The goal of this study was to characterize podocyte biology during MMF treatment in nephrotoxic serum (NTS) nephritis (NTN). NTN was induced in three-week old wild-type mice. On day 3, half of the mice were treated with MMF (100 mg/kgBW/d p.o.) for one week. On day 10, we performed proteomic analysis of glomeruli as well as super-resolution imaging of the slit diaphragm. For multiphoton imaging of Ca2+ concentration ([Ca2+]i), the experimental design was repeated in mice expressing podocyte-specific Ca2+ sensor. MMF ameliorated the proteinuria and crescent formation induced by NTS. We identified significant changes in the abundance of proteins involved in Ca2+ signaling and actin cytoskeleton regulation, which was further confirmed by direct [Ca2+]i imaging in podocytes showing decreased Ca2+ levels after MMF treatment. This was associated with a tendency to restoration of podocyte foot process structure. Here, we provide evidence that MPA has a substantial direct effect on podocytes. MMF contributes to improvement of [Ca2+]i and amelioration of the disorganized actin cytoskeleton in podocytes. These data extend the knowledge of direct effects of immunosuppressants on podocytes that may contribute to a more effective treatment of proteinuric glomerulopathies with the least possible side effects.Mycophenolate mofetil (MMF) is applied in proteinuric kidney diseases, but the exact mechanism of its effect on podocytes is still unknown. Our previous in vitro experiments suggested that MMF can ameliorate podocyte damage via restoration of the Ca Many patients with glomerulopathies can only be maintained in remission using longstanding treatment with steroids. However, the use of this drug for prolonged time results in serious adverse effects. Therefore, especially pediatricians have been investigating alternative steroid-sparing therapeutic options such as mycophenolate mofetil (MMF) in this vulnerable population2.Glomerulopathies account for 5–14% of children with chronic kidney disease and 15–29% of children with kidney failure worldwide3. The first study on kidney effects of MMF showed that it prevents the process of chronic allograft rejection by decreasing the expression of adhesion molecules and cytokines in glomeruli, thus preventing glomerulosclerosis4. In the past two decades, MMF has been established and widely used also in inflammatory glomerulopathies5.MMF, the pro-drug of the biologically active mycophenolic acid (MPA), acts as a potent, reversible inhibitor of IMPDH, the key enzyme in de novo purine biosynthesis in proliferating lymphocytes, thereby suppressing cell-mediated immune responses and antibody formation7. In diabetic nephropathy, lower apoptotic rate of podocytes and preserved expression of nephrin and podocin were observed in the MMF treated group8. This is in accordance with the results in a puromycin model where the inhibition of IMPDH restored the ATP pool, which retained nephrin and synaptopodin expression and stabilized the actin cytoskeleton9. Additionally, Fu et al. showed a direct effect of MMF in an immune-mediated kidney disease: MRL/lpr mice were treated with MMF. Subsequently, RNA sequencing performed on isolated kidney cortex showed a significant decrease of Rac1 gene expression, which disrupts the physiological stress fiber formation, when activated10. In line with that our previous in vitro experiments performing RNA sequencing of a MPA treated podocyte cell line revealed an accumulation of the terms “actin cytoskeleton” and “regulation of small GTPase mediated signal transduction”. Additionally, also the terms “calcium channel activity” and “calcium ion transport” were enriched11. The rise in intracellular calcium concentration ([Ca2+]i) in podocytes12 can lead to acute reorganization of the actin cytoskeleton or even cytoskeletal collapse and proteinuria16. In the present study, we wanted to confirm, whether MMF is also able to ameliorate podocyte damage via restoration of the Ca2+- actin cytoskeleton axis in an in vivo model of nephrotoxic serum nephritis (NTN).Beyond the well characterized, immunosuppressive effect, we know little about how MMF directly acts on the kidney tissue, especially podocytes17 are from an established colony (> 10 years) in our vivarium. All animal experiments were performed in accordance with relevant guidelines and regulations provided by the LANUV NRW . The experimental protocol was examined and approved by the LANUV NRW (VSG81-02.04.2020.A052), which adhered to the 3R principles. Each mouse was considered as n = 1. Altogether 13 mice  constituted the control group, 11 mice  the NTS + veh group and 12 mice  the NTS + MMF group. NTN was induced in three-week old mice  as follows: The animals received intraperitoneal injection of nephrotoxic serum  in a dose of 10 µl/g body weight (BW) on two consecutive days. From a pathogenic point of view, the historically coined term “nephrotoxic” is misleading since the serum induces an immune-mediated process. In detail, nephrotoxic antibodies (heterologous sheep antibodies raised against rat whole glomeruli) are deposited in the whole glomerulus including the subepithelial, subendothelial areas as well as mesangium. Subsequently, mesangial proliferation and crescent formation can be observed. Thus NTN serves as a model for immune-mediated glomerulopathy with proteinuria , Pod:cre mice4538, PodMice were genotyped using DNA isolated from ear biopsies. For Pod:cre tg/wt GCaMP3 fl/fl mice, DNA was amplified by PCR using REDTaq ReadyMix  and visualized via gel electrophoresis. All primers used and the respective fragment size detected in gel electrophoresis are listed in Suppl. Table Quantification of the urinary albumin levels was performed with a mouse albumin ELISA kit  and creatinine levels were measured with a urinary creatinine kit  according to the manufactor’s instructions.18.In kidneys, scheduled for histopathology, dehydration and embedding in paraffin was performed. Kidney tissue was cut and sections were transferred on glass slides. To assess glomerulosclerosis periodic acid–Schiff (PAS) staining was performed. Briefly, sections were deparaffinized in Xylene and subsequently rehydrated in a descending ethanol series. After incubation for 10 min each in 0.9% periodic acid , Schiff reagent  and Mayer’s haematoxylin (Merck) the sections were dehydrated in an ascending ethanol series and xylene, and covered with HistoMount . A NanoZoomer S360  was used to acquire images. To determine the extend of glomerular damage, the number of crescentic glomeruli was counted in a blinded manner. Crescents were defined as two or more cellular layers in the Bowman`s space. A minimum of 50 glomeruli were assessed per kidney0 of the therapeutic drug monitoring (TDM), followed directly by the last dose of MMF. After 30 min a terminal blood taking was conducted, which served as MPA-C30 of TDM. Accordingly, a limited sampling strategy was used. Estimated MPA–area under the concentration–time curve profiles (eMPA-AUC0–24 h) was derived from plasmatic MPA concentrations and calculated based on a bayesian-fitting approach with a two-compartment model using the pharmacokinetic software package MW/Pharm ++ (licensed version)19.On the last day of the experiment, 24 h after the latest dose of MMF, NTS + MMF mice underwent a cheek punch procedure providing the blood sample for MPA-C20. The isolated glomeruli were resuspended in 500 µL 1 × Hanks’ Balanced Salt Solution and centrifuged at 1500 rpm for 5 min at 4 °C, afterwards the supernatant was removed. Pellets were resuspended in 50 µL SP3 buffer with 5% SDS and again centrifuged at 15,000 g for 5 min at 4 °C. Afterwards, chromatin was degraded in a Bioruptor® sonicator. The reaction tubes were placed in a magnetic rack to remove the Dynabeads from the samples. The supernatant was boiled at 95 °C and then centrifuged at 15,000 g for 2 min at 4 °C. The protein concentration was determined by a standard procedure using a BCA-kit21. The concentration of each sample was adjusted to 4 µg using 1 × SP3 puffer. The glomerular proteome was analyzed by a mass spectrometry system containing a Q ExactivePlusOrbitrap and an EASY nLC 1000 with a C18 analytical column. The detailed procedure has been described elsewhere22. Statistical analysis was performed with the Perseus software (version 1.5.5.3)23 and the package limma in R24. A one-way ANOVA and a two-sample t-test were applied for the data set after filtering proteins for 3 valid values in at least one group. A detected protein was defined as valid, when at least two peptides of the protein were identified. Limma functions lmFit, eBayes and topTable were used with default settings on the filtered log2 transformed protein intensities to calculate p values and log2 fold changes. A p value of 0.05 was assumed to be significant without further correction for multiple testing. Within this, altered proteins with a log2 fold change (fc) of ≥|0.58| (fc ≥ 1.5) were categorized as relevantly altered. Proteins that reached these criteria were further analyzed. GraphPad Prism v.9.0.2, Uniprot protein knowledgebase and ShinyGO (v.0.75)25 were used for functional analysis.After sacrifice by cervical dislocation, wild-type mouse kidneys were perfused with Dynabeads to isolate glomeruli as described2 and 5% CO2. The ex vivo images were acquired using an upright multiphoton microscope . The multiphoton laser  was set to 940 nm, a 25 × water objective  was used and images were recorded applying a non-descanned hybrid detector with a FITC/TRITC (525/50) filter set. Z-stacks were taken in 2 µm steps. For analysis of Ca2+ levels in podocytes, ROIs were placed on maximum intensity projections of z-stacks of single glomeruli and the mean fluorescence intensity was quantified with ImageJ. At least twenty glomeruli per slice and two slices per animal were imaged and analyzed by an observer blinded to the treatment.Murine kidneys of Pod:cre tg/wt GCaMP3 fl/fl mice were removed directly postmortem and then embedded in 4% low melting agarose at 37 °C. The kidney in agarose was cut in slices of 300 µm thickness and kept at 4 °C. For imaging, AKS was transferred to the microscope stage containing Krebs–Henseleit-Buffer at RT infused with 95% O27. Supp. Table 27 using an ImageJ macro, which can be found in GitHub gist: https://gist.github.com/github-martin/e699d18ae6cfa5b1a6aace15d3c3544c.The protocol of optical clearing and immunolabelling were performed as previously describedAll methods are reported in accordance with ARRIVE guidelines.0–24 h was 25.6 ± 13.9 mg*h/L.All mice showed severe proteinuria on day 3 Fig. a. The avp value of < 0.05) and relevantly (linear fold change of > 1.5) when comparing NTS + MMF with NTS groups. To clarify molecular functions and biological processes affected by MMF-regulated proteins, we performed Gene Ontology (GO) analyses. Figure 2+ signaling and regulation of actin cytoskeleton .The effective exposure to MPA is indirectly confirmed by the compensatory up-regulation of IMPDH2, the main target of MPA  Fig. a. In conFig. b. ImportFig. c. AlthouFig. .Figure 62+ signaling and actin cytoskeleton regulation, which was further confirmed by direct [Ca2+]i imaging in podocytes showing an improvement after MMF treatment. This functional change resulted in a tendency towards the structural stabilization of podocyte FPs. Thus, our data indicate that MMF has a beneficial effect on NTN partially by the restoration of the Ca2+- actin cytoskeleton axis in podocytes.This is the first study to investigate the molecular effects of MMF at the level of the glomerular proteome in NTN model with a clinically relevant treatment protocol. We found that MMF ameliorated