Here, we developed biodegradable harmonophores (bioharmonophores)that consist of polymer-encapsulated, self-assembling peptides thatgenerate a strong SHG signal. When functionalized with tumor cellsurface markers, these reporters can target single cancer cells withhigh detection sensitivity in zebrafish embryos in vivo. Thus, bioharmonophores will enable an innovative approach to cancertreatment using targeted high-resolution optical imaging for diagnosticsand therapy.Opticalimaging probes have played a major role in detecting andmonitoring a variety of diseases. In particular, nonlinear opticalimaging probes, such as second harmonic generating (SHG) nanoprobes,hold great promise as clinical contrast agents, as they can be imagedwith little background signal and unmatched long-term photostability.As their chemical composition often includes transition metals, theuse of inorganic SHG nanoprobes can raise long-term health concerns.Ideally, contrast agents for biomedical applications should be degraded  To this end, we subjectedseveral peptides that have been reported to self-assemble into complexnanostructures to the emulsion–solvent evaporation method,articles .19 trileucine,20 andtriphenylalanine21) that could generatea detectable SHG signal when encapsulated in the biodegradable polymer. Transmission electronmicroscopy (TEM) analysis of the predominantly spherical nanoparticles,hereinafter referred to as bioharmonophores, revealed a diameter rangingfrom 50 to 150 nm , which was confirmed by dynamic light scattering (DLS) measurements.SHG signal from bioharmonophores can stem from (i) the bulk ofthe self-assembling peptides that form noncentrosymmetric crystallinestructures or (ii) the surface of the bioharmonophores where thereis no inversion symmetry. To ascertain that the SHG signal originatesfrom the crystalline peptide core, we performed X-ray diffraction(XRD) analysis of bioharmonophores with different peptide contents.In all cases, the peptides showed a high degree of internal orderwith distinct diffraction patterns associated with their individualcrystalline phases and self-assembling behavior . Additionally, the SHG emission patterns of FFF-based bioharmonophoresdisplayed a broad opening: one seemingly isotropic , unlike the predominantly forward-directed SHG signal of largeprotein arrays, such as collagen and myosin that require a trans-detectionapproach .10 Thisdifference in detection geometry allows exclusion of a collagen-basedSHG signal during imaging, leading to a high SNR bioharmonophore signalthat can be distinguished from large-scale collagen arrays even intissues with high SHG background. Moreover, the presence of a singlelobe suggests that the observed SHG signal originates from the bulk22 of the bioharmonophores and not from its surface,as described by nonlinear light scattering theory.23 The high intensity emission in the PPP polarization combinationfurther indicates that a coherent response originates from a noncentrosymmetriccrystalline bulk.Becausebioharmonophores based on triphenylalanine (FFF) peptides yieldedthe strongest SHG signal compared to pentaalanine and trileucine,we subjected these bioharmonophores to detailed optical characterizations.The SHG signal of FFF-based bioharmonophores was spectrally well-defined2a and coSupplementary Figure 5a,b). The measured polarimetricdiagrams  wereconsistent with the hypothesis that bioharmonophores have a self-assemblingpeptide core with a monoclinic (C2) symmetry. Indeed,the experimental curves were well fitted with the analytical expressioncalculated for this symmetry (see Supplementary Note 1). Taken together, bioharmonophores have the same photophysicaladvantages for biomedical imaging applications that have been previouslydescribed for inorganic SHG nanoprobes.6Because SHG involves only virtual energy transitions, bioharmonophoresdid not display blinking and remained stable over extended periodsof illumination, and their SHG signal intensity rose quadraticallywhen the laser intensity shone on them was linearly increased . We found that an amountof 15 mg of FFF (33 wt %) peptide provided an optimal combinationof intense SHG signal and bioharmonophore stability. Interestingly,while an FFF peptide amount of 20 mg (40 wt %) increased the overallSHG signal, it also led to bioharmonophore aggregation and decreasedcolloidal stability. Conversely, 10 mg (25 wt %) of FFF peptide generatedlittle SHG signal.To gain insight into the parameters influencing the bioharmonophorestability and signal intensity, we tested several reaction conditionsto generate bioharmonophores. Given that the SHG signal originatingfrom bioharmonophores is dependent on the amount of encapsulated peptide,we first tested whether varying the FFF peptide concentration duringproduction would improve the SHG signal intensity of generated bioharmonophores. Bioharmonophores emulsifiedin an aqueous solution with 0.3% sodium dodecyl sulfate (SDS) (40wt % of dispersed phase) yielded stable bioharmonophores with intenseSHG signal, whereas compositions employing 0.1% SDS (18 wt %) yieldedaggregated nanoparticles. Increasing the SDS concentration to 0.6%(57 wt %) diminished the SHG signal intensity, suggesting that thebioharmonophore size and hence the number of enclosed peptide moleculeswithin each bioharmonophore is influenced by the concentration ofsurfactant.Because surfactant concentration plays acrucial role in emulsificationof chloroform droplets,Supplementary Figure 8). We identified that an amount of 30 mg of poly(l-lactic acid) (PLLA) (66 wt %) resulted in an optimal combinationof intense SHG signal and bioharmonophore stability. A lower polymeramount of 10 mg (40 wt %) yielded a weaker SHG signal, whereas a higherpolymer amount  led to elongated bioharmonophore morphologies.Taken together, we identified optimal experimental conditions to generatebioharmonophores providing a high SNR along with an excellent stabilityand size distribution for biological applications.Finally, we varied the polymer quantity that encapsulatesand shieldspeptides from environmental changes and assessed its role in bothSHG signal intensity and nanoparticle morphology , indicating a successfulbiodegradation of the bioharmonophore. When characterized by TEM imaging,the structural integrity of the bioharmonophore coat started to disintegratewithin 4 h of Proteinase K treatment .Clinical imagingprobes that are biodegradable provide the significant advantage ofbeing able to be broken down in the body and removed after they haveserved their function. To demonstrate that bioharmonophores are indeedbiodegradable, we utilized the highly effective serine protease, proteinaseK, which exhibits a broad cleavage specificity.ntervals 3a. We ob26 using bioorthogonal click chemistry  and incubated them witha model cancer cell line (see below) overnight. Adherent cells werethen detached by trypsinization and centrifuged to remove excess bioharmonophoresthat did not enter the cancer cells. Following this procedure, cellswere reseeded and fixed at specific time periods to monitor bioharmonophoredegradation  using nonlinear optical imaging. Thirty hoursafter cell reseeding, a pronounced decrease of intracellular SHG signalper cell was noticeable , the drop of signal was not due to their potential accumulationin acidic endolysosomal compartments over time. Immunostaining ofuptaken bioharmonophores indeed revealed that bright SHG signal stillpersisted in lysosome-associated membrane protein 2 (LAMP2)-positiveendocytic compartments prior to degradation . In order to show that bioharmonophores can be degradedusing intracellular proteolytic degradation, we tested whether thebioharmonophores could be degraded using a cell-free lysate systembased on an established cell-free degradation assay.27 We also observed reduced SHG signal, indicating that intracellularenzymatic degradation of bioharmonophores might account for the signalloss . Importantly, bioharmonophoresdid not exhibit any short-term toxicity in vitro and in vivo  and did not induce protein aggregation ,28 rendering them safeimaging probes.To evaluate bioharmonophore degradation under physiological conditions3c, we fu2 To demonstrate the optical features of bioharmonophoresfor cancer targeting and imaging, we employed xenograft zebrafishcancer models, which offer speed, cellular resolution, and the abilityto perform large numbers of transplants for obtaining valuable informationabout several cancer types.30Among variousdiagnostic applications, bioharmonophores could be ideal imaging probesfor single-cell cancer detection due to their high SNR and photostability,which other intravital imaging modalities cannot achieve.30 By 3 days after the injection,the resulting tumors spread to various locations in the body and werefound next to blood vessels, which likely support the tumors withnutrients into the Duct of Cuvier (DoC) of zebrafish embryos at 2 dpf (dayspost fertilization) 4a.29,30 utrients 4b.31in vivo, we targeted bioharmonophores to tumorsites by taking advantage of the surface protein p32/gC1qR as a characteristicmolecular marker for MDA-MB-435-DsRed cells.32 To this end, we functionalized bioharmonophores with a p32 targetingpeptide, injected them into the DoC of zebrafish embryos at 3 dpf,1 day after the embryos were injected with MDA-MB-435-DsRed cancercells (Supplementary Note 2). In the absence of tumors, functionalized bioharmonophores didnot cause clustering at the site of injection and were localized atdifferent parts of injected zebrafish embryos , indicating good biodistribution.Without bioharmonophore injection, zebrafish as well as tumor sitesdid not produce any SHG background signal  with the exception ofminimal endogenous SHG signal localized at the zebrafish tail,33 which was excluded from assessing specificityof tumor targeting . In the case of passive targeting, zebrafish injected with PEG-coatedbioharmonophores revealed limited tumor labeling , stemming from leaky bloodvessels and enhanced permeability and retention effect (EPR).34 In contrast, we observed an increased accumulationof p32 peptide-targeted bioharmonophores within individual cancercells at tumor sites throughout the zebrafish