In vivo study showed similar results as those seen in vitro. Abivertinib in combination with venetoclax could significantly prolong the survival time and reduce the tumor burden of MV4‐11‐NSG mice compared with those of control and single‐agent groups. Our in vitro and in vivo studies have shown that the combination of abivertinib and venetoclax may benefit AML patients, especially in patients resistant to venetoclax or those that relapse. New clinical trials will be planned.B‐cell lymphoma 2 (BCL‐2), a crucial member of the anti‐apoptotic BCL‐2 family, is frequently dysregulated in cancer and plays an important role in acute myeloid leukemia (AML). Venetoclax is a highly selective BCL‐2 inhibitor that has been approved by the FDA for treating elderly AML patients. However, the emergence of resistance after long‐term treatment emphasizes the need for a deeper understanding of the potential mechanisms of resistance and effective rescue methods. By using RNA‐seq analysis in two human AML cohorts made up of three patients with complete remission and three patients without remission after venetoclax treatment, we identified that upregulation of BTK enabled AML blast resistance to venetoclax. Interestingly, we found that abivertinib, an oral BTK inhibitor, could synergize with venetoclax to inhibit the proliferation of primary AML cells and cell lines. It is worth noting that the combination of the two effectively enhanced the sensitivity of two AML patients (AML#3 and AML#12) to venetoclax. In this study, we demonstrated that combined use of the two drugs can synergistically inhibit the colony‐forming capacity of AML cells, arrest the AML cell cycle in the G0/G1 phase, and inhibit the BCL‐2 anti‐apoptotic family protein, activating the caspase family to induce apoptosis. Mechanistically, knockdown of BTK in AML cell lines impaired the synergistic effect of the two drugs.  Abivertinib could rescue AML insensitive to venetoclax‐involved therapy. AMLacute myeloid leukemiaBCL‐2B‐cell lymphoma 2BTKBruton tyrosine kinaseDLBCLdiffuse large cell lymphomaFCLfollicular cell lymphomaFLT3‐ITDFMS‐like tyrosine kinase‐3‐internal tandem duplication mutationsNSGNOD‐SCID gamma1Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the block of differentiation and clonal proliferation of myeloid precursor cells leading to the disruption of normal hematopoiesis . DespiteThe B‐cell lymphoma 2 (BCL‐2) protein plays an important role in the survival and persistence of AML blasts, as it is a key regulator of the mitochondrial apoptotic pathway , 8. Venein vitro and in vivo in combination with homoharringtonine [On the other hand, BTK inhibitors have been widely applied in hematologic malignancies, including mantle cell lymphoma, DLBCL, and FCL , 24, 25.ngtonine .in vitro and in vivo studies have shown that abivertinib and venetoclax work synergistically in treating AML, which implies a promising chemo‐free therapy for elderly AML patients and those who are not tolerant to chemotherapy.As we know, chemo‐free small‐molecule inhibitor therapy is the future trend, but the efficacy of single‐agent therapy is very limited, so the combination of small‐molecule targeted drugs may benefit more AML patients. In this AML study, we considered two drugs, a combination of two oral small‐molecule inhibitors, which have achieved good results. Our 22.1Phosphorylated and total BTK (Tyr223), AKT (Ser473), PI3K (p110α), PLCγ2 (Tyr759), IKK (Ser176/180), NF‐κB (Ser536), CDK2, CDK4, CDK6, caspase‐3, caspase‐8, PARP, Bad, Bax, BCL‐2, BCL‐XL, and GAPDH antibodies were purchased from Cell Signaling Technology . A human CD45 antibody was purchased from Abcam . Abivertinib was synthesized by Hangzhou ACEA Pharmaceutical Research Co., Ltd . Venetoclax was purchased from Selleck Chemicals .2.2MV4‐11 and MOLM‐13, human AML cells harboring FLT3 internal tandem duplication (ITD) mutations, were a kind gift from Professor R. Bhatia  and were cultured in Iscove's modified Dulbecco's medium supplemented with 10% FBS. KG‐1 and THP‐1 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. These cells were cultured in RPMI 1640 medium supplemented with 10% FBS.2.3FLT3‐ITD, NPM1, DMNT3A, IDH1, IDH2) was performed at the First Affiliated Hospital of Zhejiang University, Hangzhou, China. Cord blood CD34‐positive hematopoietic stem cells were provided by healthy donors. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University, China. The study methodologies conformed to the standards set by the Declaration of Helsinki.Bone marrow and peripheral blood samples were obtained from AML patients following written informed consent. Mononuclear cells were isolated by Ficoll‐Hypaque  density gradient centrifugation. Testing for gene mutations (2.43 cells/well) in triplicate and treated with abivertinib (156 nm), venetoclax (50 nm), or their combination for 14 days. Cell colonies were stained with 0.05% crystal violet solution for 30 min and counted.Three percent soft AGAR was diluted to 1% for the lower layer and 0.4% for the upper layer. AML cells were seeded in 6‐well plates (0.5–1 × 102.53–1 × 104 cells/well) in triplicate and treated with different drugs  for 24 h. Twenty microliters of MTS solution  (5 mg·mL−1) was added to each well followed by incubation for an additional 4 h at 37 °C. Cell numbers were assessed based on the quantification of formazan by determining the absorbance at 490 nm.Cells were seeded in 96‐well plates . After treatment with drugs for 24 or 48 h, the cells were washed twice with PBS, resuspended in binding buffer, and incubated with Annexin V‐FITC and propidium iodide (PI) for 15 min. Apoptotic cells were analyzed by flow cytometry using a FACScan™ flow cytometer .2.7−1 PI and 100 μg·mL−1 RNase A for 30 min at room temperature. Cell cycle analysis was conducted using a FACScan™ flow cytometer (Becton Dickinson).After treatment with drugs for 24 or 48 h, cells were harvested and fixed overnight with 75% ethanol at 4 °C, followed by two PBS washes and incubation in a buffer containing 50 μg·mL2.8g for 15 min at 4 °C. Western blotting was performed after 10% SDS/PAGE , with the cellular proteins transferred onto a preactivated PVDF membrane . The membranes were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies overnight at 4 °C. After incubation with primary antibodies, the blots were washed thrice with TBST buffer, and membranes were incubated with secondary antibodies  for 1 h at room temperature. The target proteins were visualized using an ECL detection kit  and analyzed using image lab™ software .Cells were lysed in radioimmunoprecipitation buffer  on ice for 30 min. The protein concentration of the cellular supernatant was determined using BCA reagent after centrifugation of the cell lysate at 12 000 2.95/well in 6‐well plates. About 100 nm siRNA and 5 μL Lipofectamine were mixed in 500 μL Opti‐MEM (Fisher Scientific) and incubated at room temperature for 20 min before adding the transfection mix to cells. Cells were then incubated at 37 °C for 72 h.The siRNAs against BTK and MCL‐1 were purchased from Invitrogen  and were transfected into THP‐1 cells using Invitrogen™ Lipofectamine™ RNAiMAX , according to the manufacturer's instructions. In brief, the cells were seeded at a density of 5 × 102.10Total RNA was extracted from the cells using TRIzol reagent according to manufacturer's instructions. Reverse transcription was performed using the RNA PCR core kit . Quantitative real‐time PCR was carried out using SYBR Green qPCR Master Mix, and GAPDH was used as an internal control. The sequences of the primers were as follows: GAPDH forward, 5′‑GGAGCGAGATCCCTCCAAAAT‑3′, and reverse, 5′‑GGCTGTTGTCATACTTCTCATGG‑3′; BTK forward, 5′‑TCTGAAGCGATCCCAACAGAA‑3′, and reverse, 5′‑TGCACGGTCAAGAGAAACAGG‑3′; and MCL‐1 forward, 5′‑TGCTTCGGAAACTGGACATCA‑3′, and reverse, 5′‑TAGCCACAAAGGCACCAAAAG‑3′.2.11Bone marrow mononuclear cells were collected from six AML patients treated with the ‘VA’ program (BCL‐2 inhibitor venetoclax combined with azacitidine). Among them, three patients had achieved complete remission and three patients had not responded to the therapy. The RNA sequencing was performed by Annuoda Company , and the expression levels related to the TEC family were analyzed.2.12–dark cycle, kept under normal room temperature, and fed standard pellet food and tap water.NOD‐SCID gamma (NSG) mice were purchased from Shanghai SLRC Laboratory Animal Center. Our animal study was approved by the Ethics Committee for Laboratory Animals of the First Affiliated Hospital, College of Medicine, Zhejiang University , and was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were exposed to a 10/14‐h light6 MV4‐11 cells were injected into the NSG mice via the tail vein. After 7 days, cell engraftment was assessed by the expression of human CD45 by PBMCs of mice each week. Mice were randomly assigned to four groups according to the expression of human CD45. The four groups were treated as follows: 0.5% methylcellulose , 50 mg·kg−1 abivertinib , 75 mg·kg−1 venetoclax , or 50 mg·kg−1 abivertinib and 75 mg·kg−1 venetoclax (administered per the single‐agent groups). The mice were humanely sacrificed after observation of typical leukemic symptoms. PBMC, bone marrow, and spleen samples were harvested for flow cytometry analysis and IHC. Human CD45 antibody was used to identify engraftments.For the MV4‐11‐NSG xenograft model , 2 × 10The study methodologies conformed to the standards set by the Declaration of Helsinki.2.13graphpad prism 6.0 software . Summary statistics (mean ± SD) are represented with statistical significance assessed using the Mann–Whitney test . Survival was analyzed using the Kaplan–Meier method and analyzed using a log‐rank test.Data were analyzed using 33.1To explore the potential mechanisms of resistance to venetoclax in AML patients, we applied paired RNA sequencing of the bone marrow mononuclear cells of six patients who received venetoclax treatment, three of whom went into complete remission and three who did not. The results demonstrated that there were multiple gene expression differences between the two sets of samples, and we noticed a significant difference in the expression levels of BTK Fig. . To furt3.2According to the results above, we were inspired to treat AML cells with BTK inhibitors and BCL‐2 inhibitors synchronously. We investigated the proliferation inhibitory effects of increasing concentrations of abivertinib and venetoclax alone or in combination on AML cell lines. As shown in Fig. At the same time, in order to evaluate the safety of the combined use of the two drugs, we treated the CD34‐positive hematopoietic stem cells derived from healthy cord blood with these two drugs. As shown in Fig. 3.3To investigate the efficacy of abivertinib combined with venetoclax on the proliferation of AML cells, we first evaluated their effect on AML clone formation ability using a soft agar colony formation assay. As shown in Fig. Next, we exposed AML cells to abivertinib, venetoclax, or their combination for 24 h followed by flow cytometry detection of cell cycle distribution. The results in Fig. Considering that venetoclax is an inhibitor of anti‐apoptotic protein BCL‐2 that targets the apoptotic pathway, we next focused on whether the combination of the two drugs could cause a synergistic induction of apoptosis. First, we used flow cytometry to detect the proportion of apoptotic cells among AML cells treated with single‐agent or combination therapy. The results Fig.  showed t3.4To further explore the mechanism of synergistic function between abivertinib and venetoclax, we measured the protein expression levels of BTK Fig.  and its We hypothesized that the synergistic effect of these two drugs is associated with the ability of abivertinib to downregulate the expression of BTK or MCL‐1, which are upregulated by venetoclax treatment. To confirm that, we knocked down BTK and MCL‐1 in AML cells (THP‐1 and MV4‐11) by siRNA technology as shown in Fig. 3.5in vivo were investigated in MV4‐11‐NSG xenograft mice. All the drug administrations referred