proteinuria and glomerular crescent formation within one week. We identified significantly changed protein expression related to Ca0–24 h, which is the first time implemented in a mouse model. By performing TDM of MPA, we were able to show effective exposure to MPA. We know from previous clinical studies that an estimated MPA-AUC reflects MPA exposure more reliably and correlates better with the clinical outcome than MPA-C0 values do30.The NTN model possesses several advantages of an immunologically triggered pathomechanism and a rapid induction phase. This enabled us to investigate young mice at the age of four and a half weeks, when the kidneys are still growing, in order to model the dynamic immunological changes in our pediatric population. Additionally, our induction and treatment protocol mirrors the clinical reality of childhood glomerulopathies, as the start of the therapy was not preventive, but held for three days after disease induction to a time point, where all experimental animals exhibited proteinuria. In order to monitor the applied doses, we established TDM via MPA-AUC2+ signal” and “actin cytoskeleton regulation” were enriched after MMF treatment, suggesting their role in improving the kidney injury in NTN. Ca2+dependent remodeling of the actin cytoskeleton is a dynamic process that enables adaptation of podocytes under physiologic and adverse conditions. Consequently, dysfunction of this dynamic process can induce detachment from the basement membrane and podocyte loss. This dynamic process is regulated by modulating the activity of members of the Rho family of small GTPases. RhoA promotes the formation of stress fibers to generate a stable foot processes. In contrast, Rac1 promotes disease-associated cell motility32. Intriguingly, some of the immunosuppressive drugs have been shown to preserve the actin cytoskeleton of podocytes34. In line with these data, we found that MMF has a direct effect on the proteome of glomerular cells , which activate Rac1, was diminished. In addition, Wasf2, a down-stream target of the Rac1 pathway, was down-regulated, indicating a lower activation of Rac1 in our model, too. Subsequently, we assume that the feedback loop between Rac1-TRPC5-Ca2+influx-Rac139 is at least partially disrupted by MMF. Altogether, MMF targets several pathways reducing the overall Ca2+influx into the cytoplasm, which further impacts down-stream signaling pathways. Accordingly, MMF may influence cell functions by changing the expression of Ca2+-dependent proteins: e.g. activation of integrin through Rasgrp2, stabilization of the extracellular matrix via fibulins and diverse cascades via copinins. In addition, potential blocking of the calmodulin-calcineurin axis may prevent the cleavage of synaptopodin and RhoA as described earlier42. Furthermore, MMF treatment resulted in expressional changes of several regulatory proteins of Rac1, all acting in the direction of Rac1 inactivation. While the decreased expression of Arhgap29 promoted the activation of RhoA. Thus, our dataset suggests that MMF mediates a reciprocal regulation of RhoA and Rac1 pathways leading to the overall preservation of stress fibers and stationary phenotype of podocytes. This was further indicated by the alterations of proteins such as Spn, Eif5a, Mylpf, Calml4 and Shank3. Our proteomics analysis revealed that MMF could beneficially influence the Ca2+ signal and facilitate the formation of stress fibers. Although these data originate from cells of the whole glomeruli, but are very likely influenced by podocytes, as they heavily rely on an intact Ca2+-actin cytoskeleton axis14. It is also in line with our previous in vitro experiments11, which we performed to exclude the impact of immune cells and study MPA’s direct effect on podocytes. Doing so, our RNA sequencing of an MPA treated podocyte cell line revealed an accumulation of terms including “calcium channel activity”, “calcium ion transport” “actin cytoskeleton” and “regulation of small GTPase mediated signal transduction”. This leads potentially to normalization of [Ca2+]i in podocytes and to amelioration of the disorganized actin cytoskeleton demonstrated also by immunofluorescence staining11.We sought to elucidate the differential molecular signaling pathways that may account for renoprotection conferred by the administration of MMF therapy in our NTN model. Comparison of proteomics profiles showed that terms of “Calls Fig. : MMF dim2+-related proteins in podocytes, we performed multiphoton microscopy on AKS of mice expressing the [Ca2+]i sensor protein GCaMP3 specifically in podocytes15. The use of AKS enables imaging of many glomeruli in three dimensions reducing the selection bias. This is particularly advantageous in our NTN model due to heterogeneous population of glomerular lesions. The rise in [Ca2+]i is an early event in podocyte injury12 and the excess [Ca2+]i can lead to acute reorganization of the actin cytoskeleton or even cytoskeletal collapse16. Therefore, the tight control of the [Ca2+]i is essential in these cells. We also detected significant elevation of [Ca2+]i in our disease model which may contribute to the activation of pathologic signaling pathways in NTN. Importantly, MMF therapy reduced the elevated [Ca2+]i in podocytes. In summary, our proteomics and imaging data were concordant: The decreased expression of Ca2+-channel triggering proteins and the indicated decreased activation rate of Rac1 in MMF treated mice, were confirmed by directly visualizing the reduced podocyte [Ca2+]i after the treatment with MMF.To validate our proteomics data on Ca27. In the present study, we detected significantly reduced SD length in the NTN group, indicating a dysfunction of the glomerular filtration barrier. Importantly, MMF therapy could slightly improve the structure of FPs. The lack of statistical significance might be explained by the short exposure to MMF or the relative low number of analyzed glomeruli in the heterogeneous NTN model. In summary, our STED data confirmed the proteomics data by showing a tendency to the restoration of the FP’ structure and suggesting the stabilization of the actin cytoskeleton in the MMF group.To validate the proteomics data related to actin cytoskeleton regulation and their consequence on the podocytes’ FP structure, we applied the STED microscopy imaging of FPs, which enables us to stain for proteins of the filtration barrier2+ signaling at the cell membrane using our cytosolic calcium indicator, as [Ca2+]i imaging via multiphoton microscopy serves rather as an indirect readout of all Ca2+-channels showing the total [Ca2+]i. However, this increase of [Ca2+]i in the whole podocyte reflects a breakdown of mechanisms to control microdomain Ca2+ signaling. In our opinion, this might reflect the general condition of the podocytes even better. (3) Since our study focused on podocytes, we cannot rule out, that MMF simultaneously improved the function of injured mesangial cells and glomerular endothelial cells, thus, the observed effects in podocytes might have also been influenced by altered glomerular cellular cross-talk. (4) Finally, it was challenging to clearly differentiate the systemic effects of immunosuppression by MMF vs. its direct effects on the glomerular cells, as they are inseparably connected in an in vivo model, though this resembles the situation in our patients. Nevertheless, we believe that the application of targeted [Ca2+]i imaging and STED studies of podocytes helped to understand the specific direct effect in podocytes.We acknowledge that our study also comes with several limitations: (1) We found that one-week MMF treatment improved proteinuria, however, not significantly. Thus, MMF has the potential to beneficially influence signal pathways and histological changes in NTN, but either the duration of therapy was too short in our reversible model or MMF alone has only limited efficacy to induce a complete remission in proteinuria resulting from the interplay of a plethora of down-stream signaling pathways. (2) We were also not able to resolve microdomain Ca2+]i and ameliorates the disorganized actin cytoskeleton in podocytes thus improving the glomerular barrier function. In conclusion, our study provides the pathophysiological rationale for utilizing MMF’s direct effects on podocytes in addition to its immunosuppressive action in proteinuric glomerulopathies to obtain a more effective treatment with the least possible side effects.In summary, we identified signaling pathways in glomeruli regulated by MPA and revealed new mechanistic understanding of how MMF reduces proteinuria in a podocyte specific manner, beyond its systemic immunosuppressive effect: MMF contributes to the normalization of [CaSupplementary Information 1.Supplementary Information 2.


A 62-year-old female was diagnosed with acute myeloid leukemia (AML) with t. She achieved complete hematological remission after induction therapy and underwent umbilical cord blood stem cell transplantation (CBT). At 150 days after the CBT, a bone marrow examination revealed relapse. We treated the patient with venetoclax plus azacitidine as salvage therapy. After five cycles of venetoclax and azacitidine therapy, the patient died due to disease progression. The prognosis of AML with t is very poor owing to the high rate of early relapse even after hematopoietic stem cell transplantation. Therefore, a novel therapeutic approach is required to improve patient outcomes. The chromosomal translocation of t16;21) shows an incidence of 1% in acute myeloid leukemia (AML) [6;21, blast cell count 84%, hemoglobin level of 11.2 g/dL (reference range: 11.6-14.8 g/dL), and platelet count of 85×103/µL (reference range: 158×103 to 348×103/µL). Bone marrow aspiration confirmed the diagnosis of AML . The conditioning regimen was composed of fludarabine (30 mg/m2 daily for six days), busulfan (4 mg/kg daily for two days), and total body irradiation (4 Gy). Graft-versus-host disease prophylaxis was administered with tacrolimus and short-term methotrexate. Fluorescence in situ hybridization of sex chromosomes showed complete chimerism on day 93. Moreover, t was undetectable in G-banding bone marrow cells. At 150 days after CBT, bone marrow examination revealed hematological relapse owing to the proliferation of t-harboring blast cells. There is no donor candidate for second transplantation. And we had no choice but to treat with gilteritinib or quizartinib as FLT3-ITD mutation was not detected. Therefore, a combination of VEN and AZA was administered as salvage therapy after low-dose cytarabine and aclarubicin plus a granulocyte colony-stimulating factor therapy. However, although five cycles of the VEN and AZA combination were administered, a hematological response was not observed. The patient died due to disease progression 15 months after the initial diagnosis.The clinical course of our case was shown in Figure Our case highlights AML harboring the t16;21), which was shown to be 0.94% in 1,277 AML patients [6;21 who are in complete remission. However, the outcomes of HSCT among AML patients with t have been mentioned to be poor . DespiteSeveral reports have shown that epigenetic therapies, such as AZA, can be used as maintenance therapy after HSCT for AML with t16;21)p11;q22) [;21 in AML cases is associated with poor prognosis. Therefore, it is necessary to establish an alternative therapeutic approach to improve patient outcomes. A combination of VEN and AZA could be administered as maintenance therapy immediately after HSCT following the first complete remission. Evidence related to AML with t should be accumulated to facilitate the development of new treatment strategies.