embryos (35 andthe continued proliferation and metastasis of cancer cells betweenbioharmonophore administration and imaging (see Supplementary Note 2).To demonstrate the specificity and efficiency of bioharmonophoresas contrast agents that can accomplish resolution down to the singlecell ercells 4c,and aSupplementary Note 2 and Supplementary Figure 20). The zebrafish cancer model injected with p32 peptide-targetedbioharmonophores had a significantly higher fraction of labeled tumorscompared with noninjected and PEG-coated bioharmonophores  at 100 mg/mLconcentration prior to experiments and diluted to 5 mg/mL final concentrationin deionized water.Diphenylalanine(FF) and triphenylalanine (FFF) (Bachem) peptide assemblies were preparedas previously described.Supplementary Figures 6–8.Forthe evaluation of different peptides and their SHG capabilities, 30mg of PLLA was dissolved in 3 mL of chloroform (Sigma) along with15 mg of triphenylalanine, 30 mg of pentalalanine (Bachem), and 30mg of trileucine (Sigma) peptides in separate glass vials. The resultingsuspension was mixed with aqueous SDS (Sigma) solution with a final0.3% SDS concentration. A macroemulsion was obtained by stirring thesamples at 1000 rpm for 1 h. Afterward, the samples were sonicated(Branson Sonifier) with a 1.5 in. tip at 70% power in a pulsed mode(30 s ON and 10 s OFF) for 2 min under ice cooling. The chloroformwas evaporated from the obtained emulsions by stirring the samplesat 500 rpm at 40 °C overnight. For the remaining experimentswith triphenylalanine peptide containing bioharmonophores, the sameprotocol was followed unless stated otherwise. For probing the optimalconditions for nanoparticle formation, FFF peptide, PLLA, and SDSconcentrations were varied as described in Produced samples were characterized usingdynamic light scattering.Nanoparticle morphology, aggregation tendency, and the SHG signalintensity were evaluated using nonlinear microscopy. XRD patternswere obtained using a PANalytical X’PERT Pro powder diffractometerin Bragg–Brentano geometry and with Cu K-α1 radiationin grazing incidence geometry between 2–60 using a step sizeof 0.0167. The samples were air-dried on silicon single crystals,and four identical scans were obtained from each sample and summedup.Supporting Information). A 1030 nm laser, pulse width 190 fs, and 200kHz repetition rate , delivered 36 mW onthe sample over a 150 μm fwhm diameter field-of-view (1 mJ·cm–2). Two non-colinear beams are incident on the sample,with a 30° opening angle. The SHG signal was detected in thephase matching direction (transmission). The image was recorded withan electron-multiplying intensified charge-coupled device (EM-ICCD)camera. Nonlinear polarimetry was performed by controlling and analyzingthe polarization state of the illuminating and emitted beams. A polarizationstate generator, comprising a half- and a quarter-wave plate, wasused. The polarization state of the emitted light was analyzed witha half-wave plate placed in the emission path, followed by a polarizingbeam splitter.The SHG polarimetry was performed ona wide-field SHG microscope (see the Supporting Information). Excitation was performed with a 1030 nm laser, pulse width 190fs, and 200 kHz repetition rate, which delivered 60 mW on the sample,a cylindrical cuvette containing the solution, over a 36 μmfocal spot (30 mJ·cm–2). The signal was detectedwith a rotating PMT and a filter  at angles between−90 and 90. Both incident and detection polarizations was controlled.SHG emissionpattern measurement was performed on a custom-build setup for thispurpose . In a typicalsetup, 1 mL of bioharmonophore in 1% Tween 80 in 1X PBS was mixedwith 25 mM phosphocreatine (Sigma), 10 μg/mL phosphocreatinekinase (Sigma), 1 mM ATP (Sigma), and 50 μL Rabbit ReticulocyteLysate. The mixture was incubated at 37 °C, and the SHG signalintensity was monitored every 2 h.Bioharmonophoreswere centrifuged for 3 min at 13500 rpmand resuspended in 1% Tween 80 in 1X PBS. In order to assess proteinaseK (Sigma) degradation, 1 mL of bioharmonophore suspension was incubatedwith 100 μg/mL final proteinase K concentration at 37 °Cand the SHG signal intensity was measured every 2 h. Similarly, inorder to assess how bioharmonophores were degraded using cellularcontent, an Candida antarctica Lipase B (Sigma) for 2 h, which hydrolyzesthe PLLA polymer to increase the number of carboxyl groups. The bioharmonophoresuspension was centrifuged at 13500 rpm for 3 min and resuspendedin 1% Tween 80 in 1X PBS and mixed with 10 mg of N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidehydrochloride (EDC) (Sigma), 10 mg of N-hydroxysuccinimide(NHS) (Sigma), and 10 mg of methoxypolyethylene glycol amine 5000Da (mPEG Amine) (Sigma) for 2 h. The suspension was centrifuged andresuspended in 1% Tween 80 in 1X PBS and stored at 4 °C priorto use.Onemilliliter of 1.5 mg/mL bioharmonophores was incubated with 1 mg of 2) (Sigma) was used as a platform forbioorthogonal click chemistry. In a similar setup, 10 mg of EDC, 10mg of NHS, and 10 mg of thiol-PEG-NH2 were incubated for2 h. The suspension was centrifuged and resuspended in 1% Tween 80in 1X PBS with methyltetrazine-PEG4-maleimide (Click Chemistry Tools)of 200 μM final concentration. The mixture was incubated for2 h at room temperature, centrifuged, and resuspended in 1% Tween80 in 1X PBS.For further functionalization experiments thiol-PEG-amine2000 Da (SH-PEG-NHtrans-cyclooctene(TCO)-PEG3-maleimide (Click Chemistry Tools) (3 mM in 200 μLof PBS) was incubated for 2 h with cysteine-containing Tat or P32targeting peptides  depending on the application.The peptides were passed through Illustra Microspin G25 columns  to remove TCO-PEG3-maleimide.The other click chemistry pair A 200 μLportionof tetrazine-modified bioharmonophores wasincubated with 20 μL of TCO-modified peptide for 2 h. The bioharmonophoresuspension was washed with 1% Tween 80 in 1X PBS to remove unboundpeptides and resuspended in 1X PBS to be immediately used for cellculture experiments.2, in high glucose DMEM withGlutaMAX , supplemented with 10% FBS  and 1X penicillin–streptomycin solution . The cells were cultured on six-well plates  until they reached ∼80% confluency and wereincubated with 400 μL Tat-derived cell penetrating peptide coatedbioharmonophores overnight. The cells were washed with 1X PBS twiceand detached using 0.05% Trypsin-EDTA  inorder to remove bioharmonophores that did not enter the cancer cells.Detached cells were centrifuged for 5 min at 500g to remove excess bioharmonophores that were not taken up, reseededor ibiTreat-coated on eight-well slides , and fixedafter 6, 30, 54, and 78 h to monitor bioharmonophores degradation.The samples were then washed three times with 1X PBS and stained withthe CellMask Orange Membrane dye (Invitrogen). The samples were washedagain and imaged subsequently. To determine cell viability after treatmentwith functionalized bioharmonophores, trypan blue exclusion methodwas used. Briefly, cells in triplicates seeded in 96-well tissue cultureplates  were exposed to varying concentrationsof functionalized bioharmonophores for 48 or 72 h. After incubation,cells were washed with 1X PBS twice and detached as described above.Ten microliters of the cell suspension was then mixed with 10 μLof 0.4% Trypan Blue, and 4 μL of this mixture was added to thecell-counting slide  and measured using CountessII Automated cell counter (Thermo Fisher). The viability was expressedas a fold difference of the untreated samples for each time point.MDA-MB-435-DsRedcancer cells were kindly gifted by Prof. R. Klemke. The cells werecultured at 37 °C, 5% CO2, in highglucose DMEM with GlutaMAX , supplementedwith 10% FBS  and 1× penicillin–streptomycinsolution . A 13 mm round coverslip was coatedwith 0.1% gelatin  for 1 h, and cells were seeded toreach around 70% confluency. The following day, 400 μL of freshlyprepared thiol-PEG functionalized bioharmonophores47 (2 mg/mL) or an equal volume of 1X PBS (control) was added to thecells and incubated for 24 h. The cells were washed with 1X DPBS  to remove theexcess of not internalized bioharmonophores, fixed with 4% PFA for15 min, permeabilized with 1X PBST (1X PBS containing 0.01% Tween20)for 15 min, blocked with 10% goat serum in 1X PBST for at least 1h, and incubated overnight with mouse anti-LAMP2  in 5% goat serum at 4 °C. The next day, cells were rinsedwith 1X PBST and incubated for 1 h with the goat antimouse Alexa488conjugated secondary antibody ,washed thoroughly with 1X PBST, and mounted in mowiol mounting mediumcontaining 2.5% v/v DABCO .HeLa cells were cultured at 37 °C, 5% COFor toxicityassay, cells were grown in 96-well plates and were incubated withbioharmonophores at different concentrations for 48 and 72 h. Afterthe incubation period, the cells were detached with trypsinization,and their viability was analyzed using Trypan Blue (Sigma) staining.For thioflavin staining, cells were seeded in an eight-well chamber(ibidi) with 50% confluency. The cells were treated with either AmyloidBeta Peptide (Bachem) or 5 μL of bioharmonophores for 24 h andextensively washed with 1X PBS to remove excess peptides and bioharmonophores.To evaluate whether bioharmonophores induce fibril formation the cellswere fixed with 4% PFA for 10 min and washed with 1X PBS three times.Afterward, 0.05% Thioflavin T (Sigma) solution was added to the samplefor 8 min, and excess dye was washed with 80% ethanol for 5 min. Thewashing step was repeated three times, and the samples were imagedusing confocal microscopy.Tg(fli1:egfp) zebrafish embryos at 2 days post fertilization (dpf). After injection,embryos were incubated for 1 h at 29 °C for recovery and celltransfer then verified by fluorescence microscopy. Fish harboringred cells were incubated at 35 °C essentially as described before.30 Fish were anaesthetized and embedded in low melting agarose as describedpreviously48 and were imaged at 5 dpf forassessing cancer cell localization.Animal experiments and zebrafish husbandry wereapproved by the “KantonalesVeterinaeramt Basel-Stadt”. MDA-MB-435-DsRed cancer cells wereinjected into the Duct of Cuvier of In vivo bioharmonophores targetingwas evaluated at 5 dpf using nonlinear laser