In humans, a GCAP2 missense mutation (G157R) has been associated with inherited retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to 3 Mg2+ with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, nonmyristoylated GCAP2 does not bind Mg2+ over the physiological range and remains as a monomer in the absence of Ca2+. Both myristoylated and nonmyristoylated GCAP2 bind Ca2+ with high affinity. At odds with guanylate cyclase–activating protein 1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations and prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 might be mostly implicated in processes other than phototransduction in human photoreceptors and suggest a possible molecular mechanism for G157R-associated IRD.In murine and bovine photoreceptors, guanylate cyclase–activating protein 2 (GCAP2) activates retinal guanylate cyclases (GCs) at low Ca Guanyla 6 (PDE) , 2. The r [Ca2+] , 5, 6 anl levels , 7.2+ or Mg2+, in the specific region defined by the helix-loop-helix motif . Up to three GCAPs isoforms have been discovered in mammal photoreceptors, and six to eight in teleost fish  and GCAP2, which are present both in rods and cones and whose biochemical and physiological features in murine and bovine photoreceptors have been deeply characterized , 21, 22. in rods , 25, GCAnditions . This monditions , 26 and nditions , 19, espasticity .GUCA1A encoding for GCAP1 have been associated with autosomal dominant cone dystrophy or cone–rod dystrophy , compatible with a decrease in the affinity for Ca2+ of that binding site. The mutant has been studied in vivo by expressing the bovine equivalent to human G157R as a transgene in a mouse model of rods GCAP1/2 KO -associated G157R variant.2+ sensitivity (B), thus pointing to a very efficient myristoylation process.NCS proteins are subjected to a post-translational modification where a fatty acid  is covalently bound to the N-terminal Gly, thus conferring the protein-specific features such as membrane binding or modulation of the Casitivity , 45. To sitivity B, thus pD), compatible with a Gly-to-Arg mutation and successful myristoylation, as judged by the substantial correspondence between measured peaks and theoretical values , thus resulting in an estimated MM of 25 and 58 kDa, respectively (see B) displayed a cation-independent elution peak at 14.7 ml, corresponding to a MM of 35 kDa, which could be related to a different oligomeric state of the protein in Mg2+ or Ca2+ saturating conditions.NCS proteins are characterized by heterogeneous oligomeric states , and it rization . We inve of Ca2+ A, thus rvely see . On the vely see B display2+ binding for both GCAP2 forms (C and D), although to a different extent. Indeed, nmGCAP2 exhibited a 0.79-nm increase in the hydrodynamic diameter , whereas mGCAP2 displayed a 1.06-nm increase upon Ca2+ binding . Nevertheless, such increase in the hydrodynamic diameter can be ascribable to a conformational change rather than to an aggregation process, as shown by the stability of the MCR over the 90-min experimental time  conveniently allows measuring the change in the hydrodynamic diameter of calcium sensor proteins resulting from the structural rearrangement upon cation binding, as well as to evaluate any potential time-dependent aggregation propensity by monitoring the mean count rate (MCR) along the time. Results from DLS experiments showed an increase of the hydrodynamic diameter upon CaP2 forms , C and Dtal time . Interesn trends  at odds 2+, the elution peak displayed a minor shift of the apparent mass from 31.9 kDa at 5 μM mGCAP2 to 34.3 kDa at concentrations higher than 10 μM (A). On the other hand, in the presence of both Mg2+ and Ca2+, the main elution peak shifted compatibly with an increase of mass from 24 kDa (at 5 μM) to 33.8 kDa at 80-μM mGCAP2 (B), moreover, each peak displayed a shoulder at higher MM with the intensity proportional to the protein concentration. By fitting the apparent MM as a function of the mGCAP2 concentration to a hyperbolic curve , thus suggesting that with physiological [Mg2+] in conditions of very low [Ca2+] mGCAP2 is a dimer. On the other hand, in the presence of both Mg2+ and Ca2+, the estimated apparent KD of dimerization shifted to 55.4 μM (R2= 0.99), thus pointing toward a Ca2+-dependent dimerization of mGCAP2.To investigate the dimerization process of the physiologically relevant myristoylated form of GCAP2 in the presence of cations, the analytical SEC profiles of mGCAP2 were collected at an increasing protein concentration. In the presence of the physiological concentration of Mgan 10 μM A. On theM mGCAP2 B, moreovic curve , inset, The same experiments performed on G157R showed that, despite having the same nominal concentration, all chromatographic profiles  were conCD spectroscopy can be used to monitor the conformational changes of NCS proteins upon metal binding, by exploiting the differential absorption of circularly polarized light of the chiral centers of the protein. Specifically, the CD signal in the near UV (250–320 nm) arises from the microenvironment of aromatic residues, thus representing a fingerprint of the protein tertiary structure, whereas in the far UV (200–250 nm), the optically active peptide bonds provide information about the secondary structure elements.A, black trace) showed a relatively intense positive signal in all three bands corresponding to aromatic residues , indicating that the protein is significantly structured even in the absence of ions. No significant changes in either intensity or fine structure could be observed after the addition of Mg2+ , while Ca2+ binding resulted in a prominent reshaping of the spectrum, as shown by the increase in ellipticity, particularly in the dichroic bands of Tyr and Trp residues . Conversely, apo-mGCAP2 exhibited an almost flat spectrum  with a nearly detectable fine structure, while the addition of Mg2+ caused a significant increase in ellipticity in all aromatic bands accompanied by a defined fine structure , suggesting that Mg2+ is required for the correct folding of mGCAP2. Finally, Ca2+ addition resulted in a slight decrease in intensity in the Phe band and a more prominent increase in ellipticity in the Trp band compatible with a small but significant conformational change.The near-UV CD spectrum of apo-nmGCAP2 ; moreover, to exclude that this observation could result from the protein being significantly aggregated at the experimental concentration (30 μM), the same experiments were repeated with halved concentration, yielding the same results.Quite surprisingly, the near-UV CD spectra of the G157R variant were flat under all tested conditions C; moreovD–F), with minima at 208 and 222 nm. The addition of Mg2+ to apo-nmGCAP2 resulted in a minor increase in ellipticity at 222 nm  of a coiled-coil secondary structure in the absence of ions, as shown by θ222/θ208 = 1.24 . Similarly, the thermal stability of mGCAP2 (B) was also slightly decreased in the presence of Mg2+  and presence of Mg2+ (ΔTm = −5 °C). Again, mGCAP2 also did not exhibit complete unfolding upon Ca2+ binding up to 96 °C, but a significantly higher percentage of the protein pool was denatured at 96 °C, with respect to nmGCAP2 , although the presence of Mg2+ slightly increased the thermal stability compared with the apo-form (ΔTm = 0.9 °C). Nonetheless, neither G157R showed a complete unfolding transition in the presence of Ca2+ , although 59.4% of the ellipticity signal was lost at 96 °C . Upon Ca2+ addition, the native band was again detectable in all GCAP2 variants; moreover, mGCAP2 exhibited a number of proteolytic fragments clustered in the ∼12- to 15-kDa region, while the pattern of both nmGCAP2 and G157 spread over a broader range of MM, with more abundant smaller-sized fragments, especially for the nmGCAP2 case (see bands at around and below 10 kDa). Overall, limited proteolysis experiments confirmed that the effects of Ca2+ on GCAP2 conformation and trypsin accessibility depend strongly on peculiar properties of each variant.All three variants displayed the residual presence of native protein in the absence of ions , either in terms of intensity or shift  to both Mg2+ . Thus, in line with DLS and analytical SEC measurements, these results suggest that the G157R mutant may be susceptible to aggregation mediated by hydrophobic interactions.ANS-fluorescence measurements highlighted that all three GCAP2 variants were highly hydrophobic, as shown by the major increase in fluorescence emission and the blue shift exhibited by the ANS probe in the presence of the proteins . In lineaddition A, either = 67.1% . In line = 67.1% B to both cations C. Thus, 2+-bound forms migrate at a lower MM , whereas addition of Ca2+ confirmed the typical shift to a lower apparent MM of NCS proteins. Indeed, Ca2+-bound nmGCAP2 presented a single band at a lower apparent MM, at odds with both WT and G157R mGCAP2, which presented two separate bands.NCS proteins are known to be subjected to differential electrophoretic mobility in SDS-PAGE experiments under different cation-loading conditions, as their Calower MM . The reat shifts . Interes2+ and Ca2+ binding to GCAP2 variants, we performed isothermal titration calorimetry (ITC) measurements by titrating apo-mGCAP2/nmGCAP2 with Mg2+ or Ca2+. Ca2+ titrations were also repeated in the presence of physiological levels (1 mM) of free Mg2+. Proteins were carefully decalcified according to a recently optimized protocol . The lack of net change in heat might result from compensating ΔH terms arising from binding and conformational change processes. However, the results obtained with ANS fluorescence (A) and near-UV CD (A) exclude conformational changes and rather suggest no binding of Mg2+ or a high-millimolar affinity binding, which would be undetectable with ITC measurements and of no physiological relevance. On the other hand, Ca2+ titrations performed with apo-nmGCAP2 (B) showed an exothermic