to started 7 days after injection of MV4‐11 cells by tail vein. All mice had equal tumor burdens, measured by testing for human CD45 in PBMCs at the beginning of therapy. In the following days, we tested the levels of human CD45 PBMCs of the mice in each group every week to evaluate the tumor burden of different treatment groups. Mice treated with a combination of abivertinib (50 mg·kg−1) and venetoclax (75 mg·kg−1) had a significantly lower leukemia burden during the treatment period compared with that of those treated with vehicle or only a single drug . The median survival time of the vehicle group was 25 days, and the abivertinib and venetoclax single‐drug groups were 28.5 and 29 days, respectively, while the combined group was 34 days , but the difference was not significant Fig. . Next, wIn recent years, in order to rescue the clinical failure of venetoclax treatment in AML patients, many medical centers have carried out clinical trials involving in venetoclax‐based therapy, particularly when combined with azacitidine. Fortunately, most of them have achieved very good results; however, some patients are still unable to achieve remission. Even more troubling is that the mechanism behind the effectiveness of this treatment regimen is unclear, and basic research in this area is still lacking. Therefore, our study suggests that a dual oral regimen may lead to a promising chemo‐free therapy for those patients.et al. [−1. Moreover, abivertinib had a well‐tolerated safety profile and promising antitumor activity in patients with NSCLC with acquired resistance to a first‐generation EGFR TKI, supporting its continued development [FLT3‐ITD mutation, one case carrying DMNT3A mutation), sensitive to the combination of abivertinib and venetoclax. The combined index ED50 < 1 also indicated a synergistic effect. Our results in cell lines and AML blasts indicate that the combination treatment shows a potential inhibitory effect in a wide‐type manner, whether they are carrying the FLT3‐ITD mutation or are hard to deal with in clinical practice. Since the safety of the two drugs alone has been clinically recognized, our study simultaneously evaluated both drugs in CD34‐positive healthy cord blood stem cells and found no significant damage. Presumably, this also paves the way for the clinical implementation of this treatment.In our study, we treated AML with BTK inhibitor in combination with venetoclax based on our preliminary results. We picked a novel BTK inhibitor, abivertinib, which is currently undergoing phase II clinical trials for lymphoma in our center. In a study by Xu et al. , they shelopment . Our groelopment , 31. In In the existing research, many scholars have also tried other drugs combined with venetoclax to treat preclinical AML. For example, Professor Michael R. Savona focused on the apoptosis family pathway (plus some others) to illustrate the synergistic effect of an MCL‐1 inhibitor and venetoclax . In thisNext, in order to verify whether the combined effect of the two agents depends on BTK, we performed a drug sensitivity test on the AML cell lines with or without BTK knockdown, and the results showed that the synergistic effect of the two drugs diminished in the AML cells with impaired BTK expression. Because of this, we proposed that the synergy between abivertinib and venetoclax in AML is dependent on the level of BTK. When BTK expression is downregulated, abivertinib fails to synergize with venetoclax to suppress AML cells. In addition, it is worth mentioning that the sensitivity of AML cells to venetoclax increases after downregulation of BTK, which may be the reason for the disappearance of the joint effect. Therefore, in order to rule out the important role of MCL‐1 in the combined action of these two drugs, we also performed MCL‐1 knockdown followed by drug susceptibility tests on AML cells, but the combined effect of the two did not change significantly. Therefore, in this study, we confirmed that abivertinib synergized with venetoclax to block AML progression in a BTK‐dependent manner.in vivo experiments have also been initially explored. According to our results, the combination of abivertinib and venetoclax can significantly prolong the survival time of MV4‐11 mice and reduce the tumor burden at the same time. Even more interestingly, the antileukemia effect in the combination group is much better than that in any single‐drug treatment group. Similar results were observed in THP‐1‐luci‐NSG xenograft mice. The results showed in Fig. in vivo experiment can also explain the significant antileukemia effect of the combination of the two drugs, and we are confident in their use for the clinical treatment of AML patients who do not respond to venetoclax treatment.In our study, 5in vivo and in vitro, especially in patients who did not achieve remission with venetoclax‐involved treatment.Our results showed that the combination of abivertinib and venetoclax can inhibit the progression of AML The authors declare no conflict of interest.SH conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript; CL, XZ, YL, JP, FL, JH, QL, WY, SM, and XH performed the experiments, analyzed the data, and participated in the manuscript preparation; and JJ supervised the study, contributed to the design of experiments, analyzed the data, and revised the manuscript.Our animal study was approved by the Ethics Committee for Laboratory Animals of the First Affiliated Hospital, College of Medicine, Zhejiang University , and was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Fig. S1. The combination curve of AML cells treated by abivertinib and venetoclax.Fig. S2. The expression of MCL1 in AML patients treated with ‘VA’ therapy.Fig. S3. The protein level of PLCγ2，ERK and NF‐κB at downstream of BTK signal was measured after different treatments in AML cells.Fig. S4. The effect of MCL‐1 impaired expression to the combination of abivertinib and venetoclax.Fig. S5. IHC of femurs and spleen of mice from different groups.Fig. S6. Leukemia burden of THP‐1‐luciferase AML mice in different treatment groups.Table S1. The combination index of AML cells treated by abivertinib and venetoclax.Click here for additional data file.


An important player of this crosstalk is the Na+/Ca2+ exchanger (NCX), however, direct pharmacological evidence was unavailable so far because of the lack of a selective inhibitor. We investigated the role of the NCX current in pacemaking and analyzed the functional consequences of the If-NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN).The exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca2+-transients were monitored by fluorescent optical method in rabbit SAN cells. Action potentials (AP) were recorded from rabbit SAN tissue preparations. Mechanistic computational data were obtained using the Yaniv et al. SAN model.Currents were measured by patch-clamp, Ca2+ level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (If) was previously inhibited and vice versa, the effect of If was augmented when the Ca2+ handling was suppressed.ORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Caf and NCX providing an important functional consequence: these currents together establish a strong depolarization capacity providing important safety factor for stable pacemaking. Thus, after individual inhibition of If or NCX, excessive bradycardia or instability cannot be expected because each of these currents may compensate for the reduction of the other providing safe and rhythmic SAN pacemaking.We confirmed the contribution of the NCX current to cardiac pacemaking using a novel NCX inhibitor. Our experimental and modeling data support a close cooperation between I LakattaM-clock) . Recent  authors .f current by caesium did not significantly influence SAN spontaneous activity arguing for mechanisms other than If  and conformed to Directive 2010/63/EU of the European Parliament. The protocols were approved by the Review Board of the Department of Animal Health and Food Control of the Ministry of Agriculture and Rural Development, Hungary (XIII./1211/2012).All experiments were conducted in compliance with the The measurements were performed in right atrial tissue obtained from young New-Zealand white rabbits from both genders weighing 2.0–2.5 kg.f pacemaker current, we isolated single cells from the SAN region of rabbit heart by enzymatic dissociation. The animals were sacrificed by concussion after receiving 400 IU/kg heparin intravenously. The chest was opened and the heart was quickly removed and placed into cold (4°C) solution with the following composition (mM): NaCl 135, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, NaHCO3 4.4, glucose 10, CaCl2 1.8, (pH 7.2 with NaOH). The heart was mounted on a modified, 60 cm high Langendorff column and perfused with oxygenated and prewarmed (37°C) solution mentioned above. After washing out of blood (3–5 min) the heart was perfused with nominally Ca-free solution until the heart stopped beating (approx. 3–4 min). The digestion was performed by perfusion with the same solution supplemented with 1.8 mg/ml (260 U/ml) collagenase . After 10–12 min, the heart was removed from the cannula. The right atrium was cut and the crista terminalis and SAN region were excised and cut into small strips. Strips were placed into enzyme free solution containing 1 mM CaCl2 and equilibrated at 37°C for 10 min. After 10 min with gentle agitation, the cells were separated by filtering through a nylon mesh. Sedimentation was used for harvesting cells. The supernatant was removed and replaced by HEPES-buffered Tyrode’s solution. The cells were stored at room temperature in the Tyrode’s solution.For measuring If current, the method of 2ATP 5, MgCl2 5, EGTA 5, HEPES 10, and GTP 0.1 (pH was adjusted to 7.2 by aspartic acid). The current was activated by hyperpolarizing voltage pulses to −120 mV from a holding potential of −30 mV. The pacemaker current was identified as ivabradine (IVA) sensitive current. The experiments were performed at 37°C.For the measurement of the I2+ transients were measured by Fluo-4 AM fluorescent dye. Isolated cells were loaded with 5 µM dye for 20 min in room temperature in dark. Loaded cells were mounted in a low volume imaging chamber  and continuously superfused with normal Tyrode solution. Fluorescence measurements were performed on the stage of an Olympus IX 71 inverted fluorescence microscope. The dye was excited at 480 nm and the emitted fluorescence was detected at 535 nm. Optical signals were sampled at 1 kHz and recorded by a photon counting photomultiplier module . Amplitudes of the Ca2+ transients were calculated as differences between systolic and diastolic values. To measure Ca2+ changes the cells were damaged by a patch pipette at the end of the experiment to obtain maximal fluorescence (Fmax). Ca2+ was calibrated using the following formula: Kd(F−Fmin)/(Fmax−F). Kd of the Fluo-4 AM was 335 nM.Isolated, spontaneously beating SAN cells were used for measurements. Caf current markedly increases as the membrane potential drops to more negative values  all of the ion channels remained intact  because of the lack of enzymatic dissociation, thus providing more precise estimates of the ratio between the currents, 2) since the SAN cells are surrounded with atrial cells having a more negative resting membrane potential, the electrotonic coupling may intimately influence the SAN cells. It may have great importance since the Ie values , (3) theSAN regions obtained from right atria were mounted in a tissue chamber superfused with oxygenated Locke’s solution at 37°C. A conventional microelectrode technique was performed as previously described . In the m/Δt between these points. The cycle length was calculated as the peak-to-peak interval between two consecutive APs. The phase 0 depolarization velocity was defined as the maximum of the first derivative of the AP during the upstroke.Action potential durations were measured at 90, 50, and 25% of repolarization. The maximal diastolic potential was defined as the most negative potential reached