scanning microscopy.For targeting experiments,p32/gC1qR ligand-functionalized bioharmonophores were injected intothe zebrafish embryos 24 h after cancer cell injection following thesame procedure. i.e., thesupernatant of the centrifuged solution) was placed on a carbon coatedgrid  previously glow-discharged for 30 s . The drop was allowed to remain for 60 s; after this interval,excess fluid was drained along the periphery using a piece of filterpaper followed by staining with 2% uranyl acetate for 1 and 15 s,respectively. Excess moisture was drained after each step, and whendry, the grid was examined in an FEI Morgagni 268 TEM operated at100 kV.Bioharmonophore sampleswere spun down to remove aggregated nanoparticles at 3000 rpm for3 min. Five microliters of the sample (Bioharmonophoreswere immobilized in low melting agarose by mixing 200 μL ofbioharmonophore with 100 μL of 1% SeaPlaque low melting agarose(Lonza) solution in eight-well imaging chambers (Lab-Tek). Imagingexperiments were performed either on a Zeiss LSM 780 microscope (CarlZeiss AG) equipped with a spectral GaAsP detector and a tunable two-photonlaser source  using an LD C-Apochromat40×/1.1 water immersion objective lens (Carl Zeiss AG) or ona Leica SP5 microscope equipped with 2 Hybrid Detectors and a tunable two-photon laser source ,using a HC PL Apochromat 63×/1.40 Oil immersion objective lens(Leica). Throughout the imaging experiments, bioharmonophores wereilluminated with 850 nm incident wavelength, and the SHG signal wascollected between 405 and 435 nm or with a GaAsP spectral wavelengthdetector for spectral measurements.n) are given in thefigure legends for each experiment. Each experiment was repeated atleast three times. Normal distribution of data sets was establishedusing the D’Agostino & Pearson omnibus normality test where P > 0.05 indicated Gaussian distribution. When all ofthedata sets had Gaussian distribution, one-way Anova was used for multiplecomparisons followed by Tukey’s multiple comparisons. Whenone or more data sets showed a non-Gaussian distribution or high degreeof variance as in the case of zebrafish tumor models, a Kruskal–Wallistest was applied along with Dunn’s multiple comparisons. Forall statistical tests, a P value was reported, n.s., P > 0.05, *, P < 0.05, **, P < 0.005, ***, P < 0.001, ****, P < 0.0001. Second-order polynomial fit and one phaseexponentialdecay values were calculated, and graphs were drawn using GraphPadPrism 6.All numerical values representmean ± s.d. Sample sizes (


Calcium, as a second messenger, plays an important role in the pathogenesis of cardiovascular diseases (CVDs). The malfunction of calcium signaling in endothelial cells and vascular smooth muscle cells promotes hypertension. In cardiomyocytes, calcium overload induces apoptosis, leading to myocardial infarction and arrhythmias. Moreover, the calcium–calcineurin–nuclear factor of activated T cells (NFAT) pathway is essential for expressing the cardiac pro-hypertrophic gene. Heart failure is also characterized by reduced calcium transient amplitude and enhanced sarcoplasmic reticulum (SR) calcium leakage. Traditional Chinese medicine (TCM) has been used to treat CVDs for thousands of years in China. Because of its multicomponent and multitarget characteristics, TCM's unique advantages in CVD treatment are closely related to the modulation of multiple calcium handling proteins and calcium signaling pathways in different types of cells involved in distinct CVDs. Thus, we systematically review the diverse mechanisms of TCM in regulating calcium pathways to treat various types of CVDs, ranging from hypertrophic cardiomyopathy to diabetic heart disease. Cardiovascular diseases (CVDs) have become common diseases and the main cause of death worldwide . In 20162+), as a second messenger, play an important role in different types of cells involved in various CVD conditions, such as hypertension, arrhythmias, and myocardial infarction (2+ concentration ([Ca2+]i) can effectively change the activity of vasodilation/contraction in vascular smooth muscle cells (VSMCs) , initiating the cardiac hypertrophy   . Endotheeostasis . In cardeostasis . Dysreguertrophy . For an hythmias . Furtherfarction , and incure (HF) . Moreoveure (HF) . Thus, c reviews .Salvia miltiorrhiza represents the most common prescription  has been used to treat CVDs for thousands of years, with antioxidative, anti-inflammatory, and protective effects on vascular endothelial cells . For exacription . A meta-oginseng . Accumuloginseng . For insmodeling . Ginsengmodeling . Based o2+/calmodulin (CaM), calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) in the cytosol and promotes NFAT translocation into the cell nucleus, which regulates gene expression during cardiac hypertrophy  activation, and to prevent phospholamban (PLN) phosphorylation -induced cardiomyocyte hypertrophy because it could attenuate the ISO-mediated upregulation of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), calcineurin, NFATc3, and β-myosin heavy chain (β-MHC) , which has been shown to play an essential role in anti-myocardial hypertrophy by targeting the Ca2+-mediated signaling cascade, including prevention of hypertrophy-related NFATc3 nuclear translocation and downregulation of calmodulin-dependent protein kinase II (CaMKII) activity  . Astragaactivity . Smilax activity . Moreoveactivity . Scutell pathway . Taken tertrophy .2+ overload in myocardial cells and generate heart rhythm irregularities and other myocardial dysfunctions , or too slow, i.e., bradycardia, which could occur in sick sinus node syndrome . There aunctions .Sophora tonkinensis was revealed in a rat arrhythmia model induced by coronary artery ligation. Matrine ameliorated ischemia-mediated abnormal reduction of intracellular Ca2+, presumably by enhancing the activity of LTCCs  and calcium currents (ICa), and changing the electromechanical properties of isolated rat cardiomyocytes (ICaL) to reduce oscillatory afterpotentials or extrasystoles , transient outward potassium current (Ito), and the downstream Ca2+/CaMKII pathway  and Ca2+ overload induced by ischemia-reperfusion and protected cardiomyocytes from arrhythmia and death  , a widely used TCM in China, prepared from five ingredients:  pathway . Ischemioverload . WXKL prnd death . Some clnd death . Shensonnd IK1) . QLQX carhythmia , and exprhythmia . In addifunction . Thus, Treatment . Indeed,reatment .Hypertension is a multifactorial disease and a known risk factor for CVDs . The morSalvia miltirrhiza , which could reduce vascular tension by inhibiting the phosphorylation of the myosin light chain (MLC) and regulate the receptor-operated channel-mediated cytosolic calcium flux induced by phenylephrine (PE) or norepinephrine (NE). Therefore, ICB can potentially be used to treat vascular hypertension  /Akt signaling pathway, and Ca2+ and K+ channels are also involved , thereby relaxing blood vessels and relieving subsequent angina  . Berberi (SERCA) . Tetrahyinvolved . Studiesinvolved . The inft angina . Therefo disease .2+ transient amplitude of cardiomyocytes is decreased, together with enhanced SR Ca2+ leakage and reduced SR Ca2+ uptake , also has the potential to treat HF because it inhibits LTCCs and protects against myocardial damage caused by Ca2+ overload  and the signaling pathways of apoptosis and autophagy (Xin-Ke-Shu (XKS), Guanxinshutong capsule (GXSTC), modified Yiqi decoction (MYQ), and other Chinese herbal compound preparations are involved in the regulation of calcium signaling during the treatment of coronary heart disease and myocardial infarction  Table 5Table 5. tability . GXSTC imyocytes . Studiesutophagy .2+ processing  and normalizing calcium release from SR  and total flavonoids from Spatholobi caulis (FSC) showed anti-inflammatory properties (Peucedani radix (CPR), as an anti-inflammatory herb, blocked the receptor-operated calcium channel (ROCC) and VDCC to inhibit calcium influx, thereby promoting vasodilation , making DS-201 a combination therapy candidate with a BK Ca2+ agonist to treat hypertension , and PKA pathways, which increase potassium outflow and promote hyperpolarization of the myocardial cell membrane, resulting in vasodilation (Ca) and ATP-sensitive K+ channel (KATP) channels and suppressing LTCCs to enhance the Akt- and NO-cGMP signaling pathways  prevented calcium release from the IP3-sensitive calcium store to promote vasodilation  reduced NCX abnormal expression to improve myocardial contractility (2+ entry (2+ overload, which is detrimental for cardiomyocytes (2+ overload and maintained cardiac histological characteristics during myocardial ischemic injury (2+ overload and endoplasmic reticulum stress (2+ overload and altered the calcineurin signaling pathway, which improved the metabolism of ischemic myocardial tissue and protected myocardial structure (2+]i and prevented apoptosis by modulating the Ca2+/CaM/CaMK signaling pathway and changing gene expression in cardiomyocytes (2+ homeostasis.The heart is responsible for pumping blood to provide nutrition for various organs and tissues, which depends on the cardiomyocytes' rhythmic contraction and relaxation . After bium ions . Ca2+ thtraction . Thus, ttraction . Indeed,traction . Varioustraction  (Figure traction . For exatraction . Magnesicurrents . In addi for TCM . FMAI fr for TCM . As a cr [Ca2+]i . Ginkgo actility . On the 2+ entry . As a remyocytes . For exac injury . Ophiopom stress . TCM cantructure . FMAI almyocytes . In summThe pathophysiology of CVDs is multifactorial and complex, involving calcium signaling as a key player. With multicomponent and multitarget characteristics, Chinese herbal medicine has shown unique advantages in CVD treatment with few side effects. Accumulating evidence suggests that TCM plays an important role in maintaining calcium homeostasis and improving calcium signaling to alleviate the symptoms of CVDs. Thus, we reviewed the positive effects of TCM and its active ingredients on a wide range of CVDs and highlighted the central role of calcium signaling in their mechanism of action in endothelial cells, VSMCs and cardiomyocytes, which may provide basic guidance for follow-up research.