pattern, which could be fitted only to a two-sequential binding site model with affinities in the low and mid nanomolar range  yielded lower affinities for both resolved binding sites, as the apparent dissociation constants were 3- to 4-fold higher . Data could be fit to a three-sequential binding site model yielding apparent dissociation constants ranging from 8.6 nM to 10.2 μM  and presence of 1-mM Mg2+ (F), both titration curves showed an inflection point that significantly exceeded the number of functional binding sites of a mGCAP2 monomer . Thus, data fitting to either a two- or three-binding site model yielded no reasonable values for apparent dissociation constant, enthalpy, and entropy changes. A possible explanation of such unusual behavior may rise from the analysis of analytical SEC (B) and near-UV CD (B) data, which showed that mGCAP2 undergoes both dimerization and conformational changes upon Ca2+ binding, therefore making impossible to dissect the different contributions to heat variation. With this notion in mind, ITC data suggest the transition from apo-, monomeric mGCAP2 to Ca2+-loaded, dimeric mGCAP2 to be exothermic. Of note, the molar ratio corresponding to the saturation of the process shifted from ∼6(Ca2+/mGCAP2) in the absence of Mg2+ to >10 in the presence of 1-mM Mg2+, suggesting that the high affinity for Mg2+ exhibited by mGCAP2 may result in a competition between the two metal cations, thus decreasing its affinity for Ca2+.Although Ca absence E and pre-mM Mg2+ F, both tical SEC B and neaar-UV CD B data, win vitro, in reconstitution experiments that confirmed the capability of human mGCAP1 to regulate the cyclase  and assessed the cGMP levels by HPLC (see 2+ (∼34-fold higher than the control) and bring the activation down to basal levels at high Ca2+ , the activation of GC1 in the presence of mGCAP2 was found to be only ∼3-fold higher than the control and substantially Ca2+ independent (2+-independent and very mild activation of GC1 (16% compared with mGCAP1). Finally, the G157R variant led to activity levels comparable with those of the controls, thus excluding any relevant activation .To confirm this unexpected result, we performed enzymatic assays, increasing the reaction time with respect to our previous experiments , in line with the high-affinity binding demonstrated also by ITC (2+ starting from the apo-form (B–E), which appeared essentially less structured compared with the nmGCAP2 variant. The latter had already a more defined tertiary structure in the absence of cations (A–D) but did not show significant Mg2+-induced conformational change.We previously showed that myristoylation of bovine GCAP2 may affect its structural properties in a rather complex manner . Indeed,2+ , the hum2+ , B and Eo by ITC  and ANS o by ITC . Indeed,apo-form , B–E, wh cations , A–D but2+ binding to nmGCAP2 and mGCAP2. In line with the present study (A), for bovine nmGCAP2, no ITC pattern compatible with Mg2+ binding was observed starting from the apo-form. However, variable and puzzling results were observed for bovine mGCAP2 , but in the same conditions, the myristoylated form promptly shifted to a dimer (B). Therefore, mGCAP2 present in the photoreceptor outer segment under conditions of very low Ca2+ corresponding to intense illumination would likely be a dimer (A). However, under Ca2+-saturating conditions, such as those typical of dark-adapted cells, a clear equilibrium between monomeric and dimeric forms was observed (B). Our analytical SEC data quantitatively match the finding in Pettelkau et al. (2+ conditions (KD = 55.4 μM), and this evidently occurs also in the copresence of Mg2+. While the copresence of monomers and dimers is in principle possible for all the tested variants, as highlighted by MALDI-TOF measurements (B), a complex relationship between cation levels and myristoylation evidently determines the actual oligomeric state of human GCAP2 in specific signaling conditions.Bovine mGCAP2 was previously shown to undergo Carization  and a moing Ca2+ . To our onomeric A, but in a dimer B. Theref a dimer A. Howeveobserved B. Our anu et al.  that GCAurements B, a comp2+ slightly destabilizes mGCAP2 . Analysis of the proteolytic patterns for nmGCAP2 , in contrast to apo-mGCAP2, which appeared to show a much lower degree of tertiary structure (B) also confirmed by the oscillations of the hydrodynamic diameter observed in time-resolved DLS (B) pointing to a dynamic molten globule state rather than to an aggregation-prone conformation.Thermal denaturation studies suggest that Mgs mGCAP2 , but thes mGCAP2 . Instead nmGCAP2 , which atroscopy A and 4A. nmGCAP2  seems to cations A, in contructure B also colved DLS B pointin2+ to a different extent (C and D) indicative of specific hydrodynamic and hydrophobic properties also revealed by ANS fluorescence , yet clearly conserving elements of secondary structure (F). Surprisingly, the CD spectra showed features of a coiled-coil assembly that, although less than the WT, responded to Mg2+ and Ca2+ stimuli (F), as confirmed by gel shift assays (C). G157R was significantly less stable than the WT under all tested conditions (C).Our tructure C, yet cltructure F. Surpri stimuli F, as cont assays  and ANS t assays C. G157R nditions , and a nnditions C.in vivo studies, a ∼50% phosphorylation of residue S201 in bovine GCAP2 (S197 in human GCAP2) causes protein sequestration at the inner segment, and the accumulation of GCAP2 variants unable to bind cations in the inner segment induced severe toxicity  for GCAP2-mediated GC1 activation as a function of the protein concentration was approximately 17-fold higher than that observed for human GCAP1 (1.13 μM), indicating a rather low affinity for the human GCAP2–GC1 complex. If the cellular concentration observed in bovine rods (approximately 3 μM for both GCAP1 and GCAP2) is taken as a reasonable reference also for human rods, this would exclude any significant role of human GCAP2 in regulating human GC1 also with the data presented in Peshenko et al. . The G157R point mutation was introduced by site-directed mutagenesis using QuikChange II Kit (Agilent), and primers  and DNA sequencing were obtained by Eurofins. Nonmyristoylated WT GCAP2 was heterologously expressed in Escherichis coli BL21 DE3 cells after transformation with pET-11a(+)-GCAP2 plasmid. Myristoylated WT and G157R GCAP2 were heterologously expressed in E. coli BL21 DE3 cells after cotransformation with pET-11a(+)-GCAP2 or pET-11a(+)-GCAP2–G157R and pBB131–yNMT (encoding for N-myristoyl transferase from S. cerevisiae) plasmids. The expression and purification protocol for GCAP2 variants was essentially the same as reported in refs.  was cloned into pET-11a(+) vector using in refs. , 70. Bri  were desalted by ZipTip C4 (Millipore), and desalted protein was eluted with 30% (v/v) acetonitrile/0.1% (v/v) formic acid. First, a sinapinic acid matrix layer was prepared by depositing a droplet (0.5 μl) of saturated solution of sinapinic acid in 100% ethanol on the target. Thereafter, the eluted sample was deposited on an MALDI target plate. MALDI analyses were performed with ultrafleXtreme MALDI-TOF/TOF MS provided by the "Centro Piattaforme Tecnologiche" of the University of Verona.2+ or 500-μM Ca2+ + 1-mM Mg2+.The apparent MM of GCAP2 variants was determined by analytical SEC using a Superose 12 10/300GL  column equilibrated with 20-mM Tris HCl, pH 7.5, 150-mM KCl, 1-mM DTT buffer, and 500-μM EGTA + 1-mM Mge) used to calculate the partition coefficient (Kav) according to the following equation:v is the void volume (8 ml) and Vt is the total column volume (25 ml). The MM was then extrapolated from the calibration curve of log(MM) as a function of Kav described in  before DLS measurements. Samples were equilibrated at 25 °C for 2 minutes, and 38 to 55 measurements were collected approximately every 2 minutes for >90 min, each consisting of 16 to 18 repetitions.The hydrodynamic diameter and the time evolution of the MCR of GCAP2 variants were measured with a Zetasizer Nano-S  at 25 °C, using the experimental setup detailed in . Protein2+ and 1-mM Ca2+, leading to approximately 500-μM free Ca2+. Far-UV CD spectra of 8-μM GCAP2 variants were collected in the 200- to 250-nm range in 1-mm quartz cuvettes in the presence of 300-μM EGTA and after sequential additions of 1-mM Mg2+ and 600-μM Ca2+, leading to approximately 300-μM free Ca2+.CD data were collected at 37 °C on a Peltier thermostated Jasco J-710 spectropolarimeter using the same parameters as described previously . The spey0 is the ellipticity value at 20 °C, a is the variation in ellipticity between 96 °C and 20 °C, b is the Hill coefficient, c is the inflection point of the sigmoid, which represents the melting temperature.The thermal denaturation profiles were recorded in the same experimental conditions as far-UV CD spectra on the same samples using the following parameters: temperature range 20 to 96 °C, scan rate 1 °C/min, response time 4 s, and wavelength 222 nm. Assuming a two-state transition process where the ellipticity signal as a function of the temperature represents the fraction of folded/unfolded protein, thermal denaturation profiles were fitted to a following 4-parameter Hill sigmoid:2+ or 1-mM Mg2+ + 5-mM Ca2+.A 15% acrylamide SDS-PAGE was performed to evaluate the cation-induced differential electrophoretic mobility of GCAP2 variants under denaturing conditions. Proteins were diluted in 20-mM Tris HCl, pH 7.5, 150-mM KCl, and 1-mM DTT buffer to 20 μM and loaded on the gel after 5 min incubation with 5-mM EDTA, 5-mM EGTA +1-mM Mg2+ or 1-mM Mg2+ + 1-mM Ca2+ was performed after incubation at 25 °C with trypsin in an enzyme:protein molar ratio of 1:60. Enzymatic cleavage was stopped after 5, 10, 15, 30, 45, 60, and 90 min through heat inactivation of trypsin, and then, samples were loaded onto a 15% SDS gel  to a two-sequential binding site model, although no