during the repolarization. The take off potential is the most negative point of the AP upstroke. DD was defined as the interval between the maximal diastolic potential and take off potential. The DD slope was calculated as ΔVet al. model of rabbit SAN cells . Statistical analysis was performed with Student’s With the exception of ORM-10962 (ORM) , and Fluo-4 AM , all chemicals were purchased from Sigma-Aldrich Fine Chemicals . ORM was dissolved in dimethyl-sulfoxide (DMSO) to obtain a 1 mM stock solution. This stock solution was diluted to reach the desired final concentration (DMSO concentration not exceeding 0.1%) in the bath.f in isolated SAN cells by applying the whole cell configuration of the patch clamp method. The selectivity of ORM on Na+-, Ca2+-, and major K+ currents was tested in a previous study from our laboratory . The preparations maintained the stable frequency in the time control experiments when DMSO was applied . In the computational SAN action potential model  as well as diastolic Ca2+ levels  between control and 3 µM IVA (versus 9.6 ± 2.3%). In contrast, ORM effect was significantly larger on the CL in the presence of 3 µM IVA, compared with the control where IVA was not applied . IVA significantly increased the CL both in 0.5 and in 3 µM concentrations . In f inhibition was varied between 0%/20%/60% block . Larger, 85% inhibition was only set in the model, since experimental application of 10 µM IVA is not feasible because of the marked IKr inhibition which can also reduce automaticity. The NCX inhibition was 41% in all cases. As f block increases, the same NCX inhibition causes larger CL prolongation) with markedly steeper correlation.In the next set of experiments, IVA and ORM were subsequently applied to study a possible coupling between If inhibition when NCX inhibition was 41% in all cases. The action potential modeling demonstrates an increased CL prolongation effect of NCX inhibition as If suppression becomes stronger, however, in contrast to the model prediction the steepness of NCX inhibition-induced CL increase was considerable flatter during experiments.f and NCX) would influence the effect of NCX inhibition. Full IKr block induced by 100 nM dofetilide (DOF) markedly increased the CL of SAN AP (control: 489.3 ± 31 ms → 100 nM dofetilide: 649.1 ± 40.2 ms). This degree of increase of CL was due to the lengthening of APD without changing the DI. The subsequent application of 1 µM ORM exerted a similar effect . However, the major difference was that the DOF-mediated increase in CL was practically entirely an APD increase-induced effect : 338.3 ± 39 ms vs. 352.7 ± 44.6 ms, n = 7) while the IVA influenced only the DI without affecting the APD90 (f inhibition by IVA increased the CL due to lengthening of the time of the DI by decreasing its slope. When ORM was applied in combination with DOF the increase of the CL was not additive.We investigated how bradycardia induced by a mechanism which does not directly involve the inward depolarizing currents (I2+ release on the effect of IVA (2+ release-induced augmentation of the forward NCX by application of 5 µM ryanodine (RYA) after the control recording. This caused a significant CL prolongation . The subsequently applied 1 µM ORM-10962 marginally but statistically significantly increased the CL . However, further 3 µM IVA markedly and significantly augmented the CL of the SAN preparations  external solution was selected to achieve this goal since in this concentration, the CL was only slightly reduced (10.3 ± 3.7%). We found that the reduced extracellular Ca2+ slightly increased the CL which was further increased after application of 3 µM IVA  and in the presence of low external Ca2+ (0.9 mM [Ca2+]o). As bar graphs in 2+ is low compared with normal Ca2+ settings . 2+ transient measurements from spontaneously contracting isolated cells. We can observe that the application of 0.9 mM [Ca2+]o significantly decreased the transient amplitude  which may reflect decreased Ca2+ influx, SR Ca2+ release which may decrease the NCX current and thus attenuate the compensating capacity of NCX. The diastolic Ca2+ also significantly decreased . We addressed this question by using mechanistic modeling (f current kinetics (middle traces), and global Ca2+ transients (lower traces) under normal condition. Upon application of 0.9 mM [Ca2+]o (right column) the CL slightly reduced, the integral of NCX current under the late phase of DD decreased while the magnitude of If current did not changed. The amplitude of the global transient decreased in similar extent as was obtained from SAN cell experiments.We further tested the coupling between Camodeling . Left coThe short term CL variability (CLV) was calculated by the analysis of CLs of N = 30 consecutive action potentials by applying the following formula:2+ release and NCX activity were suppressed  and If  or Ca2+ handling suppression  do not alter significantly the CLV while the variability was largely increased if IVA was administrated in the presence of reduced Ca2+ cycling activity .One micrometer of ORM-10962 and 3 µM IVA individually prolong the CL without considerable influence on the CL variability  3 µM IVA has moderate effects on CL (~21%) and CLV (Δ ~ 2 ms), ii) 1 µM ORM has marginal effects on CL (~8%) and no change on CLV, iii) Ca2+ cycling suppression by 1 µM ORM + 5 µM RYA has moderate effects on CL (~19%) and CLV (Δ ~ 4 ms), iv) increasing If inhibition augments the effect of a fixed ORM dose (1 µM) on CL (~ 8 to 17%), v) the effect of 3 uM IVA is enhanced when Ca2+ cycling was previously suppressed (from ~ 20 to 42%).The aim of this study was to verify and estimate the possible contribution of NCX function in SAN automaticity by direct selective pharmacological inhibition. Furthermore, we evaluated the functional consequences of the previously mentioned  If-NCX c50 values of 55 and 67 nM without changing the ICa, INa, IK1, IKr, IKs, Ito, and INa/K pump currents even at high (1 μM) concentrations. However, the If current was not investigated. The present study demonstrates that 1 µM ORM did not influence If  may influence the If indirectly  is decreased when the intracellular Ca2+ is intact . The underlying mechanism could be asymmetrical block between two modes, autoregulation of the Ca2+i or by preserved inducibility of forward NCX by elevated Ca2+i.The rate of forward NCX inhibition by using 1 µM ORM was estimated to ≈90% by applying conventional ramp protocol in the presence of ≈160 nM [Caus study . The actus study . It is ius study  we have 2+ changes compared to the Yaniv model predicted, however, in contrast with modeling, we found markedly increased Ca2+ transient amplitude which is generally expected after decreased rate of Ca2+ extrusion  a theoretical possibility exists that ORM-induced Ca2+ elevation may increase the If, however this was ruled out by a previous work  in response to NCX blockade (We can speculate that the increasing intracellular Cagulation . The gaious work . ii) It blockade .et al. claimed that partial ablation of NCX (≈70–80%) using an αMHC-inducible “Cre” transgenic line, has also a slight effect on the baseline spontaneous AP firing frequency  when Ca2+ handling was suppressed by low extracellular Ca2+ . Vice versa, when the NCX was previously suppressed (as a consequence of reduced Ca2+ release) the decreased DD current density is more sensitive to changes, which increases the bradycardic effect of IVA.We suggest that the observed NCX-Icompound . This mef cause only moderate bradycardia and rhythm disturbance. However, combined suppression of these currents acted synergistically and supports the hypothesis of mutual crosstalk between NCX and If in SAN even in multicellular tissue having important functional consequences. This means that individual inhibition of DD currents may have moderated effect on CL and variability under normal conditions because the underlying currents may be able to compensate each other. This important crosstalk may provide a considerable safety margin for SAN pacemaking.In the present study, we provide direct pharmacological evidence regarding the role of NCX in pacemaker mechanism by its selective inhibition with the novel, highly selective compound ORM-10962. We found that individual inhibitions of NCX or I1/M2 receptors—intimately changes the cAMP, PKA, CaMKII levels which have effects on the DD currents, therefore the discussed If-NCX coupling cannot be directly extrapolated to in vivo systems. The ion current crosstalk characterization during intact autonomic control requires further experiments.Our study has three important limitations. 1) The action potentials measured in our experiments do not represent the characteristics of the core SAN cells. These cells are much more “follower” cells, having AP waveforms largely influenced by the cell-to-cell coupling. 2) The applied inhibitors  are not able to cause complete block of ion channels in the applied concentrations. Therefore, the described phenomena indicate only partial effects and not able to estimate the absolute contribution of NCX during the DD. 3) Since our aim was to explore ion current cooperation, our results represent ion channel function independent from the autonomic nervous system. The activation of the sympathetic or parasympathetic nervous system—or modulation of the βAll datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by Munkahelyi Állatkísérleti Bizottság (MÁB).ZK performed ion current measurements and data analysis. NT performed fluorescent optical measurements, action potential measurements and data analysis. JS performed ion current measurements and data analysis. AL performed the computational modeling and data analysis, contributed to conception of the study as well as manuscript preparation and funding for the computational study. PG performed ion current measurements, GB and JP performed action potential measurements. NJ organized the database and ensured the financial support of the study. JL and PP contributed to the development of ORM-10962. JGYP contributed to manuscript preparation, AV and NN ensured the financial support of the study, contributed conception and design of the study, data analysis and visualization, and manuscript preparation. All authors contributed to manuscript revision, read and approved the submitted version.This work was supported by grants from the National Research Development and Innovation Office (NKFIH PD-125402 (for NN), FK-129117 (for NN), GINOP-2.3.2-15-2016-00006, the LIVE LONGER EFOP-3.6.2-16-2017-00006 project, the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (for NN), the UNKP-18-4-SZTE-76 New National Excellence Program of the Ministry for Innovation and Technology (for NN), the EFOP 3.6.3 VEKOP-16-2017-00009 (for NT), the Hungarian Academy of Sciences and by the Orion Pharma (ORM-10962).PP and JL are employed by Orion Pharma, which has been involved in the development of ORM-10962.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