Serum Ca2+ dyshomeostasis is associated with the development of insulin resistance, reduced insulin sensitivity, and impaired glucose tolerance. However, the molecular mechanisms involving Ca2+ ions in pancreatic β-cell loss and subsequently in T2DM remain poorly understood. Implicated in the decline in β-cell functions are aggregates of human islet amyloid polypeptide (hIAPP), a small peptide secreted by β-cells that shows a strong tendency to self-aggregate into β-sheet-rich aggregates that evolve toward the formation of amyloid deposits and mature fibrils. The soluble oligomers of hIAPP can permeabilize the cell membrane by interacting with bilayer lipids. Our study aimed to evaluate the effect of Ca2+ on the ability of the peptide to incorporate and form ion channels in zwitterionic planar lipid membranes (PLMs) composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) and on the aggregation process of hIAPP molecules in solution. Our results may help to clarify the link between Ca2+ ions, hIAPP peptide, and consequently the pathophysiology of T2DM.The calcium ion (Ca Although there is no sequence homology between the different proteins, the aggregation process follows a nucleation-dependent polymerization process ,2. Furth2+ [2+ [3+ [2+, in the form of heme [2+; mercury ion, Hg2+ [3+ [2+) is a metal ion involved in important physiological actions such as muscle contraction, excitability, and the regulation of several intra- and extracellular transfer processes, but it can also become toxic for the cell. Several studies have shown a link between Ca2+ and T2DM, although the results obtained seem to be contradictory. According to some researchers, the increased serum Ca2+ concentration could be a risk factor for developing T2DM, while others see no correlation. Other studies have shown that serum Ca2+ dyshomeostasis is associated with the development of insulin resistance, reduced insulin sensitivity, and impaired glucose tolerance, all hallmarks of T2DM [2+ were higher than physiological ones, together with a decrease in the activity of Ca2+ ATPase [2+ cellular homeostasis [Human islet amyloid polypeptide (hIAPP) is a hormone synthesized by the pancreatic islet β-cells, stored in the secretory granule, and secreted into the blood together with insulin. hIAPP is a peptide consisting of 37 amino acids in its native form; it has a disulfide bridge involving the amino acids Cys2 and Cys7, a highly hydrophobic amidated C-terminal (amino acid sequence 30–37) and an amino acid sequence residues 23–29) that appears to be involved in the initiation of the aggregation process ,6. hIAPP–29 that 2+ ,32; copp2+ [2+ ,34,35,36 [2+ [3+ ; iron io of heme ; cadmiumon, Hg2+ ; gold ioHg2+ [3+ ; rutheniHg2+ [3+ ,43,44,45Hg2+ [3+ ,47 with  of T2DM . Further of T2DM ,50 or freostasis .2+ in pancreatic β-cell loss and subsequently in T2DM, several studies have been conducted, although these mechanisms remain poorly understood. The results of some studies indicate that Ca2+ favors an abnormal interaction of hIAPP with negatively charged lipid vesicles, and consequent to this interaction, the peptide changes its conformation from α-helix to β-sheet. Ca2+ changes the hIAPP-mediated membrane destruction mechanisms by hindering the insertion of the prefibrillar hIAPP aggregates into the hydrophobic core of the membrane and favoring its destruction with a detergent-like mechanism. According to the authors, the effect of Ca2+ on membrane permeabilization by hIAPP is due to the effects of the ion on the biophysical characteristics of the bilayer, although they do not exclude the inhibitory effect of the ion on fibril formation as obtained from the thioflavin T (ThT) assay [2+, Zn2+, and Cu2+, on the aggregation of both the full-length hIAPP and hIAPP18-27 fragment and the ability of their oligomers to damage the membrane of unilamellar vesicles comprising a mixture of POPC:POPG = 4:1. The aggregation of the hIAPP18-37 fragment is little affected by Ca2+ ions when the peptide–metal ion ratio is 1:1 in phosphate buffer pH 7.4. Under the same experimental conditions, the ThT fluorescence intensity emitted by the full-length hIAPP oligomers in the presence of Ca2+ is slightly lower than that obtained in its absence, indicating that aggregation is little affected by the ion. The ability to damage the membrane was assessed in terms of the release of calcein from the vesicles. This ability is higher for the peptide fragment or the full-length peptide in the absence of Ca2+, indicating that the ion reduces the ability of the peptide to damage the membrane, most likely by reducing the surface hydrophobicity of the oligomers [To elucidate the molecular mechanisms involving CaT) assay . Wang anligomers .2+ ions, at hIAPP:Ca2+ molar ratios of 1:1, 1:5, and 1:10 on the aggregation process of the peptide in solution, using the ThT fluorescence assay and transmission electron microscopy (TEM) techniques, as well as the effect on the ability of the peptide to incorporate and form ion channels in zwitterionic planar lipid membranes (PLMs) composed of Palmitoyl-oleoyl-phosphatidylcholine (POPC), using the electrophysiological technique of single-channel current measurements.This study aimed to evaluate the effect of Ca2), potassium chloride (KCl), thioflavin T (ThT), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and n-decane were purchased from Sigma ; palmitoyl-oleoyl-phosphatidylcholine (POPC) was purchased from Avanti Polar Lipid . Amylin or hIAPP was purchased from Alexotech .Calcium chloride  and allowed to incubate at room temperature (RT) for 6 h. Subsequently, the solution was divided into equal portions, with each aliquot being subjected to freezing at −80 °C for 24 h followed by overnight lyophilization. The resulting lyophilized samples were then stored at −20 °C until they were ready for use.2 was prepared by dissolving CaCl2 powder (1.5 mg) in 5 mL of sterile bidistilled water via stirring for 10 min, resulting in a concentration of 2 mM. The 1 and 0.1 mM CaCl2 solutions were prepared via scalar dilution from a stock solution. These solutions were then refrigerated at 4 °C until needed.For the single-channel measurement experiments, the lyophilized sample was solubilized in sterile bidistilled water via stirring for 5 min, resulting in a concentration of 256 µM. From this solution, 5 µL was extracted and diluted in 45 µL of sterile bidistilled water via stirring for 5 min, resulting in a concentration of 25.6 µM. A stock solution of CaCl2 was prepared by dissolving CaCl2 powder (0.73 g) in 5 mL of sterile bidistilled water via stirring for 10 min to obtain a concentration of 1 M. The CaCl2 solutions at concentrations of 0.1 M, 1, 0.375, 1.875, and 3.750 mM were obtained from the stock solution via scalar dilution. The CaCl2 solutions were stored at 4 °C until use.For the TEM and ThT experiments, the lyophilized samples were dissolved in phosphate-buffered saline (PBS 50 mM at pH 7.4), achieving concentrations of 33 µM and 10 µM immediately prior to the TEM and ThT measurements, respectively. The stock solution of CaClThe investigation into hIAPP’s capacity to incorporate and form ion channels was carried out in planar lipid membranes (PLMs) composed of POPC in 1% n-decane, as detailed in prior descriptions ,55,56.cis and trans sides. The cis side, containing the hIAPP solution, defined the polarity of the voltage. A negative potential value was applied to the trans side, opposite the cis side. The two chambers held symmetrical 0.1 M KCl solutions, with pH = 7 and temperature 23 ± 1 °C.The method used in the single-channel measurements was consistent with our earlier studies . To elabAnalytical-grade salts were employed in all experimental procedures. Planar lipid membranes (PLMs) were formed using the Müller–Rudin or painted technique, following previously established protocols ,58,59. T-cis side;In the first experimental set, we added 15.6 µL of freshly prepared hIAPP solution (25.6 µM) to the -cis side of the membrane.In the second experimental set, 15.6 µL of hIAPP solution (25.6 µM) preincubated for 24 h at RT was added to the To evaluate the effect of the preincubation at RT for 24 h on hIAPP channel activity, two series of experiments were performed:The final concentration of the peptide used in this study was 0.1 µM.2+ concentrations on the peptide in solution, the following experimental series was devised:-2 (0.1 mM) was combined with 20 μL of hIAPP (25.6 µM), resulting in an equal molar ratio of hIAPP to Ca2+ (1:1);In the initial set, 5 μL of CaCl-2 (1 mM) was combined with 17.7 μL of hIAPP (25.6 µM), resulting in a molar ratio of hIAPP to Ca2+ of 1:5;In the second set, 2.3 μL of CaCl-2 (2 mM) was combined with 17.7 μL of hIAPP (25.6 µM), resulting in a molar ratio of hIAPP to Ca2+ of 1:10.In the third set, 2.3 μL of CaClTo investigate the impact of varying Cacis side of the membrane. This yielded a final hIAPP concentration of 0.1 µM, combined with the following metal ion concentrations:-2 concentration was 0.1 µM;In the initial set, the CaCl-2 concentration was 0.5 µM;In the second set, the CaCl-2 concentration was 1 µM.In the third set, the CaClAfter preparation, each mixture of metal ions and peptides was stirred for 3 min. Subsequently, in each experimental set, hIAPP was subjected to a 24 h preincubation with metal ions at RT. Following the formation of the lipid bilayer, 19.60 µL of the first mixture or 17.60 µL of the second and third mixtures, also stirred for 3 min, was introduced into the 2+ were its conductance, lifetime, frequency, and size of the single channel.The parameters considered in this study to evaluate the channel activity of hIAPP in the absence and presence of Cac ± SE) and were evaluated using analysis of variance (ANOVA–Tukey test) and Student’s t-test.To determine the conductance, we first measured, by hand, the current amplitudes of the single channels, which revealed the existence of one main conductance level. We then constructed a histogram of the amplitude distribution and fitted it with a Gaussian distribution function, giving the central value of the single-channel conductance. The results are expressed as central conductance ± standard error .c is the central conductance, σ is the specific conductibility of the solution filling the channel, π has its usual meaning, r is the radius of the channel and d is its length.To calculate the size of the hIAPP channel, the following formula was used:2+ concentrations, the ThT fluorescence assay was used. We set up the hIAPP samples in the absence and presence of the metal ion  as follows:-A 75 µL sample of hIAPP was taken from a stock solution at 10 µM;-2 at 0.375 mM was combined with 75 μL of hIAPP at 10 µM, yielding a molar ratio of