IDH1/2-mutated acute myeloid leukemia. These inhibitors decrease the stemness of the targeted IDH1/2-mutated cancer cells and induce their differentiation to more mature cells. In this review, we elucidate the mechanisms by which mutant IDH1/2 induce a block in differentiation and the biological and clinical effects of the release into differentiation by mutant-IDH1/2 inhibitors. Isocitrate dehydrogenase 1 and 2 (IDH1/2) are enzymes recurrently mutated in various types of cancer, including glioma, cholangiocarcinoma, chondrosarcoma, and acute myeloid leukemia. Mutant IDH1/2 induce a block in differentiation and thereby contribute to the stemness and oncogenesis of their cells of origin. Recently, small-molecule inhibitors of mutant IDH1/2 have been Food and Drug Administration–approved for the treatment of  Cancer stem-like cells (CSCs) are malignant cells with stem cell characteristics, specifically the potential for self-renewal and differentiation. Similar to stem cells in benign tissues that support the renewal of more mature cells, CSCs are hypothesized to replace malignant cells with a more limited lifespan and therefore enable the persistence of cancer in the presence or absence of cancer therapy and the metastasis of tumors. The CSC hypothesis is based on experimental models with immunodeficient mice in whom a small subset of cells from human tumors can be engrafted and populate the full diversity of malignant cells that were present in the original tumor, whereas other subsets of cancer cells do not have this ability. Although this definition of CSCs is thus largely based on the ability to engraft a tumor in an experimental mouse model and not necessarily clinical observations in human patients, the development of novel cancer therapies is increasingly impacted by the rationale that deep and durable remissions can best be achieved by targeting the CSCs and not just the tumor bulk.+ to NADPH. Through these metabolites, wtIDH1/2 function in the aforementioned myriad of cellular processes because α-KG is a core metabolite of the tricarboxylic acid (TCA) cycle and is also necessary for the function of α-KG-dependent dioxygenases that are involved in epigenetic regulation and DNA repair. In addition, NADPH is one of the most important sources of reducing power in most human tissues and is therefore essential in maintaining cellular redox states.4IDH1/2 mutations (IDH1/2mt) are almost always heterozygous and occur in a hotspot fashion in the enzymatically active sites, for example, IDH1R132, IDH2R140, and IDH2R172, to enable a neomorphic reaction by mutant IDH1/2 (mtIDH1/2) that converts α-KG into D-2-hydroxyglutarate (D-2-HG). Human cells have a very limited capacity to metabolize D-2-HG, which subsequently accumulates to millimolar concentrations and competitively inhibits α-KG-dependent dioxygenases.59 Therefore, D-2-HG is considered to be an oncometabolite. In addition, IDH1/2mt cancer cells have a decreased NADPH production capacity because mtIDH1/2 enzymes have lost the ability to reduce NADP+ to NADPH but instead oxidize NADPH to NADP+ to catalyze the conversion from α-KG to D-2-HG.10 Together, the D-2-HG accumulation and the decreased NADPH production capacity affect the abovementioned plethora of cellular functions, which may all contribute to oncogenesis and have been extensively reviewed before.11Isocitrate dehydrogenase 1 and 2 (IDH1/2) are enzymes that perform key roles in various cellular functions, including the regulation of carbohydrate metabolism, epigenetics, differentiation, DNA repair, and redox states. Wild-type IDH1 and IDH2 (wtIDH1/2) oxidize and decarboxylate isocitrate to α-ketoglutarate (α-KG) in the cytoplasm and mitochondria, respectively, and simultaneously reduce NADPIDH1/2mt are found in multiple cancer types, such as glioma and glioblastoma,13 chondrosarcoma, cholangiocarcinoma, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML).1618 In the origin of these types of cancer, IDH1/2mt are considered to be inaugural or at least early events.1922 In the case of MDS and AML, the data are conflicting because it has been postulated that IDH1/2mt are not necessarily early events in the formation of AML, but rather drive progression from precursor states such as MDS to full-blown AML. In tumors where IDH1/2mt are indeed inaugural or early genetic events, they are present in all, or at least the large majority of, cancer cells including the CSCs. As a consequence, IDH1/2mt that occur as early genetic events are important targets for cancer therapy because then all subclones contain the IDH1/2mt and are sensitive to mtIDH1/2 inhibitors. This appreciation of the contribution of IDH1/2mt to oncogenesis motivated the development of mtIDH1/2 inhibitors. These small-molecule inhibitors effectively reduce the production of D-2-HG by mtIDH1/2 and are also known as sidenibs.25 Examples include the mtIDH1 inhibitor ivosidenib, which is Food and Drug Administration (FDA)-approved for the treatment of IDH1mt newly diagnosed (ND), refractory or relapsed (R/R) AML and IDH1mt previously treated, locally advanced, or metastatic cholangiocarcinoma2629; the mtIDH2 inhibitor enasidenib, which is FDA-approved for the treatment of IDH2mt R/R AML31; the dual mtIDH1/2 inhibitor vorasidenib, which is being investigated for IDH1/2mt recurrent or progressive glioma; and various other mtIDH1/2 inhibitors that are still in clinical trials. In addition to the aforementioned indications, ivosidenib is currently being investigated for the treatment of IDH1mt glioma and chondrosarcoma in early-phase clinical trials35 and enasidenib is studied in IDH2mt MDS. Most clinical trials were conducted with mtIDH1/2 inhibitors as monotherapy, but ivosidenib and enasidenib have also been investigated in combination with intensive chemotherapy or azacitidine for AML.3739IDH1/2mt cancer cells and its underlying mechanisms were already appreciated. First, several hallmarks of cellular dedifferentiation were observed when IDH1/2mt were introduced into in vitro and in vivo experimental models. Second, preclinical, translational, and clinical studies with mtIDH1/2 inhibitors consistently showed that these agents reverse this stemness and promote cellular differentiation of IDH1/2mt cancer cells. Both will be discussed in this review.During the early preclinical investigation of the biological effects of mtIDH1/2 and their inhibitors, the increased stemness of IDH1/2mt, such as glioma, chondrosarcoma,42 cholangiocarcinoma, and AML. Furthermore, genomic hypermethylation and an associated block of differentiation are induced by the expression of mtIDH1/2 or the administration of D-2-HG in cell models related to these types of cancer, such as primary human astrocytes,44 human and mouse neural stem cells,4547 human and mouse mesenchymal stem cells,48 mouse hepatoblasts, and mouse hematopoietic cells,51 but also human embryonic cells and mouse adipocytes. These findings were associated with an increased number of IDH1/2mt cancerous stem cells when these mutations were induced in benign stem or progenitor cells of these tissues.4653 DNA and histone hypermethylation occurs because the accumulated D-2-HG in IDH1/2mt cells inhibits α-KG-dependent demethylases, such as the DNA demethylase TET2 and the family of histone lysine demethylases (KDMs).52 The need for IDH1/2mt cancer cells to induce epigenetic hypermethylation and suppress TET2 function is emphasized by the finding that in several IDH1/2mt AML patients who were successfully treated with an mtIDH1/2 inhibitor but whose AML subsequently relapsed, a novel TET2 mutation that was not present at baseline emerged and DNA hypermethylation was sustained despite low D-2-HG levels. In addition to epigenetic mechanisms, IDH1/2mt also blocks differentiation via metabolic pathways. For example, mtIDH1 inhibits α-KG-dependent dioxygenases necessary for succinyl-CoA production, a critical component for heme production. The resulting succinyl-CoA deficiency attenuates heme biosynthesis in IDH1mt hematopoietic cells, blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells.DNA and histone hypermethylation is a feature that is observed across most investigated types of cancer that frequently contain IDH1mt glioma xenografts grown in mice induced the expression of genes associated with both astrocytic and oligodendrocytic differentiation , and this effect was associated with reduced repressive H3K9 and H3K27 trimethylation on the promoters of these genes.56 In addition, mtIDH1/2 inhibition in IDH1/2mt AML cells induces differentiation. This was observed in an IDH2mt erythroleukemia cell line, in which erythropoietin (EPO)-induced differentiation to red blood cells was restored by mtIDH2 inhibition,58 and this was associated by the reversion of DNA and histone hypermethylation. Crucially, treatment of these IDH2mt erythroleukemia cells with an mtIDH2 inhibitor did not induce apoptosis even at high dosages, suggesting that the antileukemic efficacy of mtIDH2 inhibitors is not based on cytotoxicity but on releasing the brakes on differentiation of IDH2mt AML cells that are trapped in the stem and progenitor cell compartment. Similar findings were seen in primary human IDH1mt or IDH2mt AML cells in vitro and in mice xenografts, in which mtIDH1 or mtIDH2 inhibition induced blast differentiation as shown by an increase in cells that were positive for cell surface markers associated