One of the most fascinating aspects of mitochondria is their remarkable ability to accumulate and store large amounts of calcium in the presence of phosphate leading to mitochondrial calcification. In this paper, we briefly address the mechanisms that regulate mitochondrial calcium homeostasis followed by the extensive review on the formation and characterization of intramitochondrial calcium phosphate granules leading to mitochondrial calcification and its relevance to physiological and pathological calcifications of body tissues. Extracellularly such a high calcium and phosphate ionic environment is observed in the matrix vesicles (MV) released from cells, including osteoblasts and chondrocytes  in nature followed by the exposure of these preformed apatite material to extracellular fluid promoting the crystal proliferation and thus beginning the process of mineralization and crystal deposition.Calcification or mineralization, an accumulation of insoluble calcium salts in tissue, is a key biological process that is physiologically restricted to hard tissues, including bone, teeth, and the hypertrophic zone of growth plate cartilage . This esdrocytes . MVs conbstrates . Intracebstrates , which pSince the focus of this viewpoint is mitochondrial calcification rather than calcium regulation of mitochondria, in this section we will briefly address important aspects of mitochondrial calcium uptake and efflux including mechanisms that regulate mitochondrial calcium homeostasis . For mor2+ is known since the 1960s [2+ ions in and out of mitochondria is a concerted activity of ion transporters on outer mitochondrial (OMM) and inner mitochondrial membranes (IMM). The OMM is highly permeable to various ions, including Ca2+, whose transport is mediated by a non-selective porin, voltage-dependent anion channel [2+ (Kd around 10–20 μM) and higher conductance rates than Ca2+ uptake channels in the endoplasmic reticulum (ER), which makes them suitable to respond to large increases in cytosolic Ca2+ that occur physiologically at the calcium release or the entry points of ER and plasma membrane calcium channels and to the pathological Ca2+ overload [2+ entry into mitochondria is an electrogenic process driven by the steep mitochondrial membrane potential, Δψm,  across the IMM established by the respiratory chain or by the reverse mode ATP synthase activity [p-(trifluoromethoxyl)-phenyl-hydrazone (FCCP) that dissipate Δψm suppresses mitochondrial Ca2+accumulation. Whereas selective inhibitors of MCU, mainly ruthenium red-based compounds, and small-molecule inhibitor DS16570511 directly inhibit Ca2+ uptake [2+ levels in mitochondria are maintained at the resting levels (~100 nM), suggesting the presence of mechanisms that maintain the baseline levels of mitochondrial Ca2+ by directly regulating the activity of MCU [2+ levels, thus avoiding deleterious/futile calcium cycling and matrix overload and allowing prompt response of mitochondrial calcium uptake in situations of cytosolic Ca2+ increase. MCU, a 40 kDa protein, functions as a tetramer where a single protomer is composed of two transmembrane domains (TM1 and TM2) joined by a highly conserved short loop facing an intermembrane face (IMS) and N- and C- domains facing mitochondrial matrix. The motif between TM1 and TM2 characterized by negatively-charged residues (DIME motifs) serves as a selectivity filter of the MCU channel [2+-sensing domains, hence cannot regulate its own activity. Indeed, the activity of MCU is regulated by EF-hand-containing Ca2+ binding proteins, mitochondrial calcium uptake 1 (MICU1) and mitochondrial calcium uptake 2 (MICU2) found in the IMS, along with IMM protein essential MCU regulator (EMRE). A study suggested that by exerting opposing effects, MICU1 and MICU2 heterodimers fine-tune the activity of MCU, where at lower cytosolic Ca2+ levels the dominant inhibitory effect of MICU2 shuts down the MCU activity; however, conformational change induced in dimers by increases in cytosolic Ca2+ releases MICU2-dependent inhibition of MCU triggering MICU1-mediated augmentation of MCU channeling activity [2+ ion permeation properties of MCU [2+ by mitochondria and dysfunction and undesired cytosolic Ca2+ buffering preventing heart contractile activity. Further, induction of MCUb expression was shown to be a stress-responsive mechanism to overcome calcium overload following cardiac injury [2+ uptake sensitivities/loading capacity of tissues and implications for tissue calcification. Two additional MCU regulators are MCU regulator 1 (MCUR1) and solute carrier family 25 member 3 (SLC25A23). Silencing of MCUR1 abrogated MCU-dependent mitochondrial Ca2+ uptake in both basal and stimulated conditions [2+ influx into mitochondria following stimulation [2+ uptake, interestingly MCU-KO mice had significantly reduced but detectable levels of matrix Ca2+ with only relatively minor alterations in the functions dependent on mitochondrial influx of Ca2+: mitochondrial respiration and basal metabolism. The only substantial defect is a decrease in skeletal muscle peak performance, indicating that in vivo alterations in matrix Ca2+ are most important for adapting to needs of higher energy demands as in strenuous muscle work [2+ uptake mechanisms in mitochondria [2+ efflux mechanisms work in reverse mode, thus bringing Ca2+ into the matrix rather than exporting Ca2+, cannot be ruled out [2+ uptake and phenotypes in different cell lines and species, see reference De Stefani et al. [The direct evidence that mitochondria rapidly accumulate Cahe 1960s –13. The  channel ,15. In c channel –18. MCU + uptake . Despitey of MCU . These r channel ,25. MCU activity . EMRE [2activity , a transs of MCU . Interess of MCU ,29, comps of MCU ,29. In cc injury . Future nditions  and was nditions . SLC25A2mulation . Althougcle work ,35. Overchondria –41. In auled out . For an i et al. .2+ uptake pathways reported in mitochondria include the rapid mode Ca2+ uptake (RaM), [2+ influx through mitochondrial ryanodine receptor 1 (mRyR1) functioning in excitable cells [2+ influx mechanisms, RaM, described as a kinetic model of Ca2+ uptake, operates very rapidly , a large pore in the inner mitochondrial membrane that increases the mitochondrial permeability to solutes up to 1.5 kDa whose persistent opening can lead to cell death [2+ levels with relatively small amounts of Ca2+ [2+ uptake may be more relevant to tissues like a heart with very short but frequent Ca2+ pulses, thus protecting them against matrix Ca2+ overload the opening of mPTP but still activating Ca2+-sensitive metabolic reactions. mRyR1, mainly characterized in excitable cells like cardiac muscle cells, is another fast Ca2+ uptake pathway in mitochondria that is active in the micromolar ranges (10–50 μM) of Ca2+ and is inactivated at higher concentrations [2+ with high conductance rates, it can rapidly dissipate Δψm. This energetically unfavorable process is prevented presumably with a lower number of mRyR1 on single mitochondria, so that membrane depolarization is localized and is quickly corrected by metabolic activity [2+ dependence of various Ca2+ influx channels suggests their specific roles in different cytosolic Ca2+ environments of different tissues. However, modulation of their function in (patho) physiological conditions remains to be explored.Other potential Cae (RaM), ,42, and le cells ,44 amongle cells ,40. Of than MCU; ) and res+ levels . Fast uprylation ,16. Howell death . Hence,  of Ca2+ . Such a trations . Since mactivity ,40. The 2+ uptake via MCU seems to be a major mechanism, since RaM and mRYR1, although they have high conductivity [2+ [2+ pulses with relatively slow conductance, thus mediating large amounts of matrix Ca2+ accumulation necessary for calcification.For the mitochondrial calcification, Cauctivity , are opevity [2+  unlike M2+ can be exported from the matrix via Na+-dependent or independent mechanisms. The Na/Li/Ca exchanger (NCLX) in the inner mitochondrial membrane [+ or Li for Ca2+. Although the precise stoichiometry for NCLX still unclear, the general consensus has been an influx of 3 Na+ per 1 Ca2+ efflux, indicating that NCLX is also electrogenic [+/Ca2+ exchanger, NCX) in the plasma membrane. The unique feature only shared with mitochondrial NCLX being Li+-mediated Ca2+ transport in addition of Na+/Ca2+ exchange [2+ influx into mitochondrial matrix is driven by negative electrochemical gradient, the influx process is energetically downhill, and the efflux is uphill that would require energy. The minimum energy required for the export of 1 mole of Ca2+ from mitochondria is calculated to be 33.04 kJ/mol [2+ efflux to another ion that is moving down its electrochemical gradient or some combination of these energies. Na+ ion, whose matrix concentrations are maintained lower than cytosolic Na+ levels by a Na+/H+ exchanger [+ gradient i.e., lower inside, provides the driving force for extruding Ca2+ from matrix against its gradient through NCLX. The electrogenic feature of NCLX would indicate that in depolarized mitochondria, NCLX would function in reverse mode mediating the influx of Ca2+ rather than extrusion [2+, as shown in gene silencing and overexpression experiments [2+. This would suggest the low affinity of NCLX to Ca2+ [2+ changes to restore mitochondrial Ca2+ levels to baseline.As to the efflux mechanisms, Camembrane ,43,44, utrogenic  similar exchange , hence t4 kJ/mol . Energy xchanger , meets sxtrusion . Despite to Ca2+  and its 2+ efflux is primarily mediated by H+/Ca2+ exchanger [+/H+ exchanger, was identified as a molecule that fulfills the criteria of mitochondrial Ca2+/H+ exchanger with an electrogenic stoichiometry of 1H+/Ca2+ [+/Ca2+ for this antiport [2+ influx in an electrogenic manner (one Ca2+ in for one H+ out) when matrix Ca2+ is low, but when mitochondrial Ca2+ is high or cytoplasmic pH is low, LETM1 mediates Ca2+ efflux [In non-excitable cells, Caxchanger . In a ge1H+/Ca2+ . Howeverantiport . Interes+ efflux .2+ (mg protein)−1 min−1 compared to combined Vmax of 20 nmol Ca2+ (mg protein)−1 min−1 for efflux mechanisms [2+ selective mechanisms slower than influx? (2) How do mitochondria overcome pathological matrix Ca2+ overload that could ensue since the influx rate exceeds that of combined Ca2+ selective efflux pathways? Ca2+ accumulation by mitochondria is a function of extramitochondrial Ca2+ levels [2+ across the IMM, which will be met at the expense of increased proton conductance manifesting as a decrease in proton electrochemical gradient and hence in increased respiration, suggesting that respiratory capacity would be spent on Ca2+ recycling [Vmax and easily saturable efflux pathways would limit the energy to be spent on mitochondrial Ca2+ cycling. However, such a kinetic imbalance would expose mitochondria to a threat of Ca2+ overload, which can be overcome by the opening of a high conductance channel in the IMM such as mPTP that shows a prominent dependence on matrix Ca2+ for its activation [While the exact kinetics of these mitochondrial efflux mechanisms may vary significantly between tissues, overall, the kinetics of efflux rate is always much slower than influx, and this kinetic imbalance is apparent from Vmax values of these mechanisms. For example, initial studies established Vmax of MCU to be 1400 nmol Cachanisms . This ki+ levels . Hence aecycling . Thus, htivation ,55. The tivation . mPTP is2+ efflux. Thus, mPTP is also considered to be one of the important matrix Ca2+ efflux mechanisms [2+ efflux mechanisms, mPTP is not selective for Ca2+. Such an ion non-selectivity may facilitate a unique advantage to mPTP in overcoming the opposition by diffusion potential (−30 mV) that is generated across the IMM due to Ca2+ efflux through Ca2+ selective channels. Thus, in the absence of compensating ion transport, i.e., the influx of positive charges and efflux of negative charges, the efflux of Ca2+ through Ca2+ selective channels would be extremely slow. One way to overcome the magnitude of diffusion potential and subsequently to increase the rate of Ca2+ efflux is to increase the IMM permeability, for example, by increasing the H+ conductance. The ion non-selectivity of mPTP allows the charge compensation within a single channel itself at zero potential, thus allowing the rapid efflux of Ca2+ from matrix regulated by the modulation of the mPTP open time. Since there is no concentration gradient for Na+ and K+ across IMM, mPTP is, in a way, selective for Ca2+ transport from mitochondria [2+ binding sites (Kd 25 μM), the Ca2+ concentration required for the activation of mPTP is relatively higher than the concentration for ADP phosphorylation (20 nmol/mg vs 4 nmol/mg protein), suggesting that higher matrix Ca2+ overload is required for pore activation [2+ required for mPTP activation in pathological conditions. It should be noted that pore opening itself can also contribute to the generation of mtROS [2+ are common endogenous inhibitors of mPTP activation, including acidic pH and high membrane potential [2+ overloading promote the activation of mPTP.Interestingly, transient openings or “flickerings” of mPTP have been