hIAPP to Ca2+ of 1:1;Then, 2 µL of CaCl-2 at 1.875 mM was combined with 75 μL of hIAPP at 10 µM, yielding a molar ratio of hIAPP to Ca2+ of 1:5;Next, 2 µL of CaCl-2 at 3.750 mM was combined with 75 μL of hIAPP at 10 µM, yielding a molar ratio of hIAPP to Ca2+ of 1:10.Lastly, 2 µL of CaClTo determine the level of hIAPP amyloid aggregates in the absence and presence of different Ca2 in PBS at concentrations of 10, 50, and 100 µM were prepared as follows:After preparation, samples were stirred for 1 min and preincubated for 24 or 72 h at RT. Preliminarily, three solutions of CaCl-2, 20 µL of 0.1 mM CaCl2 (aqueous solution) was added to 980 µL of PBS;For 10 µM CaCl-2, 10 µL of 1 mM CaCl2 (aqueous solution) was added to 990 µL of PBS;For 50 µM CaCl-2, 20 µL of 1 mM CaCl2 (aqueous solution) was added to 980 µL of PBS.For 100 µM CaCl-In the hIAPP reading sample, 62 µL of hIAPP sample was combined with 246 µL of PBS and 2 µL of ThT (2 mM);-2+ (10 µM) reading sample, 62 µL of hIAPP + Ca2+ (10 µM) sample was combined with 246 µL of PBS and 2 µL of ThT (2 mM);In the hIAPP + Ca-2+ (50 µM) reading sample, 62 µL of hIAPP + Ca2+ (50 µM) sample was combined with 246 µL of PBS and 2 µL of ThT (2 mM);In the hIAPP + Ca-2+ (100 µM) reading sample, 62 µL of hIAPP + Ca2+ (100 µM) sample were combined with 246 µL of PBS and 2 µL of ThT (2 mM);In the hIAPP + Ca-2, 50 µM CaCl2, or 100 µM CaCl2 with 2 µL of ThT (2 mM).In the control samples, we combined 308 µL of PBS or 10 µM CaClAfter the preincubation time, samples were set up for the determination of the fluorescence intensity  as follows:In all reading samples, the final concentrations of hIAPP (except in control samples) and ThT were 2 µM and 10 µM, respectively.A 2 mM ThT solution in PBS was freshly prepared and added to the Eppendorf tubes holding the samples 15 min before instrumental analysis. In a 96-well black plate with a clear bottom, ThT fluorescence intensity was measured using a CLARIOstar microplate reader  at a temperature of 37 °C and a pH of 7.4. The samples were orbitally shaken for 30 s, with an amplitude of 4 mm and a frequency of 200 rpm. Emission was measured at 480 ± 10 nm and excitation was measured at 440 ± 10 nm with a 10 nm slit; data points were collected at 1 min intervals. Each experiment was carried out in triplicate.2+, was evaluated using TEM. hIAPP (10 µM) was incubated alone or with 10, 50, and 100 µM CaCl2 at RT for 30 min (T0), 24 h (T24), and 72 h (T72).The morphology of hIAPP aggregates, both in the absence and presence of Ca2 solutions at concentrations of 10, 50, and 100 µM in PBS were prepared by mixing 1, 5, and 10 µL of CaCl2 (1 mM aqueous solution) with 99, 95, and 90 µL of PBS. The samples were prepared as follows:-In the hIAPP sample, 3 µL of the hIAPP stock solution (33 µM) was combined with 7 µL of PBS, yielding a final concentration of 10 µM;-2+ (10 µM) sample, 3 µL of the hIAPP stock solution (33 µM) was combined with 7 µL of CaCl2 (10 µM) in PBS, yielding an hIAPP:Ca2+ molar ratio of 1:1;In the hIAPP + Ca-2+ (50 µM) samples, 3 µL of the hIAPP stock solution (33 µM) was combined with 7 µL of CaCl2 (50 µM) in PBS, yielding an hIAPP:Ca2+ molar ratio of 1:5;In the hIAPP + Ca-2+ (100 µM) samples, 3 µL of the hIAPP stock solution (33 µM) was combined with 7 µL of CaCl2 (100 µM) in PBS, yielding an hIAPP:Ca2+ molar ratio of 1:10.In the hIAPP + CaInitially, CaClSubsequently, 10 µL of each sample was blotted onto a carbon-coated Formvar 300 mesh copper grid for 1 min. The excess sample was removed with filter paper, and the grids were washed. The grids were negatively stained with saturated uranyl acetate for 1 min, dried at room temperature, and then observed under a Morgagni 268 electron microscope  at a voltage of 70 kV.t-test, and fitting procedures) were carried out using GraphPad Prism 3 software (GraphPad PrismTM version 3.0). A value of p < 0.05 was considered significant. The minimum and maximum number (N) and total number (Nt) of individual channel events for each experimental series were reported.Statistical analyses . After different lag times, which depended on the experimental conditions in the different sets, the channel activity of hIAPP was observed at an activation voltage of 100 mV as abrupt, discrete, nonrandom current jumps from the baseline current, consistent with the finding of our previous study [In all experiments, before peptide addition, the PLM was stabilized using voltages of ±80 and ±100 mV consecutively for 10 min each. During the membrane’s stabilization phase, the conductance and capacitance remained at constant values of 26 pS and 0.30 µF/cmus study . The chaus study , the chacis chamber, while in the second experimental set, the same volume of a stock solution of hIAPP preincubated for 24 h at RT was added. In both experimental sets, the final concentration of the peptide was 0.1 µM. After the addition of the stock solutions, the cis chamber medium was stirred for 1 min. The channel activity appeared in both experimental sets at the applied voltage of 100 mV after different lag times, which depended on the preincubation treatment.Two sets of experiments were set up to evaluate the effect of preincubation on hIAPP channel activity. In the first set of experiments, a known volume of freshly prepared hIAPP stock solution (25.6 µM) was added into the medium of the 2) and conductance (26 pS) were observed for a time of 5 h at the applied voltages of ±100 mV.Preliminarily, we tested membrane stability by performing control experiments with the lipid mixture only to exclude nonspecific effects. No changes in the parameters of capacitance . As described above, once the first channel appeared, the voltage was reduced to ±20 mV, and channel amplitude was measured. In this experimental set, the channel activity occurred in the range of applied voltages from ±20 mV to ±100 mV. At all applied voltages, there were periods of quiescence alternating with periods of channel activity.2 (immediately after the addition of the preincubated stock solution) to the value of 0.20 µF/cm2 (about 1 h after adding the hIAPP solution). Immediately after breakage, the PLM was promptly reconstituted with the operator at a voltage of 100 mV, and channel activity appeared after approximately 45 min, manifesting in some experiments as channel activity and in others as paroxystic activity that gradually disappeared and after short periods of quiescence appeared as channel activity. In this set of experiments, channel activity alternated with long periods of quiescence, and this behavior was observed at all applied voltages ranging from ±40 mV to ±100 mV. At applied voltages of ±20 mV, no channel activity was registered for long periods even after the membrane was broken and withdrawn using the operator. In the second experimental set, the channel activity appeared after membrane rupture, preceded by a decrease in capacitance from the initial value of 0.30 µF/cmc ± SE) at the different applied voltages. It is important to note that, at all applied voltages where comparison is possible, Λc values obtained after peptide preincubation are significantly higher than those obtained without preincubation except at the applied voltage of −40 mV.c at a voltage of 40 mV, obtained in the two different experimental conditions, and assuming a cylindrical channel with length (d) 5 nm (corresponding to the membrane thickness) filled with a solution having the same specific conductivity as the external medium, we calculated the channel diameters of hIAPP, which were 6.8 and 7.9 Å without and with preincubation, respectively.In Formula (1), replacing the value of ΛThe channel frequency of hIAPP after 24 h preincubation was significantly lower than that obtained using the freshly prepared peptide stock solution at all applied voltages , indicatTo evaluate the stability of the hIAPP channel in the membrane, we calculated its duration. The duration of the channel ranged from 1.25 to 3.25 s when the peptide was not preincubated, whereas it ranged from 3.5 to 10.25 s when it was preincubated for 24 h, indicating greater channel stability in the membrane.The results obtained from these two experimental sets highlight that the conductance and duration values of the preincubated hIAPP channel were higher, while the frequency was lower than those obtained without preincubation.2 for 24 h at RT.To evaluate the impact of calcium ions on the hIAPP peptide’s ability to incorporate and form channels in POPC PLMs, we conducted three sets of experiments. In each set, the peptide was preincubated with varying concentrations of CaCl2 (control experiments) to exclude the destabilization effects of the Ca2+ ions. Control experiments were conducted by adding CaCl2  into the cis chamber medium. The biophysical parameters (conductance and capacitance) of the PLM were monitored at applied voltages of ±80 and ±100 mV for a time of 10–12 h during which the conductance (25 pS) and capacitance (0.31 µF/cm2) values remained constant, and no channel activity was recorded.Preliminarily, we tested the stability of the membrane in the presence of different concentrations of CaCl2+ molar ratio was 1:1, the channel activity occurred at 100 mV following a lag time of 90 min. The channel activity was monitored in applied voltages ranging from ±20 to ±100 mV. It should be noted that at applied voltages of ±20, ±40, and ±60 mV, the channel activity alternated with periods of quiescence, while at ±80 and ±100 mV, short periods of paroxystic activity alternated with those of channel activity and quiescence. Paroxystic activity, regardless of the applied voltage, progressively disappeared, and channel activity appeared.In preincubation experiments, where the hIAPP:Ca2+ concentration was five times that of the peptide (hIAPP:Ca2+ = 1:5), the lag time was lowered to 60 min. The channel activity occurred at a voltage of 100 mV, and after the occurrence of the first channel, the applied voltage could be lowered down to ±20 mV. The characteristics of the channel activity were like those observed in the previous experimental set. with quiescent periods alternating with those with channel activity and paroxystic activity.In preincubation experiments in which the Ca2+ ion at the concentration of 1 µM (hIAPP:Ca2+ molar ratio = 1:10). The channel activity appeared at 100 mV following a lag time of about 60 min. Also, in the experiments of this set, the channel activity was recorded in applied voltages ranging from ±20 to ±100 mV, manifesting itself with the alternation of periods of channel activity and periods of paroxystic activity, which led to membrane rupture in some experiments.In the third experimental set, we monitored the channel activity of hIAPP that had been preincubated with the