binding was identified in Mg2+ titrations. Mg2+ titrations of mGCAP2 were fitted by MicroCal PEAQ-ITC Analysis Software  to a three-sequential binding site model. Data fitting yielded estimated values for dissociation constants (KDi) and enthalpy variations (ΔHi) for each (i) binding site. Data in Cation binding to GCAP2 variants was monitored by ITC using a MicroCal PEAQ-ITC  following the decalcification procedure detailed previously in refs. , 52. BriFluorescence spectra were collected at 37 °C with a Jasco FP 750 spectrofluorometer, with excitation and emission bandwidths set to 5 nm; each reported spectrum is an average of three accumulations.2+ and 1-mM Ca2+.The hydrophobic fluorescent probe ANS was diluted to 30 μM in 20-mM Tris HCl, pH 7.5, 150-mM KCl, 1-mM DTT buffer and incubated for 2 min with 2-μM GCAP2 variants. Emission fluorescence spectra were recorded in the 400 to 650-nm range after excitation at 380 nm in the presence of 500-μM EGTA and after sequential additions of 1-mM MgRecombinant human guanylate cyclase 1  was coexpressed with GFP in HEK293 cells as reported previously  to obtaiet al.  or in the presence of Ca2+ (∼30 μM). Production of cGMP was analyzed by loading the reaction mixture onto a C18 reverse phase column ). Free Ca2+ concentration was adjusted using Ca2+–EGTA buffer solutions as described previously  were energy-minimized by performing four iterations, each consisting of 65 steps with root mean square gradient for convergence set to 0.01 kcal/mol/Å.The three-dimensional model of human GCAP2 shown in ry: 1JBA ) followi , where the null hypothesis was that the mean of a population was greater than or equal to the mean of the other population.The statistical significance of the differences in hydrodynamic diameter and cGMP synthesis was assessed using a one-tailed All data contained in the article are located in within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Aberrant Ca2+ leak through ryanodine receptor 1 (RyR1) on the sarcoplasmic reticulum (SR) membrane can lead to heatstroke and malignant hyperthermia (MH) susceptibility, as well as severe myopathy. However, the mechanism by which Ca2+ leak drives these pathologies is unknown. Here, we investigate the effects of four mouse genotypes with increasingly severe RyR1 leak in skeletal muscle fibers. We find that RyR1 Ca2+ leak initiates a cascade of events that cause precise redistribution of Ca2+ among the SR, cytoplasm, and mitochondria through altering the Ca2+ permeability of the transverse tubular system membrane. This redistribution of Ca2+ allows mice with moderate RyR1 leak to maintain normal function; however, severe RyR1 leak with RYR1 mutations reduces the capacity to generate force. Our results reveal the mechanism underlying force preservation, increased ATP metabolism, and susceptibility to MH in individuals with gain-of-function RYR1 mutations.Muscle contraction depends on tightly regulated Ca In addition, adenosine 5′-triphosphate (ATP) hydrolysis by the SR Ca2+ adenosine triphosphatase (ATPase) (SERCA), which pumps cytoplasmic Ca2+ back into the SR following Ca2+ release, accounts for up to 15% of whole-body resting ATP use  is a highly specialized Ca2+ is ryanodine receptor 1 (RyR1), which forms a physical interaction with the voltage sensor of the transverse tubular (t-) system, allowing rapid transduction of excitation for precise and timely muscle contraction [excitation-contraction (EC) coupling]  under anesthesia in which volatile anesthetics trigger uncontrolled Ca2+ release and heat production  mice live to adulthood (HOM mice have a reduced life span) but show a gene dose–dependent susceptibility to volatile anesthetics and increased ambient temperature. These triggers drive further Ca2+ leakiness and additive heat production. We further show that HET mice can generate normal force during EC coupling and thus resemble humans with the same and similar mutations, who usually lack any phenotype in the absence of external triggers  , HET, and HOM RYR1 KI mice and CSQ1 null mice. HET and HOM RYR1 KI mice provided an opportunity to investigate gene dose effects of Ca2+ leak, and CSQ1 null mice provided a model of RyR1 Ca2+ leak in the absence of RYR1 mutation but lowered SR Ca2+ buffering capacity  the t-system membrane of the skinned fiber preparation continues to function physiologically, (ii) the skinned fiber allows measurements of [Ca2+]t-sys that are inaccessible to intact fiber preparations, and (iii) the sealing of the t-system provides functional isolation of the t-system membrane Ca2+-handling properties from contamination by the slow diffusion of Ca2+ with the interstitial fluid. Therefore, the skinned fiber preparation could be used to measure the physiological properties of the t-system membrane to inform our understanding of the intact muscle physiology.It is assumed that the [Ca2+] in the junctional space (JS) ([Ca2+]JS), the plasma membrane Ca2+ ATPase (PMCA) extrudes these ions across the t-system membrane, resulting in a rise in [Ca2+] in the sealed t-system ([Ca2+]t-sys) (2+]t-sys induced by cytoplasmic solutions reported by rhod-5N in WT and HOM fibers are shown (2+]t-sys during the activation of store-operated Ca2+ entry (SOCE) via SR Ca2+ depletion by caffeine, followed by establishment of millimolar-level steady-state [Ca2+]t-sys by replacing caffeine solution with a standard solution containing 200 nM [Ca2+]cyto. The introduction of tetracaine (Tet) under identical ionic conditions reduces the [Ca2+]t-sys, showing the effect of blocking RyR Ca2+ leak. The use of Tet in the same fiber where RyR1 Ca2+ leak is being determined controls for differences in t-system Ca2+ handling between fibers and genotypes, which may have different populations of Ca2+ channels. Thus, the sealed t-system provides a nanodomain sensor of nanomolar changes in [Ca2+]JS, which in turn is set by RyR1 Ca2+ leak  . Examplere shown . In both2+]t-sys following SR depletion and subsequent addition of different cytoplasmic [Ca2+] ([Ca2+]cyto) was determined for each genotype in the absence . Fitted Hill curves shifted progressively to the left as the number of RYR1 mutations increased (from HET to HOM mice) and even further in the absence of CSQ1. Physiological levels of [Ca2+]t-sys were reached at lower [Ca2+]cyto as the severity of RyR1 leak increased, because [Ca2+]JS, and therefore the activity of t-system PMCAs, was higher in these mutants. To quantify RyR1 Ca2+ leak, we calculated the difference between steady-state [Ca2+]t-sys with functional RyR1s and steady-state [Ca2+]t-sys with blocked RyR1s (Δ[Ca2+]t-sys) (2+]t-sys (RyR1 Ca2+ leak) increased for a given [Ca2+]cyto as the number of mutated RYR1 alleles accumulated between WT, HET, and HOM RYR1 KI mice  . Each da KI mice . CSQ1 nu KI mice . While wRYR1 KI mice and CSQ1 null mice display a sex-specific susceptibility to volatile anesthetics and external heat stress. Male HOM RYR1 KI  occurred 1 to 5 s after the exchange of a caffeine solution for the standard solution. WT . We conclude that RyR1 leakiness underlies the chronic increase in t-system Ca2+ extrusion capacity for each genotype. Furthermore, because there is a consistent steady-state [Ca2+]t-sys because of pump-leak balance, the t-systems of WT, HET, HOM, and CSQ1 null mice must be progressively leakier. We confirmed this by monitoring the rate of divalent ion entry into myotubes derived from RYR1 KI mouse muscle using Mn2+ quenching of cytoplasmic Fura-2. These experiments revealed that the rate of Mn2+ entry was increasingly sensitive to blocking RyR1 Ca2+ leak in the mutants in a gene dose–dependent manner (fig. S4). We did not explore t-system Ca2+ uptake rates for sex differences as our preliminary experiments indicated that the temporal resolution of our assay would not be sufficient to easily separate these groups within each genotype.We therefore determined the rate at which the t-system was able to pump (extrude) Caa2+ flux  (11). Fition. WT  and CSQ1tion. WT  fibers dar range , consistncreased . In the 2+ occurred without any change in PMCA protein expression in our two most extreme models (WT and CSQ1 null mice) (fig. S5). We can conclude that the apparent affinity of the PMCA for Ca2+ must be shifted to change the extrusion rate required to offset the influx of Ca2+ across the t-system of the resting fiber. Such a scenario could be explained by the binding of calmodulin to the PMCA, which is dependent on [Ca2+] . The raised [Ca2+]cyto in turn caused an increase to the ATP consumption rate by SERCA and, consequently, increased heat output due to an increased metabolic requirement .Having established that RyR1 leak increases [Ca2+]cyto causing mitochondria to develop a greater capacity to produce ATP. Such a mechanism would be accompanied by changes in both mitochondrial morphology and Ca2+ content . Normal SR, t-system, and mitochondria structure were observed in WT and HET muscle  in WT and mutant mouse muscles using rhod-2 and confocal microscopy (fig. S9). [Ca2+]mito increased as a function of resting [Ca2+]cyto in RYR1 KI muscles  Ca2+ content in mutated muscle fibers  (figs. S2 and S10), and mitochondrial Ca2+ was calculated as the difference between the noncaffeine releasable Ca2+ content of fibers before and after mitochondrial Ca2+ depletion. These experiments revealed that total mitochondrial Ca2+ content progressively increased between WT, HET, HOM, and CSQ1 null mice. Furthermore, muscle fibers from male mice had a greater mitochondrial Ca2+ content than those from female mice  using [Ca2+]cyto data available , respectively.As we expected that cyto, and [Ca2+]mito.The Cad HET ~1200 mice, 2+ that we observed in muscle fibers from mice with leaky RyR1s , the SR Ca2+ content reduced (blue bars). The very low SR Ca2+ content in CSQ1 null mice is due to the absence of CSQ1 buffering as well as RyR1 leak, preventing it from having comparable total