with monocytic and granulocytic differentiation  and intracellular myeloperoxidase (MPO) as marker of maturation into the neutrophilic pathway.60 Quantitatively, 20 days of mtIDH2 inhibition in primary human IDH2mt AML mouse xenografts induced differentiation of 70% of the human blood cells into the monocytic/macrophage and granulocytic lineages, which was associated with a mild to marked decrease in the percentage of human blasts (2- to 35-fold). Furthermore, functional assays during mtIDH2 inhibition in primary human IDH2mt AML cells in vitro yielded the presence of mature, functional neutrophils with phagocytic activity. Differentiation induction of primary human IDH1mt AML cells by mtIDH1 inhibition was associated with reduced histone trimethylation levels at the H3K4, H3K9, H3K27, and H3K36 loci, as well as reduced global DNA methylation.60In preclinical mechanistic studies, inhibition of mtIDH1 in primary IDH1/2mt are reversible. In primary IDH1mt glioma xenografts grown in mice, an mtIDH1 inhibitor induced demethylation of histone markers (see above) without appreciable changes in genome-wide DNA methylation. Furthermore, in some preclinical glioma models, mtIDH1 inhibition had no effect on tumor growth, histone or genome-wide DNA methylation, or the expression of genes associated with stemness or glial differentiation. In addition, long-term studies on inducible IDH1mt in immortalized human astrocytes revealed that mtIDH1 results in progressive and sometimes irreversible accumulation of methylation marks on DNA and histones because the epigenome and transcriptome do not completely return to the original state after the subsequent long-term discontinuation of mtIDH1 expression. In agreement with these findings, mouse xenografts continuously expressing mtIDH1 exhibited a tumor growth rate that was comparable to mouse xenografts with discontinued expression of mtIDH1. On the molecular level, L1CAM, a marker for glioma stem cells, was among the genes that was persistently upregulated despite long-term loss of mtIDH1 expression, and L1CAM is associated with gliomagenesis in xenograft models. In clinical studies with mtIDH1/2 inhibitors in IDH1/2mt AML patients, potent suppression of D-2-HG is sometimes observed without epigenetic demethylation. This phenomenon was associated with primary resistance against mtIDH1/2 inhibitors.However, not all epigenetic effects of IDH1/2mt malignancies that were treated with mtIDH1/2 inhibitors. Translationally, the induction of differentiation has been most thoroughly studied in the context of enasidenib treatment for IDH2mt R/R AML. Direct and morphologic evidence of myeloid differentiation during enasidenib treatment was shown in a patient with IDH2R140Q R/R AML with trisomy 8 in the majority of myeloblasts, with the persistence of trisomy 8 in promyelocytes and mature granulocytes after 4 weeks of enasidenib treatment. Of note, ivosidenib or enasidenib induces myeloid differentiation and trilineage hematopoietic recovery in IDH1/2mt R/R AML without an intercurrent period of bone marrow aplasia or hypoplasia, which is frequently seen during treatment with cytotoxic chemotherapeutic agents, consistent with differentiation as the mechanism of action.2931 This is corroborated by molecular data showing that in many IDH2mt R/R AML patients achieving a complete remission (CR) on enasidenib, the IDH2mt variant allelic frequencies (VAFs) were unchanged between pre-therapy leukemic cells and neutrophils at the time of CR, consistent with the differentiation of IDH2mt leukemia cells into mature neutrophils. Of note, some patients do achieve a decrease in IDH2mt VAF, and these patients have the most durable responses. This was further confirmed by functional assays, wherein these R/R AML patients in CR on enasidenib had IDH2mt-differentiated leukemic neutrophils with intact phagocytic activity, consistent with the restoration of normal granulocyte function. In additional translational analyses in patient samples, flow cytometry–based immunophenotyping analyses revealed that enasidenib treatment promoted differentiation of not only IDH2mt cells, but also IDH2wt cells into mature cells. The differentiation effect of enasidenib treatment on IDH2wt cells probably occurs via the abrogation of paracrine D-2-HG inhibition on differentiation of IDH2wt cells. This evidence of differentiation induction by mtIDH1/2 inhibitors can also be related to decreased DNA hypermethylation after such therapies. Genome-wide methylation is reduced in IDH1/2mt AML cells after patients have been treated with mtIDH1/2 inhibitors, but a gene expression profile associated with AML stemness persisted, suggesting that a complete reversal of the mtIDH1/2-induced DNA hypermethylation is not necessary for a clinical response. In addition to these data, a longitudinal study of patients with IDH1mt cholangiocarcinoma that was treated with ivosidenib was performed. Before-treatment and on-treatment biopsies were compared, revealing that mtIDH1 inhibition induced a more differentiated morphology of cholangiocarcinoma cells on H&E staining and the expression of hepatocyte lineage markers HNF-4α, FOXA1 (HNF-3α), FOXA2 (HNF-3β), and PPARA. These signs of differentiation were associated with a better clinical response to ivosidenib and longer progression-free survival. Translational data from IDH1/2mt glioma treated with mtIDH1/2 inhibitors are not available, probably due to the inherent difficulties of obtaining multiple tumor samples over time in glioma patients.In support of these preclinical findings, induction of differentiation has also been shown in patients with IDH1/2mt AML. The most tangible clinical proof of this hypothesis is that ivosidenib and enasidenib can induce a differentiation syndrome in ~20% of patients with IDH1/2mt AML, which can even be fatal if left untreated. This adverse effect is called IDH-inhibitor-associated differentiation syndrome (IDH-DS) and is a boxed warning for these drugs. Patients and clinicians should therefore be alert for fever, dyspnea, hypoxia, pulmonary infiltrates, pleural or pericardial effusions, rapid weight gain or peripheral edema, hypotension, lymphadenopathy, bone pain, and hepatic, renal, or multiorgan dysfunction. IDH-DS has many clinical similarities to the differentiation syndrome, or retinoic acid syndrome, that is observed during differentiation therapy with all-trans retinoic acid (ATRA) and arsenic trioxide for acute promyelocytic leukemia (APL). Once IDH-DS is suspected, corticosteroid therapy and hemodynamic monitoring should be initiated until symptom resolution.67In addition, there is supporting evidence for the induction of differentiation by mtIDH1/2 inhibitors from the clinical characteristics of these agents in the treatment of IDH1/2mt AML, with the highest CR and response rates being achieved in ND AML and the lowest rates in R/R AML  in 40–55% of patients with  AML see .28–31 Ev2974 This similarity may be relevant because at low doses, hypomethylation agents also mainly work via the induction of differentiation and only limitedly via cytotoxic effects and can require 4–6 months to induce a response. Furthermore, IDH1/2mt VAFs were studied before and during treatment with mtIDH1/2 inhibitors. Responses to mtIDH1/2 inhibitors were observed in patients with clonal IDH1/2mt and patients with subclonal IDH1/2mt, suggesting that ancestral or non-ancestral IDH1/2mt clones are both amenable to respond to mtIDH1/2 inhibitors.75 This is relevant because in ancestral IDH1/2mt clones the IDH1/2mt is likely present in the CSC compartment, whereas in non-ancestral IDH1/2mt clones the IDH1/2mt may not be present in the CSC compartment. Ivosidenib and enasidenib induced an IDH1/2mt molecular remission, defined as an IDH1/2mt VAF below the limit of detection (0.02–0.04%), in ~10–30% of patients, and all of them had a hematologic response, often a CR (~80–100%).2830 As expected, patients with an IDH1/2mt molecular remission were more likely to achieve a CR and a significantly longer overall survival than patients without a molecular remission (15 months vs 10 months for ivosidenib and 23 months vs 9 months for enasidenib), suggesting that IDH1/2mt clearance from all AML cells, including the CSC compartment, is necessary for a deep and durable remission.54In addition, the median time to first response (~2 months) and the median time to best response (~3–4 months) of ivosidenib and enasidenib are more similar to the response patterns of hypomethylating therapy with decitabine, azacitidine, or low-dose cytarabine than with intensive chemotherapy.D-2-HG level reductions have been described, but the relationship is unclear between the extent of the decrease in plasma D-2-HG levels and the likelihood of a clinical response. For example, ivosidenib generally achieves a near-complete suppression of plasma D-2-HG levels in IDH1mt R/R AML patients, but many patients with such near-complete D-2-HG reductions do not respond to ivosidenib. More specifically, plasma D-2-HG was reduced by a median of 95% in patients who achieved a response and an almost equal median of 94% in those who did not achieve a response. On the contrary, this disconcordance between suppression of plasma D-2-HG levels and clinical benefit might be disease-specific to AML because in cholangiocarcinoma, treatment duration, which may be a surrogate for clinical efficacy, appeared to be associated