diastolic mitral inflow velocity (E wave), ratio between E wave and atrial contraction flow peak (A wave), E-wave deceleration time (EDT), velocity of the mitral annulus during early ventricular filling (E′), mitral velocity annulus during atrial contraction (A′), and the ratio between filling flow peak and mitral annulus velocity during early ventricular filling (E/E′).The echocardiographic study provided data on cardiac structure and function before and after AET, i.e., after 18 and 28 weeks of experimental surgery. Commercially available echocardiography  equipped with a 5–11.5 MHz multifrequency probe was used as previously described ,31,64. R2), maximum rate of tension development , and decline . Regulator mechanisms of Ca2+ influx and L-type calcium channel activity were analyzed by Ca2+ concentration extracellular elevation maneuver (response percentage) and elevation of extracellular Ca2+ concentrations  in the presence and absence of diltiazem , a specific blocker of L-type calcium channels. A post-rest contraction maneuver (response percentage) and elevation of extracellular Ca2+ concentrations  in the presence and absence of cyclopiazonic acid , a highly specific blocker of SERCA2a, were performed to assess the potential of SERCA2a function. All variables were normalized per cross-sectional area of the papillary muscle (CSA). Seven papillary muscles with CSA >1.5 mm2 were excluded from analysis as they can present central core hypoxia and impaired functional performance [The cardiac contractile performance was evaluated by studying isolated papillary muscles from the LV as previously described ,64. The formance .Cardiomyocyte Contractility2 and 95% O2 for ~3 to 5 min. The composition of the DB solution was (mM): 130 NaCl, 1.4 MgCl2, 5.4 KCl, 25 HEPES, 22 glucose, 0.33 NAH2PO4, and pH 7.39. Afterwards, the hearts were perfused for 20–30 min with a DB solution containing 1 mg/mL collagenase type II  and Ca2+ (1 mM). Subsequently, isolated cells were placed in an experimental chamber with a glass coverslip base mounted on the stage of an inverted microscope  with an edge detection system with a 40× objective lens . After the digestion process, the supernatant was removed, and the myocytes were resuspended in Tyrode’s buffer containing (in mM): 140 NaCl, 10 HEPES, 0.33 NaH2PO4, 1 MgCl2, 5 KCl, 1.8 CaCl2, and 10 glucose. Cells were immersed in Tyrode’s solution and field stimulated at 1 Hz . Cell shortening in response to electrical stimulation was measured with a video-edge detection system at a 240 Hz frame rate , and the contractile parameters were evaluated. Sarcomere length, fractional shortening (expressed as a percentage of resting cell length), maximum shortening velocity, maximum relaxation velocity, and time to 50% shortening (time to 50% peak) and 50% relaxation (time for 50% relaxation) were measured in 6 cells per animal in each experimental group.Under anesthesia, rats from each group were euthanized, and the hearts were quickly removed by median thoracotomy and enzymatically isolated as previously described . Briefly2+ MeasurementsIntracellular Ca2+ concentration detected at approximately 510 nm. Diastolic and systolic Ca2+, time to Ca2+ peak, and time to 50% Ca2+ peak and decay were also analyzed. In addition, the ratios of systolic Ca2+ by shortening fraction and by shortening maximum velocity were performed to evaluate the responsiveness of myofilaments to Ca2+.Myocytes were loaded with 1.0 μM Fura2-acetoxymethyl (AM) ester  for 10 min at room temperature, washed with Tyrode solution, and allowed to rest for an additional 10 min to allow the de-esterification of dye. Subsequently, the cardiomyocytes were stimulated at 1 Hz, and fluorescence images were obtained using excitation of 340 to 380 nm wavelengths using a Hyper Switch system . Background-corrected Fura 2 AM ratios, reflecting intracellular Ca2+ handling was analyzed by Western Blot. Fragments of the LV were frozen in liquid nitrogen and stored at −80 °C. Frozen samples were subsequently homogenized in RIPA buffer containing protease  and phosphatase  inhibitors using a bead beater homogenizer . The homogenized product was centrifuged  at 12,000× g rpm for 20 min at 4 °C, and the supernatant was transferred to Eppendorf tubes and stored at −80 °C. Protein concentration was determined using the Pierce BCA Protein Assay Kit. SDS-PAGE was used to resolve 25 µg of protein lysate from each sample. Electrophoresis was performed with biphasic gel stacking  and resolving  at a concentration of 6 to 10%, depending on the molecular weight of the analyzed protein. The Kaleidoscope Prestained Standard  was used to identify band sizes. Electrophoresis was performed at 120 V  for 3 h with running buffer . Proteins were transferred to a nitrocellulose membrane  using a Mini Trans-Blot  system with transfer buffer . Membranes were blocked with 5% non-fat dry milk in TBS-T buffer  for 120 min at room temperature under constant agitation. The membrane was washed three times with TBS-T and incubated for 12 h at 4–8 °C under constant agitation with the following primary antibodies: SERCA2a , Phospholamban , Phospho-Phospholamban (Ser16) , Phospho–Phospholamban (Thr17) , NCX , Calcium Channel, VoltageGated Alpha 1C , Ryanodine Receptor , and GAPDH . After incubation with the primary antibody, membranes were washed three times in TBS-T and incubated with peroxidase-conjugated secondary antibodies  for 2 h under constant agitation. Membranes were then washed three times with TBS-T to remove excess secondary antibodies. Blots were incubated with ECL  for chemiluminescence detection by ImageQuant™ LAS 4000 . Quantification analysis of blots was performed using Scion Image software . The immunoblots were quantified by densitometry using ImageJ Analysis software 1.53t (NIH), and target band results were normalized to the expression of cardiac GAPDH [The protein expression of the elements responsible for the regulation of Ca2+ handling protein expression are reported as means ± standard deviation (SD), or median . Comparisons among four groups were evaluated using two-way analysis of variance (ANOVA) for independent samples or Kruskal–Wallis, complemented with Bonferroni or Dunn’s multiple comparison tests, respectively. Ca2+ elevation, post-rest contraction, and SERCA2a and L-channel blockage maneuvers are reported as means ± SD and studied using analysis of variance for repeated measures, complemented with Bonferroni’s multiple comparison test. The data were evaluated at a significance level of 0.05. The statistical analyses were performed using SigmaStat 3.5, and graphics were generated using GraphPad Prism 5.The Kolmogorov–Smirnov test was used to evaluate the data’s normality. Data from cardiorespiratory fitness tests, cardiac function, in vivo and in vitro analysis, and Ca2+ handling in animals with supravalvular AoS and ventricular dysfunction since in vitro cardiac performance analysis showed improvement in the capacity of Ca2+ recapture by the sarcoplasmic reticulum, consequent to the positive adaptations of the SERCA2a and L-type Ca2+ channel activities, and increased responsiveness of myofilaments to Ca2+, which supports the positive responses observed in echocardiograms and cardiorespiratory fitness tests. Our findings reinforce the importance of controlled exercise training not only in health promotion and disease prevention but also as a possible tool in treating heart failure. The loss of quality of life and functional capacity of individuals with heart failure is evident, and it is essential to develop strategies to improve this scenario and ensure greater longevity for this population. In addition, full knowledge of the molecular mechanisms related to the benefits of different types and intensities of exercise in failing hearts is fundamental for managing the intervention, which must be specific for each type and severity of cardiac pathology. Cardiac improvement from a macro point of view—the heart as a pump—is a product of the positive adjustments observed on a micro level—a molecular and cellular view. An enhanced heart function, along with the improvement of other organs by exercise training, generates a higher quality of life for individuals with heart failure.AET improves myocardial Ca