fiber Ca2+ to the WT. The Ca2+ content of the cytoplasm (purple bars) and mitochondria (green bars) increased with progressive RYR1 KI mutations, but was similar in HOM RYR1 KI and CSQ1 null mice, despite the twofold difference in SR Ca2+ content. The RyR1 Ca2+ leak rate and resting [Ca2+]cyto in HOM RYR1 KI and CSQ1 null mice were similar . There were no significant differences in the measured times for twitch force to develop or relax . Generally, the amount of Ca2+ released was inversely related to resting [Ca2+]cyto . Regardless, our model shows that HOM RYR1 KI fibers release less Ca2+, during a prolonged timeframe, in response to a single stimulus.To infer the Caa2+]cyto . That is2+ leak from the SR for each genotype (2+ leak into the cytoplasm was 17.1 μM s−1 (relative to fiber volume) in WT fibers. Because [Ca2+]cyto is constant in resting muscle, the leak of Ca2+ into the cytoplasm must be matched by the rate at which it is pumped back into the SR. The heat associated with the ATP splitting required to pump Ca2+ at 17.1 μM s−1 would be 0.6 mW (g whole muscle)−1, which is about 30% of the total heat production in resting mouse muscle , and CSQ1 null mice. We found that Ca2+ leak was dependent on the number of mutated RYR1 alleles, sex, and SR Ca2+ buffering capacity. RyR1 Ca2+ leak triggered a precise redistribution of Ca2+ in fibers, while the total fiber Ca2+ content remained constant in RYR1 KI compared with WT mice. We provide the first measures of mitochondrial Ca2+ content in muscle and show that this is sensitive to [Ca2+]cyto. The critical triggering event of fiber Ca2+ redistribution is RyR1 Ca2+ leak and Ca2+ entry across the transverse tubular membrane, setting [Ca2+]cyto. A functional consequence of the redistribution of fiber Ca2+ is to relieve the amount of Ca2+ release required for force generation during EC coupling. Specifically, in HET muscle fibers, the increase in cytoplasmic Ca2+ with lowered SR Ca2+ content maintained maximum force. In HOM fibers, redistribution of Ca2+ due to leaky RyR1s resulted in a further decrease in SR Ca2+ content and increase in [Ca2+]cyto compared with HET. Compensation for the reduced SR capacity for Ca2+ release in HOM fibers was only partially offset by the basal loading of cytoplasmic Ca2+, suggesting that the RyR1 itself may have been too compromised to provide the Ca2+ release required for force generation like that observed in the WT.We have investigated a broad sample of effects from a spectrum of RyR1 Ca2+ leak (2+ influx. The influx may be activated by the depletion of the SR (SOCE), raised [Ca2+]JS, or potentially the activity of the PMCA. The most likely source of the influx is SOCE, which is a Ca2+ influx across the transverse tubular membrane, graded to the near-membrane depletion of SR terminal cisternae Ca2+ due to RyR1 activity ethane-N,N,N′,N′-tetraacetic acid (BAPTA), prevents the [Ca2+]JS rise due to increasing RyR activity, but this intervention does not affect t-system Ca2+ entry activated from the SR .The redistribution of fiber Ca mutants  and 3. T2+ movements across the t-system membrane must be regulated by RyR1 leak. We suggest that RyR1 leak simultaneously generates Ca2+ nanogradients either side of the SR terminal cisternae membrane to do this. A near-membrane Ca2+ depletion and an increased [Ca2+]JS would trigger low amplitude SOCE and chronically influence PMCA activity through Ca2+-calmodulin binding (2+ must have changed (figs. S5 and S13).Coordination of Ca2+ leak, and therefore higher SR Ca2+ content than their male counterparts . In WT fibers, the first action potential in a train will trigger release of ~5 times more Ca2+ than subsequent action potentials , which cause muscle weakness  with the increased level of metabolic stress and reduced muscle force capacity. Our results thus reveal that, in healthy muscle, a stable RyR1 provides the SR with maximum Ca2+ storage capacity and a cytoplasm that is relatively free of Ca2+. This provides the muscle with not only optimal force generation capacity but also a defense against external stress–induced heatstroke or hyperthermia. The free cytoplasmic Ca2+-binding sites in resting muscle are a “safety net,” providing buffering sites for Ca2+ during uncontrolled release of SR Ca2+ that limit the increase in [Ca2+]cyto and ATP consumption.Our study describes the cost-benefit of regulated redistribution of CaRYR1  or frozen for later Western blot analysis or EM (see below).All experimental methods using mice were approved by the Animal Ethics Committees at The University of Queensland and at La Trobe University. Male and female C57/Bl6 mice WT, HET, and HOM for the p.G2435R variant of 2, 2.5 mM; MgCl2, 2 mM; fluo-5N salt, 1 mM; or rhod-5N salt, 2.5 to 7.5 mM; and Hepes, 10 mM (pH adjusted to 7.4 with NaOH). The dye solution was allowed more than 10 min to diffuse into the t-system from the surrounding bubble of solution. After this equilibration period, individual fibers that had been exposed to the solution were isolated and mechanically skinned. The final [rhod-5N] in the t-system was expected to be in the micromolar  40× Plan-Apochromat objective. Rhod-5N was excited with 543-nm HeNe laser, and the emission was filtered using a spectral detector. To track Cat) [F (t)] was collected during continuous xyt imaging during multiple internal solution changes. 2+]t-sys transient ([Ca2+]t-sys (t)) during cytoplasmic solution exchanges was tracked to determine RyR1 Ca2+ leak. The [Ca2+]t-sys (t) is first measured at 1.2 mM in response to standard resting solution with 67 nM [Ca2+]cyto . A unidirectional flux was generated by depleting the SR of Ca2+ with caffeine (to induce SOCE), which caused a rapid decline of the [Ca2+]t-sys to around 0.1 mM. Applying, in this example, 200 nM [Ca2+]cyto to the Ca2+-depleted fiber caused a rapid rise in [Ca2+]t-sys (t) to a new steady state, about 1.4 mM [Ca2+]t-sys, because the SR filled with Ca2+ to deactivate SOCE and Ca2+ filled the JS to activate t-system PMCAs. The substitution of the solution containing 200 nM [Ca2+]cyto to one containing 200 nM [Ca2+]cyto and the RyR1 blocker 1 mM Tet caused the [Ca2+]t-sys (t) to drop by ~0.3 mM. The drop in [Ca2+]t-sys is due to the reduced [Ca2+]JS as the RyR1 Ca2+ leak is blocked.T-system rhod-5N fluorescence (2+]t-sys (t) in a mechanically skinned fiber from RYR1 KI HOM. In this case, the addition of Tet caused a deeper decline of [Ca2+]t-sys than in the WT fiber, indicative of a leakier RyR1 in the HOM than the WT fiber.2+, followed by 0 Ca2+ to obtain the fluorescence maximum (Fmax) and minimum (Fmin), respectively. These values were used in conjunction with the previously determined KD of rhod-5N in the t-system of 0.8 mM  = KD,Ca * [F (t) – Fmin)/(Fmax – F (t)].At the end of the experiment, each fiber was exposed to ionophore (ionomycin from Streptomyces conglobatus) and 5 mM Ca2+ across the t-system membrane (in mM s−1) was determined as (d[Ca2+]/dt)*Bt-sys, where Bt-sys is the Ca2+ buffering power of the t-system . We have previously estimated Bt-sys to be ~1  with Tukey’s multiple comparisons was used to determine significant differences in 2+]mito in skinned fibers, preparations were incubated at 4°C for 10 min in a cytoplasmic solution containing no added Ca2+ and 10 μM rhod-2 acetoxymethyl (AM). The rhod-2 fluorescence from the mitochondria was monitored on the confocal microscope as the mitochondria were depolarized in a standard solution containing FCCP. The FCCP-induced rhod-2 spike was used to determine Fmax, and Fmin was determined as the lowest rhod-2 fluorescence signal achieved in the presence of FCCP (extended data fig. S9). These values were used in conjunction with the previously determined KD of rhod-2 in the mitochondria of 1.54 μM (2+]mito with the relationship [Ca2+]mito (t) = KD,Ca * [F (t) – Fmin)/(Fmax – F (t)].To measure ); creatine phosphate, 10 mM; total EGTA, 0.075 mM; Hepes, 90 mM; pH 7.1 and pCa (−log10 [Ca2+]) ~7.1, except where stated. Where required, the SR of the skinned fiber was totally depleted of all releasable Ca2+ by exposure to the “full release solution,” which was similar to the K-HDTA solution but with 30 mM caffeine, 0.05 mM free Mg2+ , and 0.5 mM free EGTA (pCa 8.5) present to chelate released Ca2+. Where required, the SR was maximally loaded with Ca2+ by exposing the fiber for 4 min to a load solution, which was the same as the standard K-HDTA solution but with the pCa buffered at 6.7 with 1 mM total CaEGTA-EGTA. Under these conditions, a 4-min load period was sufficient to load the SR close to its maximum capacity  at 1 mM. These two solutions were mixed in appropriate ratios to produce solutions with pCa in the range 6.7 to 4.7.Following the procedure described  to establish the exact amount of BAPTA present and was the same stock as used in our previous studies (2+ present in the mitochondria. FCCP was added from a freshly prepared 50 mM stock solution in dimethyl sulfoxide.The BAPTA solution used to pre-equilibrate fibers before lysing (see below) was similar to the standard K-HDTA solution but had no EGTA and instead had 0.1 to 15 mM BAPTA added from a 47 mM BAPTA stock solution. The BAPTA stock solution was titrated with Ca2+ relationship was determined by exposing the skinned fiber segment to a sequence of heavily buffered solutions at progressively higher free [Ca2+] , with maximum force defined as that elicited at pCa 4.7. Isometric force produced at each [Ca2+] was expressed as a percentage of the corresponding maximum force and analyzed by fitting a Hill curve using GraphPad Prism 6 software to ascertain the pCa50  and Hill coefficient (h) for each sequence.The force-Ca2+ contained in a fiber can be quantified by pre-equilibrating the skinned fiber in a solution with a known concentration of the very fast calcium buffer BAPTA and then transferring the fiber to an emulsion of 10% Triton X-100 and paraffin oil (TX-oil) to