with suppressed plasma D-2-HG levels after one cycle of ivosidenib treatment. In the context of enasidenib, a more potent reduction of plasma D-2-HG levels was achieved in IDH2R140Q than in IDH2R172K R/R AML patients (median −93% vs median −47%).31 In patients with IDH2R140Q R/R AML, the level of plasma D-2-HG reduction by enasidenib was not associated with a clinical response, with plasma D-2-HG reductions of −94%, −95%, and −90% in patients with a CR, non-CR response, or no response, respectively. In contrast, plasma D-2-HG suppression by enasidenib was associated with the clinical response in IDH2R172K R/R AML patients, with plasma D-2-HG reductions of −82%, −44%, and −38% in the abovementioned categories, respectively. These D-2-HG reductions are not complete, not even in patients achieving a CR, because of background D-2-HG production in non-malignant cells by other enzymes than mtIDH1/2. In addition, these data show that near-complete abrogation of D-2-HG production by IDH2mt AML cells is not always sufficient to induce a CR. This suggests that IDH1/2mt AML can develop resistance mechanisms that confer D-2-HG independence or that D-2-HG production is not homogeneously stopped in all malignant cells within IDH1/2mt AML with residual D-2-HG production still occurring in the CSC compartment.With respect to the pharmacodynamics of mtIDH1/2 inhibitors, variable efficacies with respect to plasma Several mechanisms of primary and secondary resistance to mtIDH1/2 inhibitors have been described. In primary resistance, a patient fails to respond to a therapy. In secondary resistance, relapse occurs after an earlier response to a therapy. In both cases, the CSC compartment may play a crucial role.IDH1/2mt cancers has not yet been described. In IDH1/2mt AML, baseline co-mutations in genes of the receptor tyrosine kinase (RTK) pathway  and hematopoietic differentiation transcription factors  are negatively associated with the occurrence of a response to mtIDH1/2 inhibitors.75 These findings can be partially extended to IDH1/2mt cholangiocarcinoma, in which gene signatures associated with activated RTK and PI3K/AKT-activity are associated with early progression upon treatment with ivosidenib. In glioma, the presence of genetic alterations in cell cycle pathway genes was associated with a shorter progression-free survival. In addition to these genetic hallmarks of mtIDH1/2-inhibitor primary resistance, leukemia stemness is associated with poor responses to mtIDH1/2 inhibitors in IDH1/2mt AML. In primary samples of patients with IDH1/2mt AML treated with mtIDH1/2 inhibitors, resistance to mtIDH1/2 inhibitors was more frequently observed in patients with versus patients without a hypermethylated genotype with a gene expression profile associated with AML stemness. In addition, a genetic CSC risk score, the LSC17, was a better predictor for response to mtIDH1/2 inhibition than the established and aforementioned molecular risk factors such as cytogenetics, RUNX1 mutation status, or RAS/RTK mutation status.With respect to primary resistance to mtIDH1/2 inhibitors, an universal mechanism across various types of IDH1/2mt cancers. First, there is the emergence of downstream mutations, such as TET2, BCOR, differentiation genes , and RTK genes .77 Second, IDH1/2mt patients who relapse on mtIDH1/2 inhibitor therapy may do so because of D-2-HG-restoring mutations.79 Restoration of D-2-HG production during mtIDH1 or mtIDH2 inhibition can result either from a second-site mutation in the inhibited IDH1/2 homolog that prevents binding of the mtIDH1/2 inhibitor or from a process termed isoform switching or homolog switching, in which an IDH2mt emerges during mtIDH1 inhibition or vice versa.79 In patients with IDH1mt R/R AML, 35% of patients who achieved a response on enasidenib developed such a D-2-HG-restoring mutation, with second-site mutations and homolog switching occurring in equal proportions of patients (~20%). Second-site mutations, such as IDH1S280F, IDH1R119P, IDH2Q316E, and IDH2I319M,78 occur outside of the catalytically active site of the IDH1/2 enzymes and are modeled to directly or indirectly prevent the binding of allosteric mtIDH1/2 inhibitors such as ivosidenib and enasidenib. Among relapsing IDH1mt R/R AML patients who exhibited isoform switching, most developed an IDH2mt that was not yet detected at baseline, either in the same clone that was previously IDH1mt or in a separate clone. A smaller proportion of patients had two co-existing clones, one IDH1mt and one IDH2mt, with the relapse driven by the IDH2mt clone during mtIDH1 inhibition. A final mechanism of acquired mtIDH1 inhibitor resistance was described in IDH1mt cholangiocarcinoma in which an IDH1R132C mutation converted to an IDH1R132F mutation, suggesting that the phenylalanine instead of the cysteine at residue 132 confers resistance to ivosidenib.With respect to secondary resistance to mtIDH1/2 inhibitors, several mechanisms have been described, and they may be more universal across the various types of  and is currently under investigation for IDH1/2mt glioma. Its dual mtIDH1/2 inhibition will likely reduce the possibility for IDH1/2mt cells to escape mtIDH1/2 inhibition via isoform switching. In addition, a novel type of inhibitor that is in preclinical testing does not target the allosteric site, but instead binds the active site of mtIDH1, thereby decreasing the possibility for IDH1/2mt to restore their D-2-HG production via a second-site mutation at the allosteric site.Currently, second-generation mtIDH1/2 inhibitors are in development, which may decrease the secondary resistance rates to mtIDH1/2 inhibition. Vorasidenib inhibits both mtIDH1 and mtIDH2 at nanomolar concentrationsIDH1/2mt ND AML,3739 and these strategies achieved higher response rates than ivosidenib or enasidenib monotherapy .Combining azacitidine and enasidenib in vitro results in greater reductions in DNA methylation and enhanced EPO-induced erythroid differentiation in an erythroleukemia cell line overexpressing mtIDH2, compared with azacitidine or enasidenib alone. This is consistent with a model wherein enasidenib-induced reactivation of TET DNA demethylase enzymes contributes to azacitidine-induced inhibition of DNA methyltransferase enzymes.IDH1/2mt cancers are sensitive to BCL2 inhibition because D-2-HG accumulation induces BCL2 dependence via inhibition of cytochrome c oxidase, also known as complex IV of the electron transport chain. This effect lowered the threshold to trigger mitochondrial apoptosis upon BCL2 inhibition by venetoclax and rendered IDH1/2mt AML cells 13-fold more sensitive to venetoclax compared with IDH1/2wt AML cells. This observation is corroborated by clinical data, in which venetoclax achieved a modest 33% CR rate when administered as monotherapy to IDH1/2mt R/R AML patients, but high and durable CR rates (57–100%) when administered in combination with azacitidine, decitabine, or low-dose cytarabine in patients with IDH1/2mt ND AML who were ineligible for intensive chemotherapy -driven APL. This was supported by experiments in primary IDH1/2mt AML mouse xenografts, in which ATRA-induced differentiation of AML blasts to more differentiated granulomonocytic cells was achieved in a similar fashion as APL, but not primary IDH1/2wt AML xenografts.In addition to mtIDH1/2 inhibitors, other differentiation agents may be used to induce differentiation of IDH1/2mt AML patients.2830 An interesting observation is that enasidenib can also induce hematopoietic differentiation independently of wtIDH2 or mtIDH2. In IDH2-deficient hematopoietic-progenitor cells, enasidenib but no other mtIDH1/2 inhibitors induced differentiation to mature erythrocytes. This process was mediated by accumulation of protoporphyrin IX, the direct precursor of heme, by virtue of off-target inhibition of enasidenib on the ATP-binding cassette subfamily G member 2 (ABCG2), a transporter highly expressed in erythroid progenitors which is responsible for efflux of protoporphyrin IX. Therefore, enasidenib may be a promising therapeutic agent for improvement of anemia in a wide array of clinical contexts outside of IDH1/2mt cancers. This mechanism is supplementary to the mtIDH1/2-inhibitor-induced restoration of the abovementioned deficiency of succinyl-CoA in IDH1/2mt myeloid progenitor cells, a critical component for heme biosynthesis.As mentioned above, mtIDH1/2 inhibitors can induce transfusion independence for red blood cells and/or platelets in IDH1/2mt are attractive therapeutic targets for various reasons, but most prominently because they are early events in oncogenesis and are therefore present in a large proportion of cancer cells, including the CSC compartment. These mutations induce a block in differentiation early in the maturation of cells in glial, cartilaginous, biliary, and myeloid tissues, and mtIDH1/2 inhibitors potently reverse the metabolic effects of mtIDH1/2 and release differentiation of IDH1/2mt cells into more mature cells. Because the mechanism of action is different and/or complementary to that of cytotoxic agents, other hypomethylating agents and other targeted agents such as venetoclax, we envisage that mtIDH1/2 inhibitors can be most efficaciously used in combination with these agents and that multiple classes of drugs can collaborate in the optimal eradication of the CSC compartment and, therefore, long-term remissions for IDH1/2mt cancer patients.