CaExamples of chart recordings of the channel activity in the three different experimental conditions are shown in c value, and the channel events counted as successful were used to calculate the frequency. c ± SE) and statistical (F ± SD) parameters of the hIAPP ion channel in the presence of the three Ca2+ concentrations. It is interesting to note that for each Ca2+ concentration, the Λc values are inversely correlated with the applied voltage, decreasing with increasing applied voltage, as found for the Λc values of the peptide alone (both without and with preincubation). For each applied voltage, the Λc values obtained in the presence of the three Ca2+ concentrations are not significantly different, and for each Ca2+ concentration, the frequency values are symmetrical for the negative and positive applied voltages.As carried out for the experiments in the presence of the peptide alone, we fitted the current experimental data with a Gaussian distribution function yielding the Λ2+ concentrations.Similar to the two previous experimental sets (without and with preincubation), we calculated the size of the channel and its duration in the presence of the three Cac at 40 mV in the presence of the three concentrations of Ca2+ in Formula (1), and assuming σ, π, and d to be unchanged, the diameter of the channel was 6.8/6.9/6.3 Å when the preincubation concentrations of the ion were 0.1/0.5/1 µM, respectively.By substituting the values of Λ2+, respectively. These results indicate greater hIAPP channel stability when preincubating with Ca2+ at 1 µM than with preincubation using the other two concentrations of the metal ion.The channel duration was in the range of 1.25–5.25 s/1.25–3.25 s/4.25–10.25 s when the peptide was preincubated with 0.1/0.5/1 µM Cac values in the presence of 0.1, 0.5, and 1 µM of Ca2+ were significantly lower than those of the peptide preincubated 24 h at all applied voltages; the frequency values, at any ion concentration, were far higher than when preincubating only the peptide; the channel diameter values calculated in the presence of Ca2+ were lower than those calculated with the preincubated peptide alone; and the duration of the hIAPP channel depended on the metal ion concentration being lower/similar to that obtained with the preincubated peptide alone when the Ca2+ concentrations were 0.1 and 0.5/1 µM, respectively. On the other hand, the biophysical and statistical parameters of the hIAPP channel obtained in the presence of Ca2+, regardless of the ion concentration used, were similar to those obtained by using the freshly prepared hIAPP solution.The results using the electrophysiological technique showed that the Λ2+ concentration used) than those obtained by preincubating the peptide alone, indicating that preincubation with metal ions favored the formation of aggregates prone to incorporation into the membrane, leading to the formation of ion channels. To verify this result, we conducted fluorescence experiments using the ThT fluorophore. ThT, interacting with the β-sheets of amyloid aggregates, is useful for monitoring the levels of amyloid aggregates and following the state of protein aggregation. We conducted two sets of experiments to measure levels of amyloid aggregates formed by the hIAPP peptide in the absence and presence of varying Ca2+ concentrations after 24 and 72 h of incubation.hIAPP channel frequency values were far higher when preincubating the peptide with the metal ion  show the presence of T0 aggregates, which appear to be more electron-dense after 24 h of incubation; at T72, image C shows the presence of fibrils and aggregates, indicating that the hIAPP aggregation process is a dynamic phenomenon where multiple species can coexist  on the shape of the aggregates is shown in images L, M, and N. It is interesting to note that the amyloid aggregates appear not very extensive and have lower electron density than those formed under the other experimental conditions. Moreover, by T0, small and scattered aggregates can be seen, which decrease in the sample by T24, almost completely disappearing in the sample at T72 where definite and electron-dense spherical structures are present.The amyloid aggregates obtained in the presence of Caon at T0 D, while on at T0 E,F than 2+ incubated with the hIAPP peptide in pH 7.4 phosphate buffer inhibits the formation of fibrils and large aggregates in favor of smaller spherical aggregates. The impact of the ion on the aggregation process seems to be concentration-dependent. Increasing its molar ratio relative to the peptide resulted in the formation of smaller spherical aggregates. These results appear to be in line with those obtained with ThT fluorescence.The results obtained with electron microscopy indicate that CaAmyloidogenic peptides are a class of small peptides that, while not exhibiting sequence homology, have the common feature of forming insoluble amyloid aggregates and fibrils, which are hallmarks of related disorders. Some examples are the insoluble aggregates and fibrils of the Aβ peptide in AD, α-synuclein in Parkinson’s disorder, prion protein (PrPSc) in Creutzfeldt–Jakob disorder (CJD), and hIAPP peptide in T2DM.2+ concentration by altering the permeability of the cell membrane [2+, which reversibly blocks the hIAPP channel at micromolar concentrations, as revealed in the study by Hirakura and collaborators [The formation of insoluble aggregates and fibrils in the absence of catalysts follows a nucleation-dependent polymerization reaction. The lag phase is characterized by the presence of monomers, which, in the subsequent phase called homogeneous nucleation, unfavorably change their conformation from random coils to β-sheets and assemble to form “nuclei”, i.e., oligomers of different sizes. In the next phase, called heterogeneous nucleation or elongation, nuclei of different sizes react with the monomers, leading to the formation of high-molecular-weight insoluble oligomers and protofibrils (growth phase), which evolve toward the formation of mature fibrils (plateau phase) ,2,5. In membrane . Some stmembrane . Severalmembrane ,66,67, aborators . Other mborators .2+, Al3+, Fe2+, and Cd2+ ions were found to promote the formation of insoluble oligomers and protofibrils, while Cu2+ and Hg2+ ions, Ru and V complexes, and some of the Au3+ complexes seemed to inhibit the fibrillation process. Studies of the effect of Ca2+ on the hIAPP aggregation process are lacking. The study conducted by Sciacca and collaborators indicates that Ca2+ promotes the elongation of hIAPP fibrils in the presence of LUVs composed of POPC:POPS = 7:3 and increases membrane fragmentation in a detergent-like manner while inhibiting the interaction of prefibrillar species with the bilayer of LUVs [The aggregation process of amyloidogenic peptides can be influenced by numerous intrinsic and extrinsic factors, including metal ions. The pro- or anti-aggregating effect of metal ions seems to depend on their molar ratio with the peptides and their chemical characteristics. Zn of LUVs .2+, at different molar ratios with the peptide, on hIAPP molecules in solution by carrying out experiments in which the peptide was incubated with the Ca2+ ions or alone (control experiments). By means of the techniques used in this study, the ability of the peptide to interact with the POPC PLMs, the levels of amyloid aggregates, and their morphology in the absence and presence of the ions were monitored.In this work, we evaluated the effect of Ca2+ for 24 h is able to incorporate into POPC PLMs and form ion channels after lag times, which are not significantly different in the experimental conditions used in this study (hIAPP preincubated without or with Ca2+). The channel conductance in the absence and presence of metal ions appears to be dependent on the applied voltage, as found in our previous study [c values of the channel, obtained by preincubating the peptide alone, were higher than those obtained in the presence of Ca2+. Conductance is the biophysical parameter correlated with the ionic current passing through the channel, which in turn is correlated with the channel size. The diameter value (7.9 Å) obtained by preincubating hIAPP in the absence of the three concentrations of Ca2+ indicates that the conductive unit is larger. Our results seem to agree with those obtained by other authors who have found that the structure of the hIAPP channels, determined in DOPC membranes using molecular modeling and molecular dynamics simulations, consists of three to five oligomeric units assembled in the membrane [The results obtained with the electrophysiological technique show that the peptide preincubated in the absence or presence of different concentrations of Caus study . The Λc membrane ,70,71. Tmembrane . The res2+, indicating lower turnover of the conductive unit in the PLM. This result indicates that fewer oligomeric structures are prone to incorporate into PLMs and assemble into conductive units. By contrast, the preincubation of the peptide with Ca2+ seems to increase the number of oligomeric structures prone to interact with the lipid bilayer. However, it is important to note that the channel frequency values obtained in the presence of the three Ca2+ concentrations are not significantly different at all applied voltages, indicating that the hIAPP channel turnover is independent of the metal ion concentration. Our results, albeit indirectly, seem to indicate a nonspecific effect of Ca2+ due to its inability to bind to specific amino acids of the primary structure of the peptide, as demonstrated by the results obtained with the CD technique [2+ ions do not influence the secondary structure of hIAPP [2+ ion, which stabilizes the secondary structure of the peptide in a random coil conformation, making the peptide more flexible and less prone to aggregate [2+ decreases the hydrophobicity of hIAPP oligomers.The frequency of the hIAPP channel was significantly lower when the peptide was preincubated alone than when it was preincubated with Caechnique ,72, as wechnique . Severalof hIAPP ,72 diffeggregate . On the ggregate , indicat2+ ions on the channel activity of hIAPP, we performed single-channel experiments by adding a freshly prepared peptide stock solution to the cis chamber medium. The channel activity occurred after a lag time of 3 h, longer than the lag times observed in the experiments of the preincubated peptide without and with Ca2+, indicating that the molecules of the peptide solubilized immediately before the addition to the cis chamber medium must organize into oligomers before incorporating into the PLM and forming ion channels. The conductance and frequency values at the different applied voltages and the channel diameter appear to be more similar