lyse all membranous compartments and release any Ca2+ from within the fiber. Briefly, the skinned fiber was first placed in the standard weakly Ca2+-buffered K-HDTA solution for 2 min to wash out all the diffusible Ca2+-binding proteins present endogenously in the cytoplasm without altering the Ca2+ content of the fiber. The skinned fiber was then equilibrated for 20 s in a solution with a set [BAPTA] before being placed in a freshly triturated emulsion of TX-oil (10% v/v). The Ca2+ released upon the membrane lysing rapidly binds to the known amount of BAPTA present within the fiber and to other sites, predominantly troponin C (TnC). If the pre-equilibrating [BAPTA] was chosen such that the fiber produced a finite, nonmaximal force response upon lysis, then the total amount of Ca2+ present in the fiber could be calculated in absolute terms from the [BAPTA] in the equilibration solution and the magnitude of the force response. Other skinned fiber segments, before the TX-oil lysing, were (i) fully depleted of their endogenous SR Ca2+ content by a 1-min exposure to the full release solution or (ii) loaded to their maximal SR Ca2+ capacity by a 4-min exposure to standard load solution  .As described previously (2+ content within the fiber at the time of lysis ([Ca2+]T), expressed in millimoles per liter total fiber volume ), as described in detail previously (2+ within a given fiber ([Ca2+]T) was calculated as follows:The total Ca2+ concentration  within the fiber at the peak of the force response elicited upon lysis was calculated from the relationship between force and [Ca2+], which was defined as being the Hill curve with values of pCa50 and Hill coefficient for that fiber of the specified mouse genotype. Mean values of pCa50 and Hill coefficient used for calculation were 5.71 and 5.2, respectively, for WT fibers; 5.81 and 6.9, respectively, for CSQ1 null fibers; 5.80 and 5.9, respectively, for RyRKI/WT fibers; and 5.83 and 6.1, respectively, for RyRKI/KI fibers. The [Ca2+] was calculated analogously to equations 1 and 2 in  (2+ (%CaBAPTA) was determined from the size of the force response, the relevant force-[Ca2+] relationship , and the known Ca2+-binding properties of BAPTA  (2+ binding to TnC (CaTnC) (in millimolar) as a function of force was calculated based on the data of  was thus calculated as being [Ca2+]T = 1.13 × [BAPTA] × %CaBAPTA/100 + CaTnC + 9.6 × [Ca2+] × 1000 – 0.015.6. Last, 0.015 mM was deducted from the total to take into account the contaminating Ca2O and incubated in Miser Antibody Extender solution  for 10 min, rinsed five times in Milli-Q H2O, blocked for ~2 hours in blocking buffer [5% skim milk in 1× tris-buffered saline with Tween 20 (1× TBST)], and washed twice for ~30 s in 1× TBST. Subsequent to blocking and washing, the membrane was cut horizontally at designated molecular weights, enabling separate probing of multiple proteins of interest at variable molecular weights and minimizing the number of primary antibody reprobes for an individual membrane section. The individual membrane sections were exposed to primary antibodies diluted in 1% bovine serum albumin in phosphate-buffered saline in Tween 20 (1× PBST) and 0.02% sodium azide overnight at 4°C and 2 hours at room temperature (RT), all with constant rocking. The primary antibodies used and the assigned dilutions were RyR1 , DHPR , PMCA , optic atrophy 1 , MFN2 , dynamic-related protein 1 , oxidative phosphorylation , MiD49 , actin , NADH:Ubiquinone Oxidoreductase Subunit A9 , and cytochrome c oxidase subunit IV , as described previously  and CSQ KO (N = 6) mice, similar to methods previously described (−1 made in tris buffer), 10 μl of acetyl-coA (6 mg ml−1 made in tris buffer), and 50 μl freshly prepared oxaloacetate acid (1.2 mg ml−1 in tris Buffer) and absorbance set to zero at 412 nm in a spectrophotometer . The blank cuvette was read for 3 min and at 15-s intervals during activity data collection to ensure that stable baseline readings were obtained. Sample cuvettes for all WT and CSQ KO mice were similarly prepared to the reference cuvette with the addition of 15 μl of muscle homogenate for each animal. For all blank and sample cuvettes, once the oxaloacetate acid solution was added, the citrate synthase activity readings began with absorbance recorded at 15-s intervals for 3 min. The overall change in absorbance (∆Abs) was determined, and the citrate synthase activity was presented as μmol min−1 total protein−1.The citrate synthase activity was measured in EDL muscle of WT (RYR1 KI (HOM and HET) mice and processed for EM using a modification of the method of (Intact EDL muscle was dissected from WT and 2+ distribution model (fig. S13) was used to determine the likely basis of the low twitch force output of RYR1 KI HOM fibers compared to the fibers from WT and RYR1 KI HET animals , ATP, and the Ca2+ indicator dye. Ca2+ is returned to the SR via the SERCA. The rate of pumping was assumed vary in a sigmoidal fashion on [Ca2+]cyto with a slope of 2 and with half-maximal rate achieved when [Ca2+]cyto was 50 μM −1  between Ca2+ pumping rate and [Ca2+]cyto and the reported values of [Ca2+]cyto in resting fibers from each of the genotypes. The projections also rely on the KM of SERCA for Ca2+ not changing between the genotypes. 2+ at 28 nM [Ca2+]cyto in each genotype, indicating that the SERCA of the mutant mice remains sensitive to low [Ca2+]; the leak from the SR is associated with a decline in SR Ca2+ content, indicating that the SERCA activity was not increased to compensate for SR Ca2+ loss  experimental values  in the presence of 5 μM calcium chloride. The digested slurry was filtered through a 100-μm cell strainer, lysed using a red cell lysis buffer containing 155 mM ammonium chloride, 10 mM potassium bicarbonate, and 1 mM EDTA at pH 7.2, and then lastly filtered through a 40-μm cell strainer. Cells were suspended in Ham’s F-10 with 20% fetal bovine serum (FBS) and pen/strep (400 U/ml) and underwent at least six preplatings to produce a pure myoblast population. The pure myoblast populations were maintained on rat tail collagen–coated (2 μg/cm2) petri dishes in a proliferative media consisting of Ham’s F-10 with 20% FBS, pen/strep (100 U/ml), and fibroblast growth factor–β (2.5 ng/ml), in an incubator at 37°C in 95% air/5% CO2. The medium was changed every 2 to 3 days. To differentiate the myoblasts into myotubes, myoblasts were plated onto Matrigel-coated 96-well imaging plates; once the cells were >60% confluent, the proliferative medium was changed to a differentiation medium consisting of Dulbecco’s modified Eagle’s medium with GlutaMAX and glucose (4.5 g/liter), 2% heat-inactivated horse serum, and pen/strep (100 U/ml) . The medium was changed every 1 to 2 days, and myoblasts were differentiated for 4 to 5 days at which point they underwent imaging experiments.Following procedures described previously -1-piperazineethanesulfonic acid (Hepes), 10 mM; pH 7.40 at 25°C. The dye was excited at its isosbestic point of 360 ± 5 nm with the emission measured at 510 ± 40 nm using 40× 1.3 NA objective on a Nikon Eclipse T2000 epifluorescence microscope at RT. Neutral density filters were used to reduce the intensity of the excitation light, thus preventing dye photobleaching and phototoxicity to the cells. Images were captured at 5 fps with a 2 × 2 bin through an intensified 12-bit digital intensified charge-coupled device  using IPLab software . The recorded images were then imported into FIJI software using the IPLab plugin . Regions of interest were drawn within individual cells, and a time series analyzer plugin  was used to determine the temporal changes in the fluorescence intensities. These data were exported to Prism 7 , which was used to fit linear regression models for the basal signal in IB, and then independently in Mn2+-containing solutions [manganese buffer (MnB)] in the absence and presence of 1 mM Tet. The MnB was similar to IB except 0.5 mM MnCl2 was used instead of the CaCl2 and MgCl2. The specific rate of Fura-2 quenching induced by Mn2+ entry was calculated by subtracting the basal slope from the slope during the MnB application  and expressed as arbitrary fluorescence units per second. The net slope was calculated in the absence and presence of Tet. Cells with a positive baseline net slope or an unstable quench following the addition of MnB were excluded from the analysis.Myotubes were loaded with 5 μM Fura-2 AM in 10% Pluronic F-127 for 15 min at 37°C and then 25 min at RT, then washed off twice before imaging in an imaging buffer (IB) consisting of NaCl, 133 mM; KCl, 5 mM; MgClP < 0.05.Nonnormally distributed data are presented as box plots showing all the data points, median, and interquartile range, with the whiskers representing the range. Statistical analyses were performed with GraphPad Prism 8 using either one-way ANOVA with a test for linear trend on data that were transformed using a log-normal transformation of the absolute values, or the Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Significance was accepted as