reported, suggesting that mPTP may also play a physiological role in Cachanisms . Unlike chondria . Given ttivation . Matrix of mtROS . Other motential . Inciden2+ has a ten-fold higher ATP affinity, ATP hydrolysis would increase the matrix Mg2+ levels [2+ and ADP), leading to pore closure restoring Δψm. This would explain the basis for transient openings of mPTP in vivo . Depend2+ are rate-limiting enzymes of tricarboxylic acid (TCA) cycle that are activated in different mechanisms: isocitrate dehydrogenase and ketoglutarate dehydrogenase are directly activated by Ca2+ binding whereas pyruvate dehydrogenase (PDH) activation depends on Ca2+-regulated PDH phosphatase [2+ also stimulates the activities of adenine nucleotide transporter [0F1 ATP synthase) [2+ in response to an increase in cytosolic Ca2+, which invariably is associated with stimulated cells, allows mitochondria to decode the energy demands of cell stimulation and adjust ATP synthesis accordingly. Since mitochondrial ETC is one of the main sites that generate cellular reactive oxygen species (ROS) in physiological and pathological conditions, Ca2+ accumulation in the matrix during cellular activation can directly contribute to mtROS by promoting mitochondrial metabolism. Mitochondrial Ca2+ also activates nitric oxide synthase, whose product nitric oxide inhibits complex IV enhancing mtROS generation [2+ overload in conjunction with oxidative stress activates the opening of mPTP. The opening of mPTP results in the rapid collapse of Δψm and membrane depolarization resulting in increased mtROS. An independent study has shown that Ca2+ induces ROS via Ca2+-mediated complex II disintegration by binding to cardiolipin, a principle IMM anionic lipid that promotes complex II stability. However, when bound by Ca2+ in the conditions of matrix overload, cardiolipin coalesces into separate homotypic clusters releasing the enzymatically competent sub-component of complex II that generates ROS by transferring electrons from succinate to molecular oxygen [2+ overload by mPTP. Available evidence shows that various calcium transport systems are sensitive to redox conditions [2+ influx into endoplasmic reticulum and extrusion from the plasma membrane via inhibition of sarco (endo) plasmic reticulum Ca2+-ATPase [2+-ATPase, respectively [2+ stores [2+ causes transient opening of mPTP to prevent cell from cytosolic overload but stimulating mitochondrial Ca2+ overload. Interestingly, in in vitro conditions, inflammation and hypoxia-induced oxidative stress were shown to regulate MCU-mediated mitochondrial Ca2+ uptake independent of cytosolic Ca2+ by relieving it from gatekeeping of MICU1/ MICU2, thus resulting in augmented mitochondrial Ca2+ at baseline cytosolic Ca2+ [S-glutathionylation) that induces a conformational change MCU promoting high order oligomerization and persistent activation even in resting conditions despite the presence of functional MICU1/MICU2 [2+, in turn, led to overproduction of mtROS, perturbed mitochondrial bioenergetics, and apoptosis. Overall, these data suggest that Ca2+ and ROS create a self-perpetuating cascade that can culminate in the mitochondrial Ca2+ overload and perturbed cell functions [+/Ca2+ exchangers, the Ca2+ efflux mechanisms function in a reverse mode promoting calcium influx rather than efflux of matrix Ca2+ [The major targets of mitochondrial Casphatase ,61. The nsporter  and compynthase) , which bneration . Matrix r oxygen . Oxidatinditions . This in+-ATPase ,67 and pectively ,69 compl+ stores ,71. The lic Ca2+ . SpecifiU1/MICU2 . The incunctions . Furtherrix Ca2+ .2+ modulates various processes, including stimulation of aerobic mitochondrial metabolism, suppression of autophagy, regulation of cell life/death processes and Ca2+-induced Ca2+ feedback, cytosolic Ca2+ buffering, and in regulating spatial restriction of Ca2+ waves  the matrix-free calcium remains invariant due to buffering by calcium phosphate [2+ levels [+ symport. The PiC transports the Pi, which is equivalent to its fully protonated form H3PO4 with H+. Since the phosphate form that interacts with matrix Ca2+ is PO43−, the phosphate has to undergo three stepwise deprotonations (H3PO4 to H2PO4− to HPO42− to PO43−) and thus the concentration of Pi in matrix is inversely proportional to the third power of proton gradient in the matrix. As we know that Ca2+ accumulation in matrix decreases Δψm which is compensated by the net expulsion of H+ by respiratory chain. If this were the only way, Ca2+ accumulation will eventually stop since entire Δψm will be converted to proton gradient (ΔpH). Note that Ca2+ influx into mitochondria is driven by Δψm component of proton motive force. However, Pi’s transport with increasing ΔpH  will neutralize the increasing matrix pH facilitating the Ca2+ accumulation and the formation of reversible Ca-P complexes by transported Pi with accumulated matrix Ca2+ [2+ mg protein−1 in the mitochondrial matrix, there is a kinetic balance between influx and efflux where the efflux pathway becomes independent of matrix Ca2+ called set point since it is at this concentration Ca-P complexes begin to form thus buffering matrix Ca2+ [Mitochondrial matrix Can et al. . Thus, tuffering –75. Net omplexes –21. Sincomplexes  do not h ability –24. In frix Ca2+ . At arourix Ca2+ .2+ accumulation induced by extramitochondrial Ca2+ levels beyond the set point causes the formation of electron-dense granules within the matrix [2+ and Pi are the major inorganic constituents of matrix Ca-P granules primarily corresponding to hydroxyapatite and whitelockite or a mixture of both as shown by the X-ray diffraction patterns of microincinerated granules . Also, significant traces of MgO presumably derived from MgCO3 were also found [2+ accumulation seems to be one of the majorfactors affecting the stoichiometry of calcium phosphate complexes, where faster Ca2+ infusion rates promote higher Ca/P ratios 2) as shown in rat liver mitochondria [2+-loaded mitochondria and the fact that Ca-P precipitates of crystalline nature are not observed in live cells reveal the indefinite amorphous nature of Ca-P complexes suggesting that crystallization is held in check within the mitochondrial matrix [nO3n+1)(n+2)−) expressed by mitochondria can also inhibit the formation of insoluble Ca-P complexes or precipitates, thus regulating the levels of free Ca2+ in the mitochondrial matrix. PolyP are negatively charged polyanions formed by the polymerization of many Pi molecules [2+ despite similar loading of Ca2+ uptake compared to wild type (independent of Ca2+ efflux rates), suggesting the buffering of matrix Ca2+ as Ca-P insoluble clusters [As demonstrated in both isolated and in situ brain mitochondria, robust Cae matrix ,79. These matrix . Chemicaso found . Similarso found , the comso found ,82,83 asso found . The ratl matrix . The amol matrix . Furtherl matrix . Thus, al matrix ,34. The l matrix –33. In aolecules , which aolecules . Accordiclusters ,90. This2+ overloaded mitochondria, intramitochondrial aggregates had both inorganic and organic components . Interestingly, the type of intramitochondrial inorganic aggregates differed based on the tissue type examined in the study. Crystalline aggregates were restricted to apparently normal muscular and myocardial cells, and granular aggregates were mainly found in swollen mitochondria of degenerated hepatic cells. However, the relationship between the state of cells and the type of intramitochondrial aggregates is less evident in literature, which requires examination at the early stages of mitochondrial calcification. In general, consistent with the presence of crystallization inhibitors, crystalline aggregates are less commonly found in mitochondria, and they have been reported in both normal and damaged cells. Granular aggregates are mostly widely reported both in the context of mitochondria overloaded with Ca2+ and in mitochondria of normal cells and cells at various stages of degeneration. In this study, although, morphologically both aggregate forms seem to be very closely associated with mitochondrial cristae there were some differences during the early stages of calcification. Crystalline aggregates were more closely situated near cristae membranes, unlike granular aggregates, which are close to cristae but lie more in the matrix. This association of crystalline aggregates with membranes is interesting considering the affinity of anionic phospholipids to Ca2+ and their potential role as organic components aiding in the deposition of inorganic material and in the initiation of mineralization [2+ and Pi overload.In a detailed study of experimentally induced mitochondrial calcification, both apatite-like crystalline, needle-shaped aggregates, and granular aggregates have been identified –93. Conslization . Furtherlization . Mitocholization –97. Accolization ,96,98,992+ overload induces mtROS generation. However, very little is known about how the formation of Ca-P precipitates affects mitochondrial function. Ca-P granules effecting mitochondrial respiration were demonstrated in a study where the activity of complex I was inhibited, thus decreasing the rate of ATP synthesis. It was proposed that Ca-P precipitates could be forming physical barriers isolating complex I from its substrate, NADH [Since both crystalline and granular aggregates are closely associated with mitochondrial cristae they could affect mitochondrial function, namely mitochondrial metabolism and ROS production. Matrix free Cate, NADH . HoweverIn an experimentally induced calcification of rat myocardium where focal areas of calcification were restricted to mitochondria, severely calcified cells generated cellular reaction . In thatThe role of mitochondrial granules in biological mineralization has been reported –108. Inc2+ uptake and storage abilities. In case of metastatic calcification, elevated levels of extracellular calcium and phosphate ions could lead to increasing levels of these ions within the cell. Although some of these ions will be exported out of the cell via efflux mechanisms on the plasma membrane, but over the time ions will accumulate in mitochondria forming Ca-P complexes, thus initiating the process of intracellular metastatic calcification. In case of dystrophic calcification, despite the presence of normal levels of calcium and phosphate ions in circulation, increased plasma membrane permeability due to injury, inflammation or hypoxia make expulsion of ions from the cell ineffective leading to their accumulation in mitochondria initiating the process of intramitochondrial mineral formation. Mechanistically, mitochondrial calcification can be the contributing factor for soft-tissue calcification of dystrophic type as observed in many pathological conditions including dermatomyositis, scleroderma, systemic lupus erythematosus, and mixed connective tissue diseases, some of in which mitochondria have been implicated [2+ overload even in baseline cytosolic Ca2+ levels has important implications for diseases like juvenile dermatomyositis in which dystrophic calcifications of muscle and skin are associated with chronic inflammation [2+ uptake and calcification.Unlike metastatic calcification, which is caused by the increased substrate availability, dystrophic calcification is secondary to the altered membrane integrity due to trauma or inflammation and as such is observed at the sites of tissue degeneration. Mitochondria could be the initial sites for intracellular calcification in both types of calcification considering their robust Caplicated ,60–65. Hammation . Incidenammation , warrantTo summarize, mitochondrial calcification is a physiological process to protect cells from calcium-induced cytotoxicity; however, dysregulated may contribute to disease and calcification of tissues. Hence, understanding mechanisms regulating mitochondrial calcification and its role in accumulation of extracellular calcium deposits in tissue may allow for identification of novel therapeutic targets in several diseases, including dermatomyositis ,102,112.