Ca2+ channels are the main actors among Ca2+ transporters that control the intracellular Ca2+ concentration in cells. It is well known that the basal Ca2+ concentration is regulated by both store-dependent and independent Ca2+ channels in BC development and progression. However, most of the literature has reported the role of store-dependent Ca2+ entry, and only a few studies are focusing on store-independent Ca2+ entry (SICE). In this review, we aim to summarize all findings on SICE in the BC progression field.Known as a key effector in breast cancer (BC) progression, calcium (Ca Indeed, Ca2+ is recognized as a universal intracellular second messenger involved in a plethora of physiological as well as physiopathological processes, such as cell proliferation, migration, invasion, apoptosis, and chemoresistance in a cancer situation [2+ signaling in cancer and especially BC, suggesting its importance in the altering processes that shape cancer cells [Cancers are a major public health problem due to their incidence and, more particularly, their mortality. Among all cancers, breast cancer (BC) is one of the most diagnosed in the world. Despite significant discoveries in treatment, some BC are currently incurable. Thereby, we still need to identify targets to treat BC. In recent years, a research field has developed around the role of ion channels and their implication in tumor progression . The invf cancer . Researcituation ,5. Many er cells . However2+ influx. The most known is the store-operated Ca2+ entry (SOCE) pathway which is regulated by the activation of the Ca2+ release-activated channels encoded by Orai channels via stromal interaction molecule (STIM) proteins [2+ entry (SICE) in regulating BC processes and several hallmarks of BC. Indeed, SICE is known to be induced either by arachidonic acid (AA) and/or the AA metabolite leukotriene C4 (LTC4) pathways and regulated by both Orai1 and Orai3 channels [2+-ATPase (SPCA2) [2+ channels (VGCC), mechanosensitive Ca2+ channels, ligand-activated Ca2+ channels, as well as constitutively activated Ca2+ channels [Several pathways, with different modes of action, permit a Caproteins . It is wproteins . Howeverchannels , or by t (SPCA2) . In addichannels ,12,13.Several reviews have focused on the description and role of SOCE through the stromal interaction molecule (STIM) protein activation ,14. Howe2+ transport [Orai channels play distinct roles in different BC subtypes . Two isoransport . Orai1 wransport ,21,22.2+ influx in BC [Interestingly, an association between Orai channels and transient receptor potential canonical 6 (TRPC6) channels has been reported. Thus, both Orai1 and Orai3 plasma membrane localization is allowed by TRPC6 in BC. Indeed, TRPC6 calcium activity permits these Orai isoforms to be translocated to the plasma membrane and thus participates in a novel way to modulate the Caux in BC .+) such as MCF-7 and T47D cell lines, but not in the estrogen-negative (ER−) basal cells such as MDA-MB-231 [+ BC cell lines such as MCF-7 [Several studies have shown an activation mode of Orai1 and Orai3 depending on the estrogenic status and/or the histological origin ,22,24. Oas MCF-7 .2+ entry through Orai1, which does not depend on the Ca2+ store depletion. Indeed, in 2010, Feng et al. demonstrated another mode of activation of the Orai1 channels [+ cells, Orai1, which is activated independently from STIM1, regulates basal Ca2+ entry and Ca2+ homeostasis. This mechanism involves the SPCA2 pumps initially located in the Golgi apparatus [2+ concentration [+) channels in ER+ cell lines and allows its trafficking from the Golgi to the plasma membrane [Orai1 is the most studied channel among the store-operated channels (SOC). Some studies have shown a modulation of the Cantration . Therefomembrane . Both SPmembrane . Indeed,membrane ,28.+ channel activity permits K+ ion efflux that induces a membrane potential hyperpolarization, therefore, increasing the driving force for Ca2+ that favors Ca2+ entry in the cell [2+-activated potassium channel (SK3) were found to work in partnership with Orai1. Indeed, these K+ channels, by regulating the membrane potential, regulate Ca2+ entry in basal-like BC cells.As seen above, when a store-independent activation of Orai1 occurs, other proteins are involved to form a functional or physical complex with Orai1. It is the case in potassium channels. Kthe cell . Both th2+ entry in BC. In fact, both Kv10.1 and SK3 functionally regulate the Ca2+ entry through Orai1 leading the cell migration regulation. Indeed, Kv10.1 was observed expressed alongside Orai1 in invasive breast tumors and lymph node metastasis, and regulates cell migration through an Orai1-dependent constitutive Ca2+ entry [For both of these channels, it has been found that Orai1 was the main actor in the constitutive Ca2+ entry . On the 2+ entry . This pr2+ entry . The SIC2+ entry ,32.− tumors (not expressing the estrogen receptor and known to be the most aggressive), TRPM7 appears to be overexpressed in the most invasive areas [+ tumors, TRPM7 is overexpressed in the non-invasive areas, but at a lower level than in the invasive areas, and regulates cell proliferation [The transient receptor potential (TRP) channels family is expressed and functional in most of the non-excitable cells in the human organism. They are known to be activated by various stimuli, such as growth factors, temperature, ligand, or mechanical stimuli, and are known to be abnormally expressed in cancers and especially BC. Many TRP channels have been identified as being up-regulated in BC and can be correlated with clinical parameters in BC. This is the case, for example, for TRP canonical (TRPC) channels , TRP melastatin (TRPM) channels , and TRP vanilloid (TRPV) channels  ,36,37,38ve areas , correlave areas , and regve areas . While i2+ (up to 10 mM) increases TRPC1 expression, via the phospholipase C (PLC) and Erk1/2 pathway in MCF-7 cells [2+ entry, and also for the proliferative effect induced by the activation of CaR. Moreover, the involvement of TRPC1 in the CaR-induced proliferation has been suggested [In addition, it has also been shown that the TRPC1 channel expression is regulated via activation of the calcium-sensing receptor (CaR) . Indeed,-7 cells ,44,45. Fuggested .2+ entry and an increase of intracellular Ca2+ concentration in a constitutive manner. Indeed, TRPC1 silencing reduces the constitutive Ca2+ entry in cells grown under a hypoxic environment [Interestingly, TRPC1 expression is upregulated in hypoxia compared to normoxia conditions through HIF1α expression due to the presence of HIF1α and HIF1β binding domains in the TRPC1 gene promoter . Silenciironment . Moreoveironment . In addiironment .2+ influx responsible for the Akt signaling pathway and a sustainable phosphorylation of FAK which could be via the Akt signaling pathway. Moreover, Akt activation is responsible for a downregulation of β-catenin and E-cadherin [In BC, Lee et al. have shown that TRPV4 calcium activity is required in cell migration via activation of the Akt signaling pathway as well as a downregulation of E-cadherin protein expression. In this study, they showed that activation of the upregulated TRPV4 by 4-α-phorbol-12,13-didecanoate (4α-PDD) triggers a Cacadherin . Moreovecadherin .2+ entry and hence the cell viability [+ BC cell line progression. In addition, they demonstrated that TRPM7 silencing decreased both Ca2+ entry and MCF-7 cell line proliferation [The melastatin family of TRP channels is also a well-known regulator of carcinogenesis processes. For example, our team showed that TRPM7 is a key regulator in BC progression. It participates in cell proliferation as well as cell migration and invasion ,41,42. Fiability . Guilberferation . Howeverferation ,49. Furt2+-permeable TRPM8 channel, was found as a regulator of BC processes. This channel was shown to be activated in BC cells and associated with an elevation of cytosolic Ca2+ concentration following the application of icilin (TRPM8 agonist) [2+ entry [Another TRPM family member, the Caagonist) . However2+ entry . Moreove2+ entry .2+ entry. Some VGCC see their expression and activity being altered in BC. This is the case in T-type Ca2+ channels, such as Cav3.1, Cav3.2, which are overexpressed in BC tissue [2+ entry following melatonin and 5α-dihydrotestosterone perfusion [A number of studies have focused on the role of VGCC, which could be activated under normal cell culture conditions. Indeed, the resting membrane potential, measured by whole-cell patch-clamp technique, varies from −40 to −20 mV in BC cell lines ,52,53. TC tissue ,55. Indeerfusion ,57,58.2+ entry. However, the application of L-type channel antagonists decreased Ca2+ entry. These results suggested that CACNG4 subunit regulates the channel in an active state resulting in the higher intracellular Ca2+ concentration leading in fine to the inhibition of processes such as cell proliferation, motility, and adhesion [2+ channel with a specific agonist BAY K8644 leads to an increase in the intracellular Ca2+ concentration responsible for filopodia stability. Indeed, treated cells with L-type channels pharmacological blockers, such as amlodipine besylate, felopidine, manidipine dichloride, and cilnipidine, lose their stable filopodia. Furthermore, it has been shown in the same study that integrin activation promotes filopodia formation through the proto-oncogene tyrosine-protein kinase Src signaling pathway, calpain activity, as well as a Ca2+ entry at the filopodia level. In addition, the L-type Ca2+ channel seems to be colocalized with myosin X (MYO10) within filopodia [L-type VGCC were found to be active at a basal level and regulated by the L-type voltage-gated calcium channel γ 4 subunit (CACNG4) . CACNG4 adhesion . Moreoveadhesion . The actilopodia .2+ channels, active at membrane potential from −50 mV and above [2+ channels reduced MCF-7 cell proliferation without altering cell viability [2+ channel antagonists) inhibited T-type Ca2+ currents and reduced cell proliferation. However, due to the poor selectivity of the pharmacological blockers used, it has been concluded that T-type Ca2+ channels are involved in the cell proliferation alongside other VGCC, such as L-type Ca2+ channels [T-type Cand above , have beiability . A similchannels .2+ channels are a protein family activated by a plethora of stimuli from store-dependent activation to the basal-activated state in BC. Based on studies demonstrated in this review, it seems that Ca2+ signaling is far from being fully understood in BC. As we schematized, the SICE constitutes a key effector in a multitude of BC cell processes, even though it is not the major actor in the Ca2+ flow as SOCE (see 2+-regulated cell processes in BC should bring answers regarding the relationship and/or the complementarity between the role of SOCE and SICE in the regulation of BC progression via Ca2+ signaling.To summarize, CaSOCE see . Neverth