High levels of apolipoprotein C3 (APOC3) can lead to hypertriglyceridemia, which increases the risk of cardiovascular disease. We aim to create APOC3-knockout (KO) rabbits and explore the effects of APOC3 deletion on the occurrence and development of atherosclerosis.An sgRNA anchored to exon 2 of APOC3 was designed to edit embryo genomes using the CRISPR/Cas9 system. The founder rabbits were sequenced, and their lipid profile, inflammatory cytokines, and atherosclerotic plaques were analyzed.When given a normal chow (NC) diet, all APOC3-KO rabbits had 50% lower triglyceride (TG) levels than those of the matched age control group. Additionally, their plasma lipoprotein lipase increased. When fed a high-fat diet, APOC3 deficiency was observed to be more conducive to the maintenance of plasma TG, total cholesterol, and low-density lipoprotein cholesterol levels, and the inhibition of the inflammatory response and the protection against atherosclerosis in rabbits.APOC3 deficiency can delay the formation of atherosclerosis-induced HFD in rabbits, indicating this is a novel therapeutic target to treat atherosclerosis.The online version contains supplementary material available at 10.1186/s12944-021-01605-7. Oryctolagus cuniculus) continue to play significant roles in the study of lipid metabolism and histopathology [APOC3 is a key regulator of plasma triglycerides (TG) and shows significant correlation with plasma very low-density lipoprotein (VLDL) levels , 2. Hypeathology .CRISPR/Cas9 is machinery for editing genomes that forms a natural part of the bacterial defense mechanism and has been developed as a tool for genetic manipulation in mice , 11, dogNew Zealand White rabbits were sourced from Yangzhou University’s Animal Genetic Engineering Laboratory. The animals were kept in a barrier facility with a 12-h light–dark cycle at 24 °C and 55% humidity, and they were provided ad libitum access to water and fed twice a day with normal chow (NC) diet or high-fat diet (HFD) . Yangzhou University’s Animal Care Committee approved all animal studies in this work.http://www.ncbi.nlm.nih.gov/) and designed CRISPR/Cas9 single-guide (sg) RNAs using the tool provided on the website: http://crispr.mit.edu. Then, we chose one sgRNA targeting the loci of rabbit APOC3 by anchoring to exon 2, as shown in Fig. To design mutant loci of the rabbit APOC3 gene, we obtained sequences from NCBI  and sgRNA (40 ng/μl) in solution. The processed embryos were placed in M2 cushion fluid and incubated at 38 °C, 5% CO2 for 30 min, and the oviducts of pseudo-pregnant female rabbits received 10–15 of the resulting zygotes.Tail biopsy specimens were taken for the extraction of genomic DNA using the phenol-chloroform method. PCR amplification of sgRNA target sites utilized the primers F: 5′-GCTCACCCAGCTGAGATCCAT-3′ and R: 5′-CAAAGTGCTTACGGGCAGAGG-3′. After purifying with agarose gel electrophoresis, the PCR products were cloned into a pMDTM 19-T vector , and Lasergene software  was employed for the sequencing and analysis of positive clones.TRIzol reagent  was used to extract the total liver and small intestine RNA, which was reverse-transcribed with the iScript cDNA Synthesis Kit . In qPCR, the Mini Opticon Real-Time PCR detection system (Bio-Rad) was used with GoTaq qPCR Master Mix  to amplify specific cDNA. The relative mRNA quantity was calculated using the 2 − ΔΔCq method and normalized to GAPDH. We completed triplicate qPCR reactions on three separate samples.The sequences of the primers were as follows: APOC3: For 5′′-CCTCCCTTCTCAGCGTCATG-3′, Rev. 5′-GTCCCAGAACCCAGAGAACT-3′ and GADPH: For 5′-CATGTTTGTGATGGGCGTGAACAA-3′, Rev. 3′-TAAGCAGTTGGTGGTGCAGGAT-3′.http://crispr.mit.edu/), then selecting the top five POTS for each sgRNA, which were PCR amplified with the primers shown in Table The CRISPR/Cas9 system is used to efficiently alter genes in living cells and organisms allowing any subsequent changes to the phenotype to be studied. However, certain off-target effects of this technology should be considered. The APOC3-KO rabbits were checked for off-target mutations by first predicting potential off-target sites (POTS) in the sgRNAs with the CRISPR Design Tool , triglyceride (TG), LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) levels were measured enzymatically with commercially available kits . Western blotting was used to detect plasma APOC3, APOA1, APOE, and PCSK9; the following primary antibodies were used: goat anti-APOC3 , sheep anti-apoA1 , goat anti-APOE , and mouse anti-PCSK9  polyclonal antibodies. Transferrin  was included as a loading control. The secondary antibodies were horseradish-peroxidase-conjugated donkey anti-goat IgG , donkey anti-sheep IgG , and goat anti-mouse IgG .Plasma lipoprotein fractions were determined by agarose gel electrophoresis. Plasma (2 μl) samples were run on 1% agarose gel  electrophoresis and the neutral lipids stained with Fat Red 7B.The activities of hepaticlipase (HL) and LPL were assayed using commercial kits .Plasma levels of interleukin (IL)-1β and tumor necrosis factor alpha (TNF-α) were ascertained using anti-rabbit ELISA . Complete blood counts were measured using a BC-2800Vet auto hematology analyzer .The rabbits were fasted for 12 h, weighed, and orally gavaged with olive oil (Aladdin) at 10 ml.per kg body weight using a 20 ml syringe. Blood was collected before and after 1, 3, 5, and 7 h of.gavage, and the plasma TG levels at each time point were measured as already described. Oral fat tolerance tests (OFTT) were evaluated in terms of the accumulation and clearance of TG.We analyzed the atherosclerotic lesions of the aorta using a previously published protocol . In brieIn subsequent histological analysis, the aortic root samples were paraffin-sectioned, treated with hematoxylin-eosin (HE) and Masson’s trichrome (MT) stains, and incubated with monoclonal antibodies specific for macrophages (Mφ)  or the α-smooth muscle actin in smooth muscle cells .In accordance with the method described in a published report, we sectioned the formalin-fixed hearts into six blocks and embedded these in paraffin . The lefP < 0.05 was used as statistical significance threshold.The data were expressed as mean ± SD and compared by Student’s t test. All statistical analyses were conducted on GraphPad Prism, and We designed one sgRNA targeting the APOC3 anchored to exon 2 to generate mutant rabbits. Following in vitro transcription from the sgRNAa, the mRNAs and Cas9 mRNAs were co-injected into the rabbit zygotes. As shown in Tables A pair of primers was designed to amplify the target fragment of APOC3 , which was sequenced to determine whether the gene was mutated; then the PCR products were sequenced by TA-clone to further determine the mutation type and efficiency. The PCR results in Fig. Western blotting analysis showed that APOC3 was undetectable in the serum of the APOC3-KO rabbits Fig. D. FurtheEvidence shows that APOC3 can effectively increase the level of circulating TG –21. WhenTo understand the effects of APOC3 on lipid metabolism in more detail, APOC3-KO and control rabbits were put on an HFD supplemented with 3% cholesterol and 10% soybean oil for 12 weeks at 6 months of age, and their fasting plasma lipid profiles were measured in the 6th and 12th weeks. The plasma of the wildtype (WT) rabbits appeared cloudy due to the high lipid content following high-fat feeding, whereas in the sera of the APOC3 KO rabbits, no obvious chylomicron (CM) aggregation was observed  and immune cell counts in the rabbits after 12 weeks of HFD. The APOC3-KO rabbits had fewer leukocytes and monocytes in their peripheral blood compared to the WT rabbits in addition to significantly lower serum inflammatory cytokine levels Fig. A and B. Hypertriglyceridemia is a sign of abnormal lipid metabolism and an independent risk factor in atherosclerotic development . APOC3, APOC3 mainly comes from the liver, although the intestine is a secondary source, and it can be secreted into the blood . We desiThe plasma lipoprotein profiles of the WT and APOC3 KO rabbits showed significant differences; while APOB and APOE were significantly reduced in the APOC3 KO rabbits. APOA1 is the main apolipoprotein of plasma HDL , so the Atherosclerosis is a chronic inflammatory disease, and the inflammatory process is important in both the initiation and progression of lesion development –34. AfteAfter 12 weeks of HFD, the aortic tree lesion coverage of the WT rabbits was as high as 21%, while that in KO rabbits was much lower (only 3%). HE, MT, and immunohistochemical signals showed the WT rabbits had obvious atherosclerotic lesions, as well as increased intima thickening and collagen content, accompanied by the accumulation of macrophages and the proliferation of smooth muscle cells, while KO rabbits only had small early atherosclerotic lesions. However, there were no obvious pathological changes in the coronary arteries of the two groups. For the formation of coronary artery plaques in rabbits, higher blood lipids (such as seen in Watanabe heritable hyperlipidemic rabbits or LDLR KO rabbits) or other additional conditional interventions, such as hybridization or surgical ligation, are usually required. Taken together, the results suggest a lack of APOC3 restrains atherogenesis, although the exact mechanistic basis needs to be elucidated.Statins are often used to treat dyslipidemia, especially in controlling LDL-C elevation; although clinicians generally believe that the benefits of statins are exaggerated, while the potential side effects are underestimated . In addiAPOC3 KO rabbits have previously been successfully constructed using the ZFN technique with adenine inserted into exon 3 of APOC3 gene . In thisFor the first time, APOC3 KO rabbits were produced using the CRISPR/Cas9 system, and the models showed lower lipid levels and fewer atherosclerotic lesions after an HFD. However, only three APOC3 KO primary rabbits were obtained, and their genotypes were not completely consistent, even though, in the end, APOC3 was not detectable in the plasma, liver, or small intestine. The small sample size and different genotypes used were not conducive to the statistical analysis of the experimental data.An APOC3-KO rabbit model was constructed using CRISPR/Cas9 gene editing technology. APOC3 deficiency alleviated cholesterol-induced hyperlipidemia and reduced atherosclerotic plaque formation. As the lipid metabolism profile of rabbits is similar to that of humans, this study provides an animal model for basic research on the anti-atherosclerotic effects of APOC3 inhibition therapy, in addition to providing information on the condition for drug development and clinical application.Additional file 1: Supplement Fig. 1. Results of body weight of rabbits before and after HFD.