However, this is more challenging for Ca2+-ATPase modulators due to the physiological role of Ca2+ in cardiac dynamics. Over the past two decades, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) modulators have been studied as potential chemotherapy agents because of their Ca2+-mediated pan-cancer lethal effects. Instead, recent evidence suggests that SERCA inhibition suppresses oncogenic Notch1 signaling emerging as an alternative to γ-secretase modulators that showed limited clinical activity due to severe side effects. In this review, we focus on how SERCA inhibitors alter Notch1 signaling and show that Notch on-target-mediated antileukemia properties of these molecules can be achieved without causing overt Ca2+ cellular overload.P-type ATPase inhibitors are among the most successful and widely prescribed therapeutics in modern pharmacology. Clinical transition has been safely achieved for H This ef binding . A second Leu260 , 175. Ho2+ homeostasis plays critical roles in key processes that regulate cellular survival, growth, differentiation, metabolism, and death in normal and cancer cells. Thus, it is not surprising that several anticancer agents suppress pro-survival and activate pro-apoptotic pathways through modulation of Ca2+ signaling-dependent mechanisms. This is, for example, the case for chemotherapeutics such as cytotoxic alkylating agents [2+ signaling component to induce cancer cell death [2+ concentration and to participate in apoptotic signaling pathways. Among them, SL, such as thapsigargin, has been long regarded as target compounds for drug development. Thapsigargin has a broad spectrum of growth-suppressing activity in several tumor types including poorly dividing cells [2+ overload [NOTCH1 appears more sensitive to SERCA suppression compared to wild-type isoform or other proteins more broadly. One hypothesis to explain NOTCH1 and SERCA functional dependency is by mechanisms of co-regulation. It has been previously shown that PSEN and SERCA co-localize in the ER [2+ mediated one. In fact, Malecki and colleagues previously demonstrated that clinically relevant activating NOTCH1 HD mutations destabilize the NOTCH1-negative regulatory region and have deleterious effects on NOTCH1 folding and maturation. Because EGF and LNR repeats of NOTCH1 rely on Ca2+ for folding and activation, it may be possible that changes in ER Ca2+ may preferentially impair unstable NOTCH1 mutant proteins [2+-mediated transcriptional activation of inhibitors of furin-like proteases. This would explain for example why CAD204520 efficiently targets cancers with isolated PEST deletions that would not be predicted to be unstable given a normal LNR and HD protein sequence.Modulation of intracellular Cag agents  or antimll death . Similarng cells . Howeverng cells . Importaoverload . An impon the ER . Since Pn the ER . The secproteins  compared2+ toxicities.In conclusion, the transient disruption of SERCA activity can be leveraged for targeting Notch1 in cancers. Legitimate concerns associated with SERCA inhibition can be overcome by the identification of druggable modalities to lock ATPase function without causing dose-limiting ER Ca


Acute myeloid leukemia (AML) is a heterogeneous group of clonal disorders characterized by abnormal proliferation of undifferentiated myeloid progenitors, impaired hematopoiesis, and variable response to therapy. To date, only about 30% of adult patients with AML become long-term survivors and relapse and/or disease refractoriness are the major cause of treatment failure. Thus, this is an urgent unmet clinical need and new drugs are envisaged in order to ameliorate disease survival outcomes. Here, we review the latest therapeutic approaches  for AML treatment. A specific focus will be given to molecularly targeted therapies for AML as a representation of possible agents for precision medicine. We will discuss experimental and preclinical data for FLT3, IDH1, BCL-2, Hedgehog pathway inhibitors, and epitherapy. Despite the progress in treatment and supportive therapies, the outcome of patients with adult acute myeloid leukemia (AML) remains dismal with only about 30% of patients becoming long-term survivors ,2.Globally, AML accounts for the highest percentage of leukemic deaths and every year 4–5 per 100,000 persons are expected to develop this disease with a peak of incidence over the sixth decade of life and a slight male predominance . During In the last 10 years, the U.S. Food and Drug Administration (FDA) approved several new pharmacologic agents for the treatment of AML, enhancing the plethora of treatments in the new millennium scenario. A full description of the numerous drugs and clinical trials of the recent years is outside the scope of this manuscript; however, we highlighted the crucial role of potential direct and/or indirect therapeutic targets  for AML treatment.Fms-related tyrosine kinase 3 (FLT3) is a cytokine receptor regulating cellular differentiation and proliferation through multiple activation pathways  . In 1996Two generations of FLT3 inhibitors have been developed and explored so far in preclinical and clinical settings . The firMidostaurin is the first FDA approved FLT3 inhibitor. This first-generation compound has a broad kinase activity profile and was originally developed for solid tumors. Midostaurin has a potent antiblast activity and is now part of the treatment of FLT3-mutated patients during 3+7 induction scheme and post remission regimens. The Randomized AML Trial in FLT3 Young patients (RATIFY) is a multicenter, randomized, double-blinded, and placebo-controlled phase 3 pivotal trial enrolling 717 patients with de novo AML and age less than 60 years. The study compared midostaurin 50 mg BID to placebo in combination with the standard induction regimen 3+7, followed by consolidation and maintenance in patients harboring both FLT3-ITD and TKD mutations. This combination resulted in a better OS and event free survival (EFS) for the group receiving the drug, yielding to FDA approval . However2. It is a current practice to use anthracycline ranging from 45 to 90 mg/m2, according to the fitness of patient, mostly dependent on the age and many studies have been conducted so far to understand the best dosage. However, it has to be noted that midostaurin and daunorubicin share common toxicities  suggesting more precautions and low anthracycline dose if used in combination [Stone et al.  administbination . Moreovebination .The second and more selective generation of FLT3 inhibitors includes quizartinib, crenolanib, and gilteritinib. Quizartinib AC220) is a potent FLT3 inhibitor with a weak non FLT3-ITD TKI activity. Thus, TKD mutant clones represent a well-known escaping mechanism after quizartinib treatment 0 is a po. A signiSimilar to quizartinib, crenolanib is another potent and selective FLT3 inhibitor, although it retains activity against TKD mutations, overcoming the known mechanism of resistance of the previous second generation drug. Resistance to crenolanib is acquired in the case of F691L gatekeeper mutations, targeted by other TKI inhibitors such as ponatinib or new compounds still under investigation such as PLX3397 and cabozantinib ,26. CrenIn 2018, the FDA approved gilteritinib for the treatment of R/R FLT3-mutated AML after the promising results of the ADMIRAL trial, a randomized, open label, multicenter phase III study comparing gilteritinib or conventional chemotherapy for R/R FLT3-mutated AML patients . This neDespite the development of all these new compounds, there is still room to improve survival outcomes in FLT3-mutated AML. Although FLT3 inhibitors are known to provide a viable blast reduction and maintain remission state, many patients are resistant or relapsing after treatment due to various possible mechanisms.The first remarkable result is that FLT3 inhibitors are very useful for reducing the peripheral disease burden although this does not necessarily translate into a similar effect on bone marrow (BM) disease burden. Many studies have proved how the BM microenvironment plays an important role in mediating resistance to FLT3 inhibitors due to the expression of CYP3A4 by stromal cells, providing protection against these new selective drugs by affecting their pharmacokinetics . MoreoveFurthermore, FLT3 constitutive active signaling converges to downstream activation of STAT5, also associated with concomitant phosphorylation of FOXO3A (retained into the nucleus) and AXL upregulation, a tyrosine kinase receptor which is higher expressed under quizartinib treatment and hypoxia conditions of the hematopoietic niches . STAT5 aTherefore, targeting these pathways with Bcl-2 inhibitors such as venetoclax may represent a way to overcome FLT3-mutated R/R AML. Recent studies have also shown the important role of FOXM1 in R/R AML and particularly in FLT3-ITD-mutated cases where the expression of Forkhead box (FOX) proteins correlates with OS. In vitro studies have shown that inhibition of FLT3-ITD by quizartinib/AC220 also downregulates FOXM1 expression in FLT3-mutated cells suggesting that FOXM1 may be a potential prognostic marker and therapeutic target in AML. Ultimately, secondary mutations in the FLT3 gene often involving single amino acid substitutions have been postulated as a putative mechanism of resistance. These mutations are known to occur specifically in relapsing patients after type II inhibitors treatment and can be targeted by broader TKI such as ponatinib or sorafenib . Most liIsocitrate dehydrogenases (IDHs) are metabolic regulatory enzymes, recognized as mutated in various types of cancer  ,40,41,42In particular, isocitrate dehydrogenases 1 (IDH1) is the cytoplasmatic isoform of a class of dehydrogenases involved in the Krebs cycle whose role is converting isocitrate, generated in the tricarboxylic acid cycle (TCA), to alpha-ketoglutarate (α-KG) . The mutMarcucci et al.  reportedIDH1 mutation treated with 500 mg oral ivosidenib resulting in an overall response rate (ORR) of 41%, 21% complete response (CR), and 30% hematological improvement [Recently FDA approved ivosidenib (AG-120), a first-in-class IDH1 inhibitor  and enasrovement . Enasiderovement . Similarrovement .Despite IDH inhibitors development, multiple resistances have been described with poorly elucidated mechanisms. As higher 2-HG levels are required for blasts’ proliferation, one peculiar mechanism to maintain its level under IDH selective isoform blocking, is the “isoform switching” from IDH1 to IDH2 and vice versa . MoreoveFurthermore, other IDH1 inhibitors, such as olutasidenib, an oral highly potent and selective inhibitor of IDH1 and vorasidenib (AG-881) a pan IDH inhibitor, are now under investigation . In partLastly, other nontargeted strategies for IDH1 mutant AML may include PARP inhibitors such as olaparib and talazoparib, already tested in IDH positive gliomas and in studies using AML cellular models . Since gB-cell lymphoma 2 (BCL-2), a member of the BCL-2 family of genes and an integral part of the intrinsic mitochondrial apoptotic pathway, was first discovered in follicular lymphoma harboring t  abnormality . This paBCL-2 expression has been found deregulated in AML . ParticuL (B-cell lymphoma-extra large) with consequent side effects such as thrombocytopenia [Since the development of the antisense nucleotide oblimersen (G3139) in 2003, many efforts have been made to discover new drugs targeting BCL-2 pathway. However, many compounds have been tested in AML preclinical and clinical setting without promising results until the discovery of ABT-199 (venetoclax) . The preytopenia . On the ytopenia . 