to those obtained from preincubation experiments in the presence of Ca2+ than to those in the absence of the ion. These results indicate that the lack of preincubation or preincubation with Ca2+ favors the incorporation of the peptide into the PLM and the formation of smaller-sized conductive units with a higher frequency than those obtained by preincubating the peptide alone. This result, albeit indirectly, indicates that Ca2+, in an aqueous solution with a peptide, helps to stabilize the smallest oligomers prone to interact with the PLM, disfavoring the elongation process.To evaluate the effect of preincubation and Ca2+ ions, as found by Sciacca and colleagues, who showed that Ca2+ did not significantly reduce the rate of fibril formation [2+ ions on the aggregation process tended to increase, even if not significantly, by increasing the peptide–metal ion ratio from 1/0.33 to 1/1 [2+ ion on the interaction of ThT with the hIAPP peptide cannot be excluded.The results obtained with the electrophysiological technique appear to be in line with those obtained with the ThT assay. The formation of amyloid aggregates was inhibited by Caormation . A simil3 to 1/1 . However2+ depends on the incubation time more than on the peptide–Ca2+ molar ratio. Indeed, after 72 h of incubation, the values of the fluorescence intensity percentage obtained in the presence of the three concentrations of Ca2+ were not significantly different from each other. This result seems to confirm the nonspecific effect of Ca2+, as the results of CD [2+ concentrations. The peptide incubated alone forms amyloid aggregates which over time evolve toward the formation of fibrils coexisting with electron-dense amyloid aggregates (2+ show that the morphology of the aggregates changes drastically, passing from a fibrillar morphology and amyloid aggregates at 10 µM of Ca2+ to an approximately spherical morphology and with small, low electron-dense aggregates at 100 µM of Ca2+ after 72 h of incubation (2+, Zn2+, and Cu2+ [2+ at concentrations of 50 and 100 µM [2+ stabilizes hIAPP aggregates in the form of low-molecular-weight oligomers.Our results show that the antiplatelet effect of Cats of CD ,72 and sgregates A–C. Simicubation D–N. The and Cu2+ . Abedinid 100 µM . Conside2+ and T2DM; they support the notion that Ca2+ could be a risk factor in T2DM as the metal ion promotes the stabilization of small aggregates, which are more cytotoxic than mature fibrils.The results obtained in this study show a link between Ca2+; the morphology of the aggregates observed at the concentrations of Ca2+ 50 and 100 µM was significantly different from that obtained by incubating the peptide alone or at the metal ion concentration of 10 µM; the channel frequency values obtained in the presence of Ca2+ were significantly higher than those obtained by preincubating the peptide alone; and the biophysical and statistical parameters obtained by preincubating the peptide with Ca2+ were similar to those found using the freshly prepared peptide solution.The results obtained in this study using different investigation techniques revealed that the levels of amyloid aggregates decreased when the peptide was incubated with Ca2+ influences the hIAPP aggregation process in solution, and those obtained with the electrophysiological technique indicate that the effect of the metal ion is nonspecific since no correlation was found between the biophysical parameters and the Ca2+ concentration in the concentration ranges used in this study.The results obtained with the ThT assay and TEM indicate that Ca2+ affects the surface hydrophobicity of hIAPP by stabilizing it in the form of small aggregates rather than interacting with specific amino acids of its primary structure, as reported in the literature.Our results, albeit indirectly, seem to support and strengthen the hypothesis that Ca2+ ions, hIAPP peptide, and consequently the pathophysiology of T2DM.The results obtained in this study may help to clarify the link between Ca


However, to be clinically relevant, biomarkers should not only correlate with pathological changes but also with disease stage and progression. Therefore, we aimed to investigate the correlation between topology of [18F]PI-2620 uptake and symptomatology in 4-repeat tauopathies.In recent years in vivo visualization of tau deposits has become possible with various PET radiotracers. The tau tracer [18F]PI-2620-PET. Principal component analysis was performed to identify groups of similar brain regions based on 20–40 min p.i. regional standardized uptake value ratio z-scores. Correlations between component scores and the items of the PSP Rating Scale were explored.72 patients with possible or probable 4-repeat tauopathy, i.e. 31 patients with PSP-Richardson’s syndrome (PSP-RS), 30 with amyloid-negative CBS and 11 with PSP-non-RS/CBS, underwent PI-2620 tau-PET topology to correlate with symptomatology in 4-repeat tauopathies. Longitudinal studies will be needed to address whether a deterioration of signs and symptoms over time can be monitored by [18F]PI-2620 in 4-repeat tauopathies and whether [18F]PI-2620 may serve as a marker of disease progression in future therapeutic trials. The detected negative correlation of tracer binding in the caudate nucleus and thalamus with the signs disorientation and arising from chair may be due to an increasing atrophy in these regions leading to partial volume effects and a relative decrease of tracer uptake in the disease course. As cerebral regions correlating with symptomatology differ depending on the clinical phenotype, a precise knowledge of clinical signs and symptoms is necessary when interpreting [18F]PI-2620 PET results.Our data reveal [ Dependiathology . The cliathology . Due to hies PSP  and CBS hies PSP . Howeverhies PSP . Our pre0 in PSP  and CBS 0 in PSP  indicate22.1The study was performed according to the Declaration of Helsinki (1991). PET data analysis was approved by the local ethics committee . Written informed consent was obtained from every participant.The CBS study cohort is embedded in “Activity of Cerebral Networks, Amyloid and Microglia in Aging and Alzheimer’s Disease (ActiGliA)”, a prospective cohort study at Ludwig-Maximilians-Universität München (LMU), Munich, Germany, approved by the local ethics committee (project number 17–755).2.218F]flutemetamol and [18F]florbetaben PET respectively. The threshold for Aβ42/40 ratio was set at < 5.5% and the threshold for Aβ42 concentration was set at < 375 pg/ml according to standardized laboratory diagnostics at Ludwig-Maximilians-Universität München, Munich, Germany. The control group used for generation of regional tau-PET z-scores consisted of 14 age-matched healthy subjects whose demographic data are provided elsewhere . The protecting group was cleaved under the radiolabelling conditions. The product was purified by semipreparative HPLC. Radiochemical purity was 99%. Non-decay corrected yields were about 35% with a molar activity of 8∙106 GBq/mmol at the end of synthesis.Radiosynthesis of [2.3.218F]PI-2620 PET was performed in a full dynamic 0–60 min setting initiated upon intravenous injection (∼10 s) of 185 ± 10 MBq of the tracer in most of the patients and short imaging windows were used for a subset due to severe disability (11%). PET data were reconstructed iteratively  with a matrix size of 336 × 336 × 109/400 × 400 × 148, a voxel size of 1.018 × 1.018 × 2.027/1.018 × 1.018 × 1.500 mm3 / and a slice thickness of 2.027/ 1.500 mm. The previously evaluated single 20–40 min frame . A low-dose CT scan preceded the PET acquisition and served for attenuation correction. [in frame  was used2.3.318F]PI-2620 images were normalized to MNI space by applying a non-linear transformation . The cerebellum (excluding the dentate nucleus and superior layers) was used as a reference region for scaling of [18F]PI-2620 images. Standardised uptake value ratios (SUVr) of all 246 supratentorial volumes of interest of the Brainnetome atlas were extracted. For further analysis regional SUVrs were converted into z-scores using tau-PET quantification of the healthy control cohort in order to ensure standardized region comparability. Initial judgement of tau-positivity was performed by a visual red of an experienced reader and supervision by an expert reader considering tau-PET signals in the frontal cortex, basal ganglia, substantia nigra and dentate nucleus. Basal ganglia positivity or positivity ≥ of the remaining regions defined the scan as positive.All image data were processed and analysed using PMOD . For spatial normalisation, a tracer-specific 20–40 min template in the MNI space were created as described previously . Based u2.4post hoc Bonferroni corrected ANOVA or Kruskal-Wallis test as appropriate. Chi-square analysis was used to check for significant differences in sex. Standard statistical significance level was set at p < 0.05.Data were analysed using IBM SPSS Statistics for Windows . Demographic as well as clinical data were compared between PSP-RS, CBS and PSP-non-RS/CBS patients by post hoc according to the distribution of cerebral regions. Individual regression-based component scores were calculated  with direct oblimin rotation. Following the advice of Stevens  factor llculated . To visuFalse discovery rate-corrected Spearman’s tests (q < 0.05) were used to explore significant correlations between the calculated individual component scores and the items of the PSPRS in the groups of PSP-RS and CBS patients as well as the whole study sample. Due to the comparatively low sample size and the mixture of clinical phenotypes, no correlation analysis was performed for the subgroup of PSP-non-RS/CBS patients.2.5The data that support the findings of this study are available from the corresponding author upon reasonable request and submission of a formal project outline. Data are not publicly available due to privacy restrictions.3post-hoc Mann-Whitney tests did not show significant pairwise differences. All but 8 patients  were tau positive on visual inspection. Data on β-amyloid status were available in 16/31 PSP-RS patients. In 14 patients a primary AD pathology could be excluded by evaluation of β-amyloid in cerebrospinal fluid or [18F]flutemetamol and [18F]florbetaben PET respectively. Two patients showed a reduced Aβ42/40 ratio.Clinical scorings of the study population are provided in 3.1PCA with direct oblimin rotation applied to the tau-PET data revealed the presence of 26 components with eigenvalues above 1.0. However, 13 of these components contained<3 variables and were consecutively excluded from the analysis, leaving a 13-component solution which explained 85.2% of variance. 