All-optical controlof nonlinear photonic processes in nanomaterialsis of significant interest from a fundamental viewpoint and with regardto applications ranging from ultrafast data processing to spectroscopyand quantum technology. However, these applications rely on a highdegree of control over the nonlinear response, which still remainselusive. Here, we demonstrate giant and broadband all-optical ultrafastmodulation of second-harmonic generation (SHG) in monolayer transition-metaldichalcogenides mediated by the modified excitonic oscillation strengthproduced upon optical pumping. We reveal a dominant role of dark excitonsto enhance SHG by up to a factor of ∼386 at room temperature,2 orders of magnitude larger than the current state-of-the-art all-opticalmodulation results. The amplitude and sign of the observed SHG modulationcan be adjusted over a broad spectral range spanning a few electronvoltswith ultrafast response down to the sub-picosecond scale via differentcarrier dynamics. Our results not only introduce an efficient methodto study intriguing exciton dynamics, but also reveal a new mechanisminvolving dark excitons to regulate all-optical nonlinear photonics. In previous works, all-optical control of SHG2 has been demonstrated in semiconductors3 as well as in metallic4 and hybrid structures,7 primarily relying on opticallyinduced electric fields and hot electrons. However, the reported modulationof SHG in noncentrosymmetric materials is generally very weak . This lack of efficient all-opticalmodulation strategies represents a major bottleneck toward the developmentof emerging and future applications, such as quantum photonics andon-chip nonlinear devices.Second-harmonic generation(SHG), a nonlinear optical process originatingin the second-order response of noncentrosymmetric materials, is arguablythe most commonly used nonlinear optical effect.8 including nonlinearoptics.9 Specifically, excitons introducestrong resonances in the optical response of TMDs, which dominatetheir linear and nonlinear optical properties aided by the extremequantum confinement and reduced screening in these materials.12 As a consequence, significant research efforts have been devotedto investigate and exploit the enhancement of the nonlinear opticalresponse in TMDs,21 which is fascinating from both fundamental and applied perspectives.Interestingly, excitonic Rydberg states exhibit general characteristicsof hydrogen-like atoms, possessing a series of discrete opticallyaccessible and optically forbidden  states, as determined by optical selection rules.22 Through strong resonant enhancement of brightexcitons, SHG can be actively tuned using several methods, such aselectrical and chemical doping.27 However, the influence of dark exciton states on nonlinear opticshas remained largely unexploited.28In recent years, two-dimensional(2D) transition metal dichalcogenides(TMDs) have emerged as a powerful platform for applications in photonicsand optoelectronics,Here,we demonstrate giant all-optical modulation of SHG withina broad spectral range in monolayer TMDs at ultrafast speed (downto ∼500 fs). Our results confirm that SHG modulation is stronglyrelated to dark excitonic states, with the SHG modulation being enhancedby the creation of dark excitons and suppressed by bright excitons.The measured enhancement of SHG reaches a factor as large as ∼386.By combining bright- and dark-exciton resonances, we achieve a dramaticmodulation of the SHG amplitude, sign, and response time over a widespectral range. We explain our results by performing first-principlescalculations supporting the leading role of optically pumped darkexcitons. Our study emphasizes time-resolved SHG spectroscopy as anefficient way to investigate high-order excitonic states and theirdynamics in 2D materials and their heterostructures. Additionally,our demonstration of a giant enhancement in the nonlinear opticalprocesses of TMD materials holds great potential for applicationsin all-optical devices.2.All experiments are performed at ambient conditions . We present a typical SHG modulation result in c ≈3.1eV (λc ≈ 400 nm wavelength) above the C-excitonpeak is chosen. The SHG signal at ℏωSHG ≈2.36 eV (λSHG ≈525 nm) generated by the seedlight at ℏωs ≈ 1.18 eV (λs ≈ 1050 nm) is immediately enhanced by the controllight with a single-exponential rising time constant are similar to those shown in Pw/Pwo, where Pw and Pwo are thesecond harmonic (SH) powers measured with and without the controllight, respectively. We find that γ is highly dependent on theincident light power. 2). We find that γ increases linearlywith the control light power (Pc) andis slightly saturated for Pc > 0.4μW pair density of>5.7 × 1014 cm–2 when consideringthe measured absorption of ∼7.1% at ∼3.1 eV). In 29 We remark that the control light intensity is only∼4.29 GW/cm2, that is, ∼5 times less thanthe seed light intensity of ∼21.9 GW/cm2. This isnotable as the control light power is typically larger than the seedlight power in the previously reported results.30 Similar enhancement phenomena  are observed when the control lightenergy is changed to ∼1.55 eV (λc = 800 nm).The maximum achievable γ at this lower photon energy of thecontrol light is 75, that is, ∼5 times smaller than the resultsobtained with the 3.1 eV control light , confirming the valley selection rule fromthe Dh3 crystal symmetry.21 When switching the control light on and off, the polarizationdirections of SHG after passing a quarter-wave plate are almost thesame with only ±2° variation  seed light at ∼1.18eV, the SH spectra filtered with σ– and σ+ polarizationsshow ∼96% helicity contrast (see PSHG = (Pw – Pwo)/Pwo) at differentseed energies (ℏωs from −0.92 to 1.44eV) with a fixed control light energy of 1.55 eV. We note that thenormalized time-resolved SHG dynamics with control light at 1.55 eVis similar to that observed at 3.1 eV . The former one allows us to precisely determinethe zero-delay time by sum frequency generation in MoS2 .To explore the modulation mechanism, we measure the temporallyand spectrally resolved SHG fractional power changes (ΔPSHG ≥ 0) by the control light when ℏωSHG (λSHG) lies in the ∼2.07–2.56 eV(∼598–485 nm) range. We denote this spectral range asthe enhancement region. A representative result forℏωSHG ≈ 2.27 eV is plotted in SHG is in either the ∼2.64–2.88eV  or the 1.84–2.07 eV  range, the SHG is reduced  by the control light. We referto this spectral range as the suppression region . Then, ΔPSHG recovers withbiexponential time constants τ1 ≈ 590 fs andτ2 ≈ 96 ps . Within the range lyingin between the above-mentioned enhancement and suppression regionsin SHG ≈ 2.58eV is offered in transition region.n region 2a. A repFigure S3 in the SI), whereas the relative SHG change (ΔPSHG) is linearly related with the control light powerin all three regions using the 3.1 eV control light. Therefore, wecan rule out an exciton–exciton interaction effect , which would commonly exhibita nonlinear excitation power dependence. We can thus attribute theSHG modulation effects  to variousexcitonic transition processes  in monolayer MoS2.In our measurements 2, the tiPSHG asa function of the SHG photon energy  inthe linear absorption spectrum of monolayer MoS2 , which holds great interest for on-chipelectrically tunable all-optical nonlinear device applications. Ourresults demonstrate that electrical doping suppresses optical modulation,in analogy to electrically tunable SHG.21 This further confirms that the optically suppressed SHG effect isrelated to the bright excitons.To gain furtherunderstanding, we plot Δn energy 3a for fiyer MoS2 3c. We thelow see . The blen region 2c. The sFigure S8 in the SI). Furthermore, as shown inthe time-resolved results of 0 ≈ 600 fs, 34 At the sametime, we do not observe any change in SHG modulation at 2.27 eV whenapplying electrical doping . This indicates that electrical dopingdoes not influence the SHG enhancement. In addition, by comparingthe normalized SHG polarization dependence in monolayer MoS2 with and without the control light , we can exclude the possibility of a phasetransition during the SHG enhancement process.Additionally, we observetwo strong enhancement peaks at ∼2.27and 2.36 eV in 2 . We observe similarenhancement (with a measured γ reaching ∼70) and suppressioneffects in monolayer WS2, further corroborating the reportedall-optical modulation as a general phenomenon in exciton-supportingTMDs. PSHG results at a delay time of2.8 ps . By comparing with the optical absorptionspectral profile in sA state, which also matches well with the PL measurements . In addition, the enhancementregion in the ΔPSHG spectrum rangingfrom ∼2.0 to 2.67 eV displays a strong peak at ∼2.11eV and two small peaks at ∼2.43 and ∼2.58 eV, all ofwhich are not visible in the linear interband absorption spectrum. We then introduce opticalpumping through an effective depletion of electrons within an energyinterval Δ at the top of the valence band, accompanied by thecorresponding filling near the bottom of the conduction band (1–D4), whose real-space wave functionsare potted in To understand the observed optically driven SHG