2, days 1–7 subcutaneuosly) or decitabine  schedules. Half of the patients harbored poor-risk cytogenetics and median age was 74 years. The ORR was 68% with a CR rate of 73% in the venetoclax 400 mg cohort and was of 67% in the whole cohort. The treatment was well-tolerated and common adverse events were mostly gastrointestinal and hematological and in particular neutropenia, probably responsible for early mortality within 30 days after initiation (despite the paucity of events). This latter event has been in part counteracted by the quick median time to best response of 1.8 months if compared with the average four months with HMA-treated historical controls [FLT3-positive patients and in secondary AML, as well as in TP53-mutated cases (despite the lower incidence—47%—and median duration—5.6 months—of CR). Conversely, IDH1/2-mutated patients were particularly sensitive to venetoclax and reached a median survival of 24.4 months. However, venetoclax shares some downsides with other AML targeted therapies; it is metabolized by CYP3A4 hepatic cytochrome alerting the use of concomitant drugs utilized in fungal prophylaxis. Nevertheless, the incidence of fungal infections in this trial was similar to other previous reported studies demonstrating the need of reaching adequate neutrophil counts in a short time as it occurs with this combination therapy. Preclinical data suggested that this new drug targeting BCL-2/tp53 apoptotic pathway may be useful also for AML as a single agent or in combination with other backbone therapies . Konoplecontrols . MoreoveFLT3 inhibitors or glasdegib- NCT03625505 and NCT03735875) to overcome AML resistance and apply a better personalized pharmacologic approach [Venetoclax is now on study with conventional chemotherapy (NCT03709758), as well with other multi-targeted combinations  [Hedgehog (HH) signaling pathway is fundamental in embryonic development and homeostasis of adult tissues . HH intectively) . Thus, Hctively) . This agctively) . The firctively) . The proctively) . The resctively) . OS for ctively) . The expctively) . The mosctively) . Glasdegctively) . As aforementioned, GLI expression has been related to AML survival outcomes and therefore may be a useful biomarker of HH pathway inhibitors response/resistance. Acquired mutations in SMO gene may cause resistance as suggested by many observations derived by the clinical experience of basal cell carcinoma (BCC) patients. In particular, some case reports showed that arsenic trioxide (ATO), well-known for APL treatment, may overcome HH inhibitors resistance in combination with itraconazole in patients with BCC . FinallyEpigenetic and genetic anomalies cooperate in AML initiation and progression, as demonstrated by the presence of frequent mutations in genes encoding proteins that control the epigenome and/ or by the abnormal expression of epigenetic proteins. Given the reversible nature of epigenetic changes, targeting epigenetic alterations by using epigenome-influencing agents has represented a powerful approach in the treatment of AML with the promise of restoring the function of affected genes and reprogramming the state of the cells from abnormal to normal ,87. ThisIn the late 1970s, reverse epigenetic agents amenable to reverse DNA methylation were discovered. However, these agents only reached clinical trials about twenty years later specifically in MDS. The therapeutic strength of epigenetic therapies was empowered by the fact that these agents improved patient tolerance at much low doses. DNA methyltransferase inhibitors were indeed internationally approved by the FDA for the treatment of patients with high-risk MDS and AML less than 30% blasts. More recently these drugs were also approved by EMA (European Medicines Agency) for AML with more than 30% of blasts and in combination with venetoclax for the management of elderly AML ineligible for high-intensity chemotherapy ,89. DNMT3A through somatic mutations are frequent in AML and independently associated with a poor outcome. Recently, DNMT3A mutations have been identified also in individuals carrying clonal hematopoiesis of indeterminate potential, a condition associated with risk of developing blood cancers including AML. DNMT1 is involved in cellular differentiation by methylating hemi-methylated sites after DNA replication and leading to hypo or hypermethylation. In fact, DNMT1 is a target of treatment in AML [®) and 5-aza-2′-deoxycytidine (DAC or Dacogen®) irreversibly inhibit the enzymatic functions of DNMTs and cause their proteasomal degradation by freely incorporating into DNA (DAC) and RNA (5-AZA) and depleting DNMTs. The DNA methylation process depends on the function of five enzymes called DNA methyltransferases  which are known to add an extra methyl group to DNA sequences. These enzymes are key players in AML pathogenesis. In particular alterations of t in AML ,91. NuclThe second class of epigenetic drugs are histone deacetylase (HDACs), a group of enzymes capable of removing acetyl groups on lysine residues in the N-terminal tail and on the nucleosome-core of histones and representing the ‘’epigenetic erasers’’ together with TET family of proteins. Deacetylated histones condense the chromatin structure reducing gene transcription levels. Additional drugs targeting epigenetic modifiers are the inhibitors of histone methyltransferases (HTMs) EZH2 and DOT1L, the histone demethylases LSD1, and proteins interacting with acetylated histones (BETs) see also . INK4), inducing apoptosis by upregulating TRAIL receptor-1 (TNF-related apoptosis-inducing ligand), repressing co-stimulatory molecules and molecules functioning in immunological recognition  and blocking T-cell response through increasing the expression of ligand for checkpoint inhibitor receptors [Given the exciting results in cellular models of AML, all these drugs have started early-phase clinical trials . Althougeceptors .2 5-AZA for the first seven days of each 28-day cycle, SC or IV according to individual tolerability. This study showed an overall composite CR of 52% . A phase 3 trial with both agents is now ongoing (NCT03151408). Combination of HMAs with the LSD1 inhibitor NCB059872 (NCT02712905) or with the PRTM5 inhibitor GSK3326595 (NCT03614728) is now under evaluation in clinical trials. HMAs have also been combined with immunotherapy agents as the case of DAC plus avelumab targeting DNMT and PD-L1 (NCT03395873) or 5-AZA plus nivolumab targeting DNMT and PD-1 (NCT0385367).In fact, combination strategies with other epigenetic drugs have been tested in the clinic; for instance, HDAC inhibitors in combination with HMAs. However, to date randomized studies have showed challenges including an antagonist effect possibly due to lack of clinical benefits. Given also the complexity of the epigenome, drug pharmacodynamics and schedule of administration might represent the biggest barriers to overcome in order to get the maximum efficacy of the agents. Examples of these combinations are exemplified by 5-AZA combined with HDAC inhibitors (entinostat or vorinostat) which showed similar results without leading to any benefits compared to single-agent 5-AZA. This might be due to increased toxicity attributable to combination therapy or to the fact that HDAC inhibitors have a wide number of biological targets which can generate diverse effects. Additional trials are ongoing with the next generation of HDAC inhibitors belinostat, panobinostat, and pracinostat ,97. The Histone acetyltransferases (HATs) small molecule inhibitors are also being tested. HAT are enzymes capable of transferring an acetyl group from acetyl coenzyme A to the ε-amino group of lysine residues in histones. Type A HATs consist of three families of enzymes  while type B HATs consist of KAT1. HAT inhibitors include bisubstrate inhibitors, natural compounds, and low molecular weight inhibitors. This is the case of the HAT paralogs p300 and CREB binding protein (CBP) inhibitors. Both p300 and CBP have oncogenic roles in AML and other malignancies. CCS1477 is a small molecule which binds selectively to p300/ CBP, decreases cellular proliferation of blast cells and upregulates differentiation-promoting markers . In AML cell lines derived xenografts, CCS1477 (20 mg/Kg) reduces tumor burden . CCS1477KAT6A plays key roles in normal hematopoiesis and is target of chromosomal translocations in AML.Inhibitors of the lysine acetyltransferases (KATs) have also been studied. Among several KATSs CDKN2A. Similarly, KAT6B has also being selected for targeted approaches as indicated by the development of potent KAT inhibitors (WM-8014 for KAT6A) and WM-1119 (KAT6B) [Studies have shown that KAT6A blocks senescence by regulating  (KAT6B) .MET, also called APR-246, specifically target mutant tp53, reactivating its function and showing an antileukemic activity in vitro models [Tp53 signaling pathway is essential for cell cycle regulation, senescence, and apoptosis . About 5o models . Chemicao models . A firsto models . In anoto models . Based oOnly about 30% of AML patients become long-term survivors. Still there is a paucity of pharmacologic agents for patients who relapsed or become refractory to treatment. Especially in case of unfit-for-chemotherapy patients, a new paradigm is impelling: A comprehensive targeted molecular study is needed to better choose the best tailored therapy to avoid unnecessary toxicity. Thus, not only younger patients but also elderly patients may be suitable of NGS-based approaches and benefit of a deeper molecular characterization of their disease. The availability of these new orally antileukemic compounds is rendering feasible treating also this setting of frail patients where the aim is now also a hematological improvement with transfusion independence and not only the achievement of a CR. Moreover, new insights are giving the rationale for studies on maintenance therapy in AML and this field may be of paramount interest in elderly patients, characterized by more aggressive AML phenotype and possibly taking advantage from a less intense and more prolonged treatment schedule. A lot of efforts have been made to test new drugs and their combination with existing agents see also . The res


In view of the well-maintained skeletal muscle found in Daurian ground squirrels (Spermophilus dauricus) during hibernation, we hypothesized that hibernators possess unique strategies of intracellular Ca2+ homeostasis. Here, cytoplasmic, sarcoplasmic reticulum (SR), and mitochondrial Ca2+ levels, as well as the potential Ca2+ regulatory mechanisms, were investigated in skeletal muscle fibers of Daurian ground squirrels at different stages of hibernation. The results showed that cytoplasmic Ca2+ levels increased in the skeletal muscle fibers during late torpor (LT) and inter-bout arousal (IBA), and partially recovered when the animals re-entered torpor . Furthermore, compared with levels in the summer active or pre-hibernation state, the activity and protein expression levels of six major Ca2+ channels/proteins were up-regulated during hibernation, including the store-operated Ca2+ entry (SOCE), ryanodine receptor 1 (RyR1), leucine zipper-EF-hand containing transmembrane protein 1 (LETM1), SR Ca2+ ATPase 1 (SERCA1), mitochondrial calcium uniporter complex (MCU complex), and calmodulin (CALM). Among these, the increased extracellular Ca2+ influx mediated by SOCE, SR Ca2+ release mediated by RyR1, and mitochondrial Ca2+ extrusion mediated by LETM1 may be triggers for the periodic elevation in cytoplasmic Ca2+ levels observed during hibernation. Furthermore, the increased SR Ca2+ uptake through SERCA1, mitochondrial Ca2+ uptake induced by MCU, and elevated free Ca2+ binding capacity mediated by CALM may be vital strategies in hibernating ground squirrels to attenuate cytoplasmic Ca2+ levels and restore Ca2+ homeostasis during hibernation. Compared with that in LT or IBA, the decreased extracellular Ca2+ influx mediated by SOCE and elevated mitochondrial Ca2+ uptake induced by MCU may be important mechanisms for the partial cytoplasmic Ca2+ recovery in ET. Overall, under extreme conditions, hibernating ground squirrels still possess the ability to maintain intracellular Ca2+ homeostasis.Intracellular calcium (Ca Skeletal muscle inactivity can trigger Ca2+ homeostasis disturbance, often characterized by cytoplasmic Ca2+ overload  and TM BCA Protein Quantitation kit . The supernatants were mixed with 1 × SDS loading buffer  at a 1:4 v/v ratio, followed by boiling and then storage at −20 °C for further analysis.Muscle samples (~0.1 g) were weighed and fully homogenized with 1 mM RIPA Lysis Buffer , 1% protease inhibitor cocktail , and 1% phenylmethylsulfonyl fluoride . After 15 min of centrifugation at 4 °C and 15,000 rpm, the supernatants were removed and placed into new tubes, with soluble protein