64 cerebral regions were excluded as their factor loadings were below 0.4. A detailed description of the different components is provided in In summary, the resulting components comprised the following regions: component #1 = right lateral temporal lobe  (variance explained 41.1%), component #2 = mesial frontoparietal lobes (variance explained 16.9%), component #3 = caudate nucleus/thalamus (variance explained 6.5%), component #4 = medial superior frontal gyrus and adjacent anterior cingulate cortex/orbital gyrus (variance explained 3.9%), component #5 = parahippocampal gyrus (variance explained 3.3%), component #6 = hippocampus/thalamus (variance explained 2.9%), component #7 = inferolateral frontal lobe (variance explained 2.1%), component #8 = left parietotemporal junction (variance explained 1.8%), component #9 = parietooccipital junction (variance explained 1.6%), component #10 = lateral frontoparietal lobes (variance explained 1.5%), component #11 = medio ventral occipital cortex (variance explained 1.4%), component #12 = superior and middle frontal gyrus (variance explained 1.3%), component #13 = basal ganglia (variance explained 1.1%). Regions of confluent tau-PET signal as identified by PCA do not necessarily reflect the regions of highest tau load compared to the control group. For comparison, 3.2sr = 0.299, p = 0.011 and sr = 0.310, p = 0.008) and #4 . Furthermore, postural instability was positively correlated with component #2 . While the signs disorientation and bradyphrenia showed a positive correlation with component #9 , disorientation and arising from chair were negatively correlated with component #3 . A schematic illustration of the correlation between regional tau-tracer uptake and clinical signs is given in Correlation analyses performed within the whole study sample showed a positive correlation of the signs arising from chair and gait respectively with component #2  and a trend towards a negative correlation with component #3  but did not survive the significance threshold for multiple comparisons .Fig. 5Cosr = 0.354, p = 0.002) and #13  and negatively correlated with component #3 . The MoCA showed a positive correlation with component #3  while the SEADL showed a negative correlation with component #2  and a positive correlation with component #3 . No significant correlation with the calculated component scores could be detected for disease duration and the duration of education.Furthermore, age was positively correlated with component #1 , could be detected. Furthermore, apraxia of hand movement was negatively correlated with component #3  and #8 . Arising from chair showed a positive correlation with component #1  while gait showed a positive correlation with component 4 .In PSP-RS patients a negative correlation of the sign disorientation with component #7 , gait , postural instability  and sitting down , as well as with total PSPRS  could be detected. Arising from chair was also positively correlated with component #4 .In CBS patients a negative correlation of component #3  with the signs arising from chair .18F]PI-2620  under Germany’s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy – ID 390857198).Sonja Schönecker: Conceptualization, Formal analysis, Writing – original draft. Carla Palleis: Resources, Writing – review & editing. Nicolai Franzmeier: Writing – review & editing, Visualization. Sabrina Katzdobler: Resources, Writing – review & editing. Christian Ferschmann: Resources, Writing – review & editing. Sebastian Schuster: Resources, Writing – review & editing. Anika Finze: Resources, Writing – review & editing. Maximilian Scheifele: Resources, Writing – review & editing. Catharina Prix: Resources, Writing – review & editing. Urban Fietzek: Resources, Writing – review & editing. Endy Weidinger: Resources, Writing – review & editing. Georg Nübling: Resources, Writing – review & editing. Jonathan Vöglein: Resources, Writing – review & editing. Marianne Patt: Methodology, Writing – review & editing. Henryk Barthel: Methodology, Writing – review & editing. Osama Sabri: Methodology, Writing – review & editing. Adrian Danek: Resources, Writing – review & editing. Günter U. Höglinger: Resources, Writing – review & editing. Matthias Brendel: Conceptualization, Methodology, Writing – review & editing, Funding acquisition. Johannes Levin: Resources, Writing – review & editing, Funding acquisition, Supervision, Project administration.Johannes Levin reports speaker fees from Bayer Vital, Biogen and Roche, consulting fees from Axon Neuroscience and Biogen, author fees from Thieme medical publishers and W. Kohlhammer GmbH medical publishers, non-financial support from Abbvie and compensation for duty as part-time CMO from MODAG and being beneficiary of the phantom share program of MODAG GmbH, outside the submitted work. Matthias Brendel received speaker honoraria from Roche, GE healthcare and Life Molecular Imaging and is an advisor of Life Molecular Imaging. Sabrina Katzdobler has received travel support from Life Molecular Imaging. Osama Sabri receives research support from Life Molecular Imaging. Henryk Barthel received speaker honoraria from Novartis/AAA and reader honoraria from Life Molecular Imaging. Nicolai Franzmeier received research funding from Avid Radiopharmaceuticals and consulting fees from Merck.


V1.1 serves as the voltage sensor for both excitation‐contraction coupling (ECC) and L‐type Ca2+ channel activation. We have recently adapted the technique of action potential (AP) voltage clamp (APVC) to monitor the current generated by the movement of intramembrane voltage sensors (IQ) during single imposed transverse tubular AP‐like depolarization waveforms (IQAP). We now extend this procedure to monitoring IQAP, and Ca2+ currents during trains of tubular AP‐like waveforms in adult murine skeletal muscle fibers, and compare them with the trajectories of APs and AP‐induced Ca2+ release measured in other fibers using field stimulation and optical probes. The AP waveform remains relatively constant during brief trains (<1 sec) for propagating APs in non‐V clamped fibers. Trains of 10 AP‐like depolarizations at 10 Hz (900 ms), 50 Hz (180 ms), or 100 Hz (90 ms) did not alter IQAP amplitude or kinetics, consistent with previous findings in isolated muscle fibers where negligible charge immobilization occurred during 100 ms step depolarizations. Using field stimulation, Ca2+ release did exhibit a considerable decline from pulse to pulse during the train, also consistent with previous findings, indicating that the decline of Ca2+ release during a short train of APs is not correlated to modification of charge movement. Ca2+ currents during single or 10 Hz trains of AP‐like depolarizations were hardly detectable, were minimal during 50 Hz trains, and became more evident during 100 Hz trains in some fibers. Our results verify predictions on the behavior of the ECC machinery in response to AP‐like depolarizations and provide a direct demonstration that Ca2+ currents elicited by single AP‐like waveforms are negligible, but can become more prominent in some fibers during short high‐frequency train stimulation that elicits maximal isometric force.In skeletal muscle, Ca 2+ release, and Ca2+ ionic currents evoked by transverse tubular action potential (AP)‐like waveforms in skeletal muscle fibers. They show constant Q(t) activation but decreased Ca2+ release and in some fibers increasing Ca2+ currents during short high‐frequency bursts of AP‐like depolarizations.Bibollet et al. investigate voltage‐sensing charge movement (Q(t)), sarcoplasmic reticulum Ca Skeletal muscle cells are electrically polarized at rest, with an intracellular negative voltage with respect to the extracellular environment. During skeletal muscle activity, when the AP is initiated by a local depolarization at the neuromuscular junction of a skeletal muscle fiber, it propagates both longitudinally along the fiber length via the sarcolemma (surface AP) and inwardly into the fiber via the radial spread of the transverse tubule (TT) system (tubular AP), invaginations of the surface membrane  within the TT membrane  membrane  of skeletal muscle  to characterize the AP‐like induced Ca2+ influx in mammalian muscle. Here, we evaluated the properties of the ECC‐related voltage‐sensing current, the Ca2+ transient and the underlying Ca2+ release flux that it evokes from the RyR1s, and unexpectedly, demonstrate Ca2+ current activation only in a fraction of the fibers studied during trains of tubular AP‐like waveforms.Until recently  were used. A total of 35 C57BL/6J male mice were used. All mice were between 30 and 60 days of age. Environmental conditions were maintained with a 12‐h light/dark cycle and constant temperature (21–23°C) and humidity (55 ± 10%). The cages contained corncob bedding (Harlan Teklad 7902) and environmental enrichment (cotton nestlet). Mice were supplied with dry chow  and water ad libitum.2.22 followed by cervical dislocation. FDB muscle was isolated and enzymatically dissociated with collagenase type I  in S‐MEM  with 10% FBS, and 50 μg/mL gentamicin for 3 h ± 15 min at 37°C in 5% CO2. Muscle was then gently triturated to separate fibers. Individual fibers (20–50 fibers per dish) were plated in MEM media  with 10% FBS on glass‐bottomed dishes  coated with laminin . Fibers were maintained in culture for 14 to 18 h at 37°C, 5% CO2 to facilitate attachment to substrate and studied within the first 20 h after isolation to avoid dedifferentiation effects  was carried out as previously described  with the potentiometric dye Di‐8‐ANEPPS in intact non‐clamped fibers, which were activated by field stimulation. Importantly, the trajectory of di‐8‐ANNEPS line scan signal monitors voltage changes across the TT system, which is characterized by its own repertoire of ion channel and passive properties when compared to surface sarcolemma : 150 TEA‐CH3SO3, 10 HEPES, 1 MgCl2, 0.001 tetrodotoxin (TTX), 0.5 4‐aminopiridine (4‐AP), 0.025 BTS (N‐benzyl‐p‐toluene sulphonamide), pH adjusted to 7.4 with CsOH, osmolarity of 295 mOsm. Where noted, 0.5 mM Ca2+ plus 1.5 mM Cd2+ or 2 mM Ca2+ were added to the external solution to measure non‐linear currents or Ca2+ currents, respectively. This external solution was used for impalement. The current injecting electrode (V1) and voltage sensing electrodes (V2) were filled with a solution consisted of (in mM): 100 Cs‐CH3SO3, 20 EGTA, 6 MgCl2 (free [Mg2+] ~ 1 mM), 11.5 CaCl2 (free [Ca2+] ~ 100 nM), 5 ATP disodium, 5 phosphocreatine disodium, 10 HEPES, pH 7.4 (CsOH); osmolarity of 314 mOsm. Microelectrode V1 was placed at the middle of the selected fiber, and V2 was positioned halfway between the middle and the end of the selected fiber. This arrangement allows for symmetrical current injection via V1, and V2 measures average Vm between the middle