enhancement, weelaborate a theoretical interpretation of our experimental resultsin monolayer MoSion band 4a. The oion band . This alion band 4b. Specte-h composition of the excited dark excitonicstates, which acquire a substantial transition strength, thus playinga leading role in SHG  and mid-infrared intraband absorption measurements (seedetails in Table S1 in the SI).31 We find that the two enhancement peaks at ∼2.27and ∼2.36 eV are likely associated with the 2p and 3p excitonic states, respectively.Supported by these theoreticalcalculations, we attribute the SHGenhancement to the modified exciton oscillation strength created bya redistribution of excited carriers. In the enhancement region, wepropose that some of the carriers in the quasi-continuum of statesexcited by the control light scatter into low-energy bands and modifythe e in SHG 4b, c. WeFigure S7g in theSI). The results confirm that the time-resolved SHG dynamics . We assign those peaks in the enhancement region as 2p, 3p, and 4p dark statesby comparing with the energies of dark states from refs (Table S3 in the SI). This suggests that all-optical modulationof SHG is indeed applicable to other TMDs as well as their heterostructures.We also note that similar modulation effects are possible in othertypes of nonlinear optical processes, such as third harmonic generation,optical comb generation, and high harmonic generation, which deservefurther investigation.The generality of the dark-excitonmechanism and the dynamics unveiledin this work is further supported by SHG experiments in monolayerWSrom refs  and 36)2  have been well studied already, dark excitons remain largelyunexplored. This is because they are optically forbidden when relyingon traditional interband absorption/emission-based pump–probespectroscopy due to the optical selection rules.28 Here, thanks to our time-resolved SHG modulation method,we can access dark excitonic states and study their properties . The demonstrated method features two additionaladvantages for carrier dynamics exploration: First, its sensitivityis extremely high because the detection parameter of the modulationor change of the SHG signal can be extremely strong. For example,our modulation amplitude  is 4orders of magnitude larger than the variation in the linear absorption; secondly, the background noise is low becausethe detection signal is SHG, thus avoiding the strong probe signalbackground that is commonly encountered in traditional pump–probespectroscopy.Although bright excitons . We have identified three observed regionswith completely different SHG modulation responses that can potentiallyenable versatile photonic devices with different functionalities.In particular, an enhancement region that could be utilized for all-opticallyenhanced nonlinear processes with giant enhancement ratios by applyinga relatively low control power. Also, a transition region, in whichthe large fractional SHG change |ΔPSHG|  and the ultrafastfall (<150 fs) and rise (∼600 fs) response times could beused for ultrafast all-optical photonic devices, such as all-opticalnonlinear modulators. Such a fast response time corresponds to a modulationspeed of ∼1.4 THz, which is ∼14 times faster than thatof state-of-the-art electro-optic modulators.40We have demonstratedgiant all-optical modulation of SHG mediatedby excitons in monolayer TMDs. The transient dynamics of excitonicdark and bright states in monolayer MoS2 is grown on a SiO2/Si substrate by using the chemicalvapor deposition method with an ∼10 mg sulfur (at 170 °C)and ∼0.5/15 mg NaCl/MoO3 mixture (at 750 °C)for 5 min in high purity argon.41 Opticalcharacterization of MoS2, including Raman, photoluminescence,and reflection spectra, can be found in Figure S2 in the SI. A similar method is used to synthesize WS2, for which characterization is presented in Figure S14 in the SI.Monolayer MoSFigure S1 in the SI). The combined beams arefocused on the sample by a 40× objective of NA 0.75. The full-width-at-half-maximumbeam diameters of the control light at 400 nm (800 nm) and the seedlight are ∼2.5 and ∼2.2 μm, respectively. Thegenerated SHG signal is then collected by a monochromator (Andor 328i).Different filters are used to remove the control and seed light beforethe monochromator. A photomultiplier tube  connectedto a lock-in amplifier is used to detect and monitor the SHG signal.To calibrate the photon energy dependence, we extract the data afterconsidering the whole system loss within the broad range of used photonenergies and the optical reflectance/absorption of both MoS2 and the substrate.In the all-optical modulationexperiment, the control and seed light pulses (2 kHz repetition rate)are generated by an optical parametric amplifier  and divided into two parts using a dichroic mirror. The pulseduration of both control and seed pulses is ∼230 fs. The seedlight goes through an optical delay line and is then combined withthe control light by using another dichroic mirror (see 42 We then use thePerdew–Burke–Ernzerhof (PBE) version of the generalizedgradient approximation (GGA) for the exchange-correlation functional,43 combined with norm-conserving, fully relativisticpseudopotentials of the Pseudo-Dojo database.44 The plane-wave energy cutoff is set to 90 Ry for the ground-statecalculations. We use the supercell method and include 45 atomic unitsof vacuum space between two periodic images of the semiconductor layerin order to minimize interactions between adjacent cells. Quasiparticleself-energy corrections to the KS eigenenergies are calculated withinthe many-body G0W0 approximation46 as implemented in the YAMBO code.47 Theabsorption spectrum and excitonic effects are obtained by solvingthe Bethe–Salpeter equation49 (BSE) on topof G0W0. The excitonic wave functions are describedas |ΦS⟩ = ΣvckAvckS|vck⟩, where v and c denote valence and conduction band indices, k runsover wave vectors, AvckS areexpansion coefficients, and S is the exciton index.The excitation energies are determined by solving the BSE equations(Eck – Evk)AvckS + Σv′c′k′⟨vck|Keh|v′c′k′⟩Av′c′k′S = ΩSAvckS,where ΩS is the exciton eigenvalue, Evk and Eck denote the quasiparticle energies of valenceand conduction electron band states, respectively, and Keh is the electron–hole interaction kernel. Weemploy a wave vector grid consisting of 30 × 30 × 1 k points for both G0W0 and BSE calculations.The Coulomb cutoff technique is used at the edges of unit cells inthe out-of-plane direction.47 We computethe self-energy and dynamical dielectric screening using 200 bands.The four highest valence bands and four lowest conduction bands aretaken into account in the calculation of excitonic states.50We modelthe pumping-dependentchange in the exciton transition strengths from first principles assumingan effective depletion of the valence band produced by the controllight. We obtain Kohn–Sham (KS) wave functions and eigenvaluesby performing density-functional theory (DFT) calculations using theQUANTUM ESPRESSO code.S using the expression fS = |⟨G|r⃗|Φ̃S⟩|2/|⟨G|Φ̃S⟩|2, where |G⟩ denotes the ground state, whereas|Φ̃S⟩ = ΣvckAvckSfvk(1 – fck)|vck⟩is the exciton wave function obtained from its electron–hole-pairdecomposition coefficients AvckS and modified by electron redistribution according to the holeand electron occupations fvk and fck thatfollow the band filling scheme shown in CBM′CBM+Δ dEckρEck = ∫VBM−ΔVBM dEvkρEvk, where VBMand CBM correspond to the valence band maximum and conduction bandminimum, respectively, and ρEck and ρEvk arethe conduction and valence band densities of states, respectively.The depletion Δ is approximately related to the pumping lightintensity I0 through the expression ∫CBM′CBM+Δ dEckEckρEck – ∫VBM−ΔVBM dEvkEvkρEvk = I0Aτeff, where A is the absorbance calculatedat the pump energy ℏωp = 3.1 eV and τeff is an effective electron–hole recombination time,which we set to an estimated value of 4 ps.51We simulate the optical transition strengthin the presence of optical pumping by introducing an effective electrondepletion near the top of the valence band, and correspondingly, anoccupation near the bottom of the conduction band that preserves chargeneutrality. More precisely, we calculate the pumping-dependent transitionstrength of exciton


The intracellular Ca2+ changes of SR101 labeled astrocytes and the SD-related arteriole diameter variations were simultaneously visualized by multiphoton microscopy in anesthetized mice. Post-SD astrocyte Ca2+ oscillations were identified as Ca2+ events non-synchronized among astrocytes in the field of view. Ca2+ oscillations occurred minutes after the Ca2+ wave of SD. Furthermore, fewer astrocytes were involved in Ca2+ oscillations at a given time, compared to Ca2+ waves, engaging all astrocytes in the field of view simultaneously. Finally, our data confirm that astrocyte Ca2+ waves coincide with arteriolar constriction, while post-SD Ca2+ oscillations occur with the peak of the SD-related vasodilation. This is the first in vivo study to present the post-SD astrocyte Ca2+ oscillations. Our results provide novel insight into the spatio-temporal correlation between glial reactivity and cerebral