We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca Direct ionic communication between electrically excitable cells was discovered in the early 20th century ,2, but f2+ [2+i]) caused by Ca2+ influx played a role in the regulation of gap-junctional communication. Soon after, the Ca2+-role in cell uncoupling was confirmed by evidence that cell–cell communication was lost in cells subjected to treatments that increase the [Ca2+]i i ,23,24,252+]i effect on gating has been questioned for more than four decades. Early studies suggested that a [Ca2+]i as high as 40–400 µM is needed [2+]i, ranging from ~100 nM to low µM is effective. Data for the effectiveness of nanomolar [Ca2+]i were first reported in a study [2+]i of 251 nM (at pH 7.4), ~400 nM (at pH 7.0) and 2.5 µM (at pH = 6.5) were proven effective in uncoupling cardiac cell pairs, one of which was mechanically perforated to allow the influx of extracellular solutions well buffered for Ca2+ and H+. More recently, a treatment with ionomycin and gramicidin of arterially perfused rabbit papillary muscle uncoupled the cells at ~685 nM or greater [Ca2+]i [2+]i proved effective on gating in many other cells, such as crayfish giant axons [2+]i only from 180 nM to 860 nM [2+]i.The i ; these d [Ca2+]i . Low i and [H+]i in crayfish septate axons uncoupled by low pHi i by approximately one order of magnitude, from resting values of 100–300 nM, and greatly increased Rj, indicating that that crayfish gap junctions are sensitive to low µM [Ca2+]i [2+]i matched well, while that of Rj and [H+]i did not i . The tim did not .2+]i effective on gating, we studied Novikoff hepatoma cell pairs by double whole-cell clamp [2+i-gating sensitivity was tested by monitoring the decay of junctional conductance (Gj) at different [Ca2+]i (buffered with BAPA), at pHi = 7.2 or 6.1 . Channel gating was activated by [Ca2+]i ranging from 500 nM to 1 µM, irrespective of pHi [2+]i = 0.5–1.0 µM, the Gj dropped to ~25% of the initial values with mean τ’s of 5.9 and 6.2 min, at pHi = 6.1 and 7.2, respectively. With [Ca2+]i = 3 µM, the cells uncoupled in <1 min (τ = ~20 s) [2+]i on gating was confirmed in the same cells with brief (20 s) exposures to 20 µM arachidonic acid I < 0.5–1 µM blocked the cell–cell diffusion of Lucifer Yellow in chicken-lens-cultured cells [2+]i drastically reduced the Gj in pancreatic β-cells, with a temperature drop from 37° to 30° and an external [Ca2+]o rise from 2.56 mM to 7.56 mM [2+]i was also reported in astrocytes injected with Lucifer Yellow and Ca2+ [2+]i prevented cell–cell dye transfer independently of pHi; the dye transfer was blocked by [Ca2+]i ranging from 150–600 nM [2+]i. Dye coupling was also blocked in cultured astrocytes treated with ionomycin, which increased the [Ca2+]i to 500 nM [2+-influx and reduced the Gj by 95% [2+]i increased from ~80 to ~250 nM. All of these data confirm the idea that Ca2+i is a fine modulator of gap-junctional coupling.Similarly, a i was carefully buffered with BAPTA i to a level sufficient for channel gating. In these Cx36-expressing cells, channel gating occurred with alkalinization, which may result from a high pHi-induced increase in [Ca2+]i [2+]i and uncoupled the cells at pHi > 7.8 [The study of Burr and coworkers  revealed to ~6.5 . A possi [Ca2+]i . Indeed,Hi > 7.8 .Xenopus oocyte pairs the CO2 gating sensitivity of channels made of chimeras and mutants of Cx32 and Cx38, two connexins whose channels are at the opposite end of the spectrum in chemical gating sensitivity i and induced a 95% drop in the Gj; in contrast, in Cx40-expressing N2a cells ionomycin did not significantly change the Gj. The apparent gating incompetence of human Cx40 i; rev. in i (~50 nM) i rise above the resting values and in most cases would require either a connexin mutation i rise above resting levels (>~50 nM) causes a CaM lobe (most likely the N-lobe) to block the channel’s mouth (vestibule) by electrostatically and hydrophobically interacting with a receptor site located at CL2 or NT (CaM-gating site) (2+]i (~50 nM), the CaM is believed to be anchored to each of the six connexins by one of its lobes (most likely the C-lobe), while the other lobe is likely to be free, but unable to access the channel’s mouth at resting [Ca2+]i (The Ca-CaM-cork gating envisions that a [Cang site) . This in [Ca2+]i .2+-affinity constant of the C-lobe’s EF-hand pair is almost one order of magnitude greater than that of the N-lobe’s EF-hand pair (KD(app) = 5.6 μM and 32 μM for C-lobe and N-lobe, respectively) [2+]i above the resting values (>~50 nM) (The Cactively) ,126. The>~50 nM) .2+]i, while the latter may result from a greater increase and/or a more prolonged [Ca2+]i rise. We believe that the ultrastructural correlate of the “irreversible Ca-CaM locked gate” is reflected by gap junctions displaying “crystalline”  channel arrays i (~50 nM) as the mutations might have caused the channel’s mouth to be unprotected, making it accessible to the CaM’s lobe. The CaM lobe would electrostatically interact with the positively charged channel’s mouth so that it could be displaced by Vj positive at the mutant side [2+]i, while the N-lobe is free to interact with the channel mouth of the mutant’s hemichannel.The gating behavior of Cx32/mutant heterotypic channels ,114, Cx4ant side ,114. Indant side , one fin2+]i rise  into the channel’s mouth at resting i rise sufficient to activate the N-lobe of the CaM, how many N-lobes are expected to participate in the channel gating? If indeed the CaM is anchored to each of the six connexins of a connexon, could all of the six N-lobes simultaneously interact with their receptor for gating the channel? This might be possible, but we think it is unlikely due to steric hindrance and because the negatively charged CaM’s N-lobes would electrostatically repel each other. More reasonable, in contrast, is the possibility that channel gating is caused by the interaction of just one N-lobe with the receptor site, either in the same connexin (trans-domain interaction) or in another connexin (trans-subunit interaction) of the connexon  display tightly packed  arrays with ~150 Å center-to-center spacings  Figure , which a2+]i rise would cause a CaM’s N-lobe to reversibly close the channel  and Ca2+-activated (holo-CaM). In a human kidney cell line, they found that the [CaM]i at basal [Ca2+]i is 8.8 ± 2.2 µM, but a [Ca2+]i increase dramatically decreases the [CaM]i to ≤200 nM because of the extensive buffering of the free CaM by the large increase in the available CaM-binding sites. The same phenomenon with [Ca2+]I elevation was observed in rabbit cardiac-myocytes, as the CaM reversibly migrated into the nucleus [2+]i rise, the CaM migrates from the Z-line to the Ca2+-CaM-binding sites with higher affinity. The same phenomenon may to occur in gap junctions, a possibility being that with a great increase in [Ca2+]i the non-gating CaM molecules, linked to the connexins by just one lobe, are released from the connexins.If this were the case, what would cause the release of CaM molecules from gap junctions at high i in the high nanomolar range indicates that the fine modulation of direct cell–cell communication provides cells with the means for regulating tissue homeostasis. In addition, and more importantly, the field should be encouraged to test the effect of recently discovered disease-causing CaM mutants on gap junction function [One may ask: why is it important to understand in detail the gating mechanism of gap junction channels? It is important not only because cell–cell uncoupling is more than just a safety mechanism for protecting healthy cells from damaged neighbors , but because the gating sensitivity to [Cafunction .2+ channel. However, other membrane channels, potential targets of CaM mutants, have also been suggested. Curiously, however, in spite of strong evidence for the direct CaM role in gap junction channel regulation, the potential consequences of these CaM mutants on direct cell–cell communication—a mechanism fundamental for the function of virtually all vertebrate and invertebrate organs—have not yet been addressed. Therefore, it is clear that future efforts should be aimed at testing the effect of these CaM mutants, and the future discovered CaM mutants, on gap junction mediated communication [Indeed, recent evidence of diseases caused by CaM mutations ,140,141 nication .