and end of the fiber.To measure non‐linear capacitive currents, Caout) and charge moving in (Qin) during AP repolarization were quantified as previously reported , HS‐9A x1 headstages were used for V1 and V2, and borosilicate glass . The resistance of the current injecting electrode was 3–8 MΩ and 10–15 MΩ for the V‐sensing electrode when filled with this internal solution. Once the fibers were impaled with both microelectrodes, cells were held at −80 mV. Fibers with signs of clamp error, such as unstable holding current (or >−20 nA) or rapid drifts on holding potential, were rejected from the analysis. Measurements started 10 minutes after TEVC clamp configuration was established. Voltage protocols were generated, and current responses were digitized and stored in Clampex and Clampfit . Command pulses were delivered at 15 s intervals to the levels and duration indicated in each figure from a holding potential of −80 mV, unless otherwise indicated. Currents were typically low‐pass‐filtered at 3–10 kHz (3‐pole Bessel filter). Currents were sampled at 50 kHz. Linear capacitive and ionic currents were routinely subtracted by a P/−4 protocol , a membrane‐permeable non‐ratiometric high affinity Ca2+ indicator , and 4‐[2‐(6‐(dioctylamino)‐2‐naphthalenyl) ethenyl]‐1‐(3‐sulfopropyl)‐, inner salt (di‐8‐ANEPPS), a membrane‐impermeable potentiometric dye . Fluo‐4 am and di‐8‐ANNEPS were dissolved in Pluronic F‐127  2.5% solution in DMSO. Stock solutions were prepared for TTX , 4‐AP , CdCl2 , and BTS . Drugs and toxins were added to the TEA‐based or Ringer's solution from the stock solutions. The final DMSO and ethanol concentrations were 1:1000 or 1:2000.We used Fluo‐4 acetoxymethyl (fluo‐4 2.5am (2 μM for 60 min at 22°C) in 1 mL of L‐15 media  supplemented with 0.25% w/v bovine serum albumin . Then, the fibers were washed thoroughly with plain L‐15 media to remove residual fluorescent dye. Di‐8‐ANEPPS dye staining and AP recordings were performed as previously described . Confocal imaging of fluo‐4 (20 μs/line) and di‐8‐ANEPPS (20 μs/line) were carried out independently using high‐speed confocal system LSM 5 Live system (Carl Zeiss). Fluo‐4 was excited with a 488 nm laser, and the fluorescence emitted >505 mm was detected. Di‐8‐ANEPPS stained fibers were excited with a 532 nm laser; emitted light was collected with a 550 nm LP filter. Fibers stained or loaded with di‐8‐ANEPPS or fluo‐4, respectively, were viewed on a Zeiss Axiovert 200 M inverted microscope and confocal imaging was performed in line scan xt mode as previously described . Average intensity of fluorescence within the selected optical field was recorded and background corrected by subtracting an average value recorded outside the cell. The average F0 value in each trace before electrical stimulation was used to scale fluorescent signals  in the same field as ΔF/F0 or −ΔF/F0. Line scan was positioned perpendicular to the main axis of the fiber, as close as possible to its center. Region of interest (ROI) used to obtain F vs. time profiles was placed in the center of the line scan with a × dimension of 42 pixels at 0.20 μm/pixel.Electrical field stimuli were applied via two parallel platinum wires closely spaced (5 mm). Application of each stimulation protocol was synchronized relative to the start of fluorescence acquisition. Each stimulating pulse  was delivered alternating the polarity using a custom‐made pulse generator. Only fibers responding with action potential‐induced twitch responses for both polarity stimulation before and after staining were studied  and parvalbumin‐like Ca2+‐Mg2+ sites (P‐sites), as well as fast Ca2+ binding to ATP was calculated using binding site concentrations, rate constants, and approximations adopted from previous reports , and uptake] were summed, and the Ca2+ release flux was calculated as the time derivative of the sum  were used to estimate the time course of the free [Ca2.8F/F0 or −ΔF/F0 values. Data were then analyzed and plotted using OriginPro 2021 (OriginLab Corporation). F (t) records from di‐8‐ANEPPS and fluo‐4 were acquired at 50 KHz and were smoothed using adjacent‐averaging function with a 25 points window. The precision and synchronization between the LSM 5 Live confocal and voltage clamp amplifier was corroborated as previously reported . Visual Basic (Microsoft) macros were used to systematically determine the Δ2.9t = 0 to t = 10–30 ms elicited by single AP or repetitive AP waveforms. Width of both non‐linear current and charge was quantified as the width at 50% of its maximum amplitude. Qout was quantified by integration of the non‐linear current signal, beginning at the initiation of the depolarization, passing by the maximum outward part of the signal and ending when non‐linear current was equal to zero, just before becoming a negative value. Qin was evaluated from the running integral, from its maximum to the time when it crossed the x‐axis.Data analysis was performed using Clampfit 8.0 (Molecular Devices). Further data evaluation, non‐linear fitting, and statistical analysis were conducted using OriginPro 2021 software. Q‐t relationship of each individual fiber was obtained by integration of the capacitive current from 2.10FDB in vivo force measurement was assessed as previously described  was used through the analysis.To avoid one source of systematic bias, experimental and control measurements were alternated in a randomized manner. Experimenters were not blinded to the source of the cells or recording conditions evaluated. Normal distribution of data was assessed using the Kolmogorov–Smirnov test. Summary data were reported as mean ± SD. Summary plots displayed as box and whiskers show mean (white square), standard deviation (whisker), median , 1st and 3rd quartile (box delimitation), and individual data (black dots). Statistical significance was assessed using either parametric two sample 33.1V1.1 functions as a Ca2+ ion channel, but because of its slow activation kinetics its contribution to ECC appears to be irrelevant  in skeletal muscle has not been previously explored. In neurons and cardiac cells, Ca2+ currents are not measurable during the rising phase of the AP. Instead, it is during the falling phase of the AP when the Ca2+ current (deactivation) can be measured  or in the presence Cd2+  to suppress the Ca2+ currents. Note that this tubular AP‐like depolarization tracks the averaged signal obtained from tubular di‐8‐ANEPPS measurements in multiple fibers  and in (Qin) in response to AP‐like waveform were similar whether or not the Ca2+ channel blocker Cd2+ was included in the recording chamber  continues to be increasingly negative after the AP reflects the presence of a very small but maintained negative non‐linear ionic current continuing after AP termination . Note the slow activation kinetics of the CaV1.1 ionic current  by the peak release elicited by the first AP during the train (Rel1st). Fractional inactivation was calculated as 1 − (RelAP#/Rel1st)  external field stimuli  Figure . Fractio) Figure . Next, t) Figure . The for3.42+ release in response to trains of APs via field stimulation, we then asked whether non‐linear currents and charge movement are also affected by repetitive AP‐like waveforms. Charge movement reduction or immobilization could, in principle, account for the suppressed Ca2+ release seeing during repetitive firing , and 10th AP in the train (blue) for each frequency evaluated.Having characterized the properties of the Ca2+ release during short trains of APs as seen in Figure When doing the running integral for the first 10 ms of these signals  in the external solution. While we showed in Figure 2+ current activation is negligible during a single AP‐like waveform, we next investigated if repetitive AP‐like activation could lead to Ca2+ current facilitation  measured in the presence of 2 mM Ca2+ without Cd2+ For comparison, non‐linear current recordings in presence of Cd2+ are shown in Supporting Information Figure p = 0.233, Scheffe post hoc), while decay at 100 Hz was slower and significant when compared to 10 (p = 5.87E‐5 Scheffe post hoc), or 50 Hz . The average decaying time constant was 1.37 ± 0.40 ms at 10 Hz, vs. 2.66 ± 1.74 ms at 50 Hz and 5.63 ± 2.74 ms at 100 Hz. Subsequently, we measured the non‐linear current 10 ms after each AP‐like depolarization to track the ionic current evolution within the train  or maximal force (100 Hz). This observation corroborates previous findings showing that maximal force is obtained at approximately 80–150 Hz, in other predominantly fast‐twitch locomotor muscles , or derived from differences in muscle fibers type    of APs. However, the amount of Ca2+ released with subsequent APs was progressively decreased when compared to that evoked by the first AP in the train, being more pronounce when using higher frequency stimulation. Repeated activation of muscle can affect the AP features ‐like waveforms in skeletal muscle fibers. They show constant Q(t) activation but decreased Ca2+ release, and in some fibers, increasing Ca2+ currents during short high‐frequency bursts of AP‐like depolarizations. Our results confirm previous observations related to charge movement and Ca2+ release using step depolarizations in skeletal muscle but also provide new information about multiple early steps critical for ECC and Ca2+ homeostasis using more physiological stimuli, the muscle fiber AP‐like waveform. Determining these parameters using AP‐like waveforms instead of step depolarizations could be of high relevance for a fundamental understanding of skeletal muscle excitability, Ca2+ release, and Ca2+ homeostasis and establishing more accurate numerical models aiming to predict changes in ECC caused by physiological adaptations or disease conditions.We investigated voltage‐sensing charge movement (Q(t)), sarcoplasmic reticulum CaH. Bibollet designed and performed most of the experiments, analyzed data, and edited the manuscript. E.L. Nguyen, D.R. Miranda A.A. Voss, and C.W. Ward designed and performed experiments, and analyzed data. E.O. Hernández‐Ochoa and M.F. Schneider conceived the project and designed the research. A. A. Voss, M.F. Schneider, and E.O. Hernández‐Ochoa wrote the paper.The authors declare no competing financial interests.All vertebrate animal experiments were performed in accordance with the Animal Welfare Act and the Office of Laboratory Animal Welfare regulations, NIH, USA. The University of Maryland Institutional Animal Care and Use Committee approved all animal protocols.Supplemental Figure S1.Click here for additional data file.Supplemental Figure S2.Click here for additional data file.Supplemental Figure S3.Click here for additional data file.Supplemental Table S1.Click here for additional data file.