concentrations then detected using a PierceWestern blotting procedures were as described by Zhang et al. (2017) . In brieBriefly, 10-μm thick frozen cross-sections were cut from the muscle mid-belly at −20 °C with a CM1850 cryostat . After fixing samples in 4% paraformaldehyde for 30 min, slices were permeabilized in 0.1% Triton X-100 for 30 min, blocked with 1% BSA in PBS at room temperature for 60 min, and then incubated at 4 °C overnight with an anti-ORAI1 antibody . On the second day, after washing samples three times with PBS (10 min/time), the sections were incubated at 37 °C for 2 h with an Alexa Fluor FITC-conjugated secondary antibody . After again washing samples three times with PBS (10 min/time), the slices were incubated at 4 °C overnight with an anti-STIM1 antibody . On the third day, after washing samples three times with PBS (10 min/time), the sections were incubated at 37 °C for 2 h with a 647-labeled IgG secondary antibody . The slices were then washed three times with PBS (10 min/time) and dried and treated with anti-fade mounting medium . Images were visualized and captured via confocal laser scanning microscopy  at a 40× objective magnification with krypton/argon laser illumination at 488 and 647 nm and captured at 526 and 665 nm. As previously described ,35, the p < 0.05 was considered statistically significant.Data are presented as means ± SEM. SPSS Statistics 17.0 was used for all statistical tests. Group differences were determined via one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) post hoc test. When no homogeneity was detected, ANOVA-Dunnett’s T3 method was applied. A value of Changes in skeletal muscle morphology were observed by analyzing the muscle mass (MM) B and mus2+ level in the single PL and AM muscle fibers increased significantly during hibernation, and almost recovered to SA levels post-hibernation. During the torpor-arousal cycle, the cytoplasmic Ca2+ level partially recovered when animals re-entered the torpor state. Compared with the results in the LT group, significant decreases in cytoplasmic Ca2+ levels were observed in the PL (24%) and AM muscles (32%) of the ET group   .2+ levels during different periods. Firstly, the opposite changes in cytoplasmic and SR Ca2+ suggest that SR Ca2+ participates in fluctuation of the cytoplasmic Ca2+ level during hibernation. In addition, the slight elevation in mitochondrial Ca2+ during hibernation may result from increased cytoplasmic Ca2+ or SR Ca2+ leakage. Further studies were subsequently carried out to explore the mechanisms involved in intracellular Ca2+ fluctuations during hibernation.We comprehensively analyzed the changes in cytoplasmic, SR, and mitochondrial Ca2+ transport proteins/channels located in the cytoplasm, SR, and mitochondria, including STIM1, ORAI1, RyR1, LETM1, PMCA3, SERCA1, MCU, MICU1, MICU2, and the free Ca2+ binding protein CALM, as well as the Pearson correlation coefficients for the co-localization of ORAI1 and STIM1, were detected to explore the potential mechanisms involved in intracellular Ca2+ fluctuation during hibernation. It should be clarified that, as mRNA expression is very sensitive to both internal and external environmental factors, large variability in the size of the error bars occurred in the mRNA statistical results. Therefore, we set a fold-change of ≥2-fold as the threshold for the biological significance of mRNA expression.The mRNA and protein expression levels of several major Ca2+ level fluctuation during hibernation. In the PL muscle, compared with that in the SA group, the mRNA expression levels of both STIM1 and ORAI1 increased during IBA  in the IBA and ET groups. In the AM muscle, compared with that in the SA group, the mRNA expression of LETM1 was dramatically elevated in the POST group  were first detected. The results showed that, compared with the SA and PRE groups, the mRNA level of PMCA3 in the PL muscle increased significantly in the IBA and ET groups  of skeletal muscle fibers during different hibernation stages. The results showed that the cytoplasmic Ca2+ levels in the PL and AM muscles were elevated to varying degrees during hibernation, suggesting that, similar to the phenomenon found in non-hibernators, prolonged skeletal muscle disuse during hibernation was accompanied by elevated cytoplasmic Ca2+ levels [2+ levels were observed during the torpor-arousal cycle, with an increase during LT and partial recovery when the squirrels re-entered torpor after transient arousal. Therefore, periodic arousal from torpor may be a strategy employed by hibernators to adjust and stabilize cytoplasmic Ca2+ levels to effectively avoid excessive Ca2+-induced skeletal muscle loss or damage. Our previous study found that, compared with pre-hibernation levels, serum Ca2+ concentrations in ground squirrels increased significantly during hibernation and recovered after post-hibernation [2+ concentrations during hibernation influence cytoplasmic Ca2+ levels. SOCE is the main channel of extracellular Ca2+ influx. Higher co-localization coefficients of its two major components, i.e., STIM1 and ORAI1, represent a higher activation probability of SOCE [2+ influx mediated by SOCE may be one reason for the increased cytoplasmic Ca2+ level during LT and IBA. In addition to extracellular Ca2+ influx, Ca2+ release from intracellular Ca2+ storage can also cause elevated cytoplasmic Ca2+ levels. RyR1 is the main Ca2+ release channel located in the SR. The results showed that its protein expression level was up-regulated to varying degrees during different periods of hibernation. Combined with the phenomenon that the Ca2+ concentration in the SR showed the opposite change to that of cytoplasmic Ca2+ during hibernation, the increased release of SR Ca2+ was likely a major reason for the increased Ca2+ level in skeletal muscles of ground squirrels during hibernation. In addition to SR Ca2+ release, several proteins also mediated Ca2+ efflux from the mitochondria to the cytoplasm. The present study showed that the protein expression of LETM1, a major channel that mediates Ca2+ efflux in the mitochondrial membrane, was increased to varying degrees during different periods of hibernation, intimating that Ca2+ release mediated by LETM1 in the mitochondria may also be involved in the increase in cytoplasmic Ca2+ levels during hibernation.During prolonged hibernation, hibernators experience various stressful conditions, including prolonged inactivity, hypoxia, fasting, and repeated ischemia-reperfusion from the torpor-arousal cycle. Here, after several months of hindlimb inactivity during hibernation, slight decreases in muscle mass and single muscle fiber CSA (15–20%) were observed, suggesting that only limited skeletal muscle loss occurs in the PL and AM muscles, consistent with our previous report on PL and gastrocnemius muscles . Obvious+ levels ,38,39,40ernation . This ra of SOCE . Our res2+ pump located in the cell membrane, PMCA3 can pump intracellular Ca2+ out of the cell. In the current study, however, the PMCA3 protein expression levels showed no significant differences during hibernation. Therefore, PMCA3 did not appear to play a substantial role in the fluctuation of cytoplasmic Ca2+ in hibernation. In contrast, the protein expression levels of SERCA1, a major Ca2+ uptake channel in the SR, were significantly up-regulated during different stages of hibernation, contrary to the decrease in SERCA activity found in non-hibernating animals under disuse conditions [2+ into the SR, thus avoiding an excessive increase in cytoplasmic Ca2+ and related skeletal muscle injury. In addition, the protein expression of MCU, a major Ca2+ absorption channel located in the mitochondrial membrane, was elevated during hibernation, and may therefore be another important mechanism for the absorption of Ca2+ into the mitochondria and alleviation of the Ca2+ level in the cytoplasm. However, we know that when the concentration of mitochondrial Ca2+ reaches a certain threshold, mitochondrial depolarization is triggered and, as a consequence, the pro-apoptotic protein Bax is activated, translocated, and inserted into the outer membrane via Bax/Bax-homo-oligomerization [2+ only increased slightly during hibernation. Therefore, the simultaneously higher expression of MCU and LETM1 may be a strategy employed by hibernating ground squirrels to control the balance between cytoplasmic and mitochondrial Ca2+, and thereby avoid mitochondrial Ca2+ concentration-induced skeletal muscle damage, such as cell apoptosis. As a free Ca2+ binding protein, CALM can relieve the elevation of cytoplasmic Ca2+ by binding to free Ca2+ in the cytoplasm. Our results showed that the protein expression of CALM in the PL muscle increased significantly, and the enhanced free Ca2+ binding capacity mediated by the elevated protein expression of CALM may thus be another important mechanism in hibernating ground squirrels to alleviate cytoplasmic Ca2+ levels during hibernation.As a Canditions . Our prenditions . Other snditions ,44. Suchrization . This isrization . In the 2+ level showed increased-decreased fluctuation. Compared with that during LT and IBA, the cytoplasmic Ca2+ level in the skeletal muscles of the Daurian ground squirrels was down-regulated to varying degrees after re-entry into torpor (ET). Compared with levels in LT, the co-localization coefficients of STIM1 and ORAI1 decreased to varying degrees, representing decreased Ca2+ influx mediated by SOCE, which may be another critical mechanism used to avoid the intracellular Ca2+ increase caused by persistent Ca2+ influx during hibernation. In addition, we found that the higher protein expression of Ca2+ uptake channel MCU in ET may be another vital mechanism through which PL muscle can relieve cytoplasmic Ca2+ overload. It is worth noting that, after arousal from hibernation, the intracellular Ca2+ levels  returned to the levels found in the SA and PRE groups, thus reflecting a strong and rapid Ca2+ recovery ability of these ground squirrels. We further found that the expression of Ca2+ transport proteins/channels or free Ca2+ binding protein also returned to the levels observed in the SA and PRE groups. This may be due to the sampling time of the POST group, which occurred 3 d after the squirrels aroused from torpor, and all physiological states had returned to normal.During the torpor-arousal cycle, the intracellular Ca2+ transporter protein, we found that the levels were not always consistent in the same stage. In particular, during LT, the mRNA levels of most Ca2+ transport proteins/channels were decreased or showed no significant change, whereas the corresponding protein expression levels were significantly increased. In view of these results, we checked the transcription and proteomic analysis results of the skeletal muscles during different hibernation stages (unpublished data) and found that the predicted results were consistent with our experimental findings. Previous studies have demonstrated that transcriptional elongation and initiation are essentially arrested during hibernation [2+ transport proteins/channels in skeletal muscles of Daurian ground squirrels during LT. In other words, the inconsistent changes in mRNA and protein expression levels may be the result of total inhibition at the transcriptional level during LT, although the corresponding protein expression was not affected during hibernation.By comparing and analyzing the mRNA and protein expression levels of each Caernation ,48, whic2+ operation ability. Here, by regulating the activity and protein expression of Ca2+ pumps, Ca2+ channels, and Ca2+ binding proteins in the cytoplasm, SR, and mitochondrial membrane, the dynamic balance of intracellular Ca2+ homeostasis was well-maintained during hibernation. Therefore, maintaining intracellular Ca2+ homeostasis and avoiding skeletal muscle injury caused by its disturbance appear to be priority strategies employed by hibernating squirrels to cope with the various stresses induced during the torpor-arousal cycle. In summary, despite experiencing stresses such as a low temperature, low metabolism, and prolonged hindlimb inactivity during hibernation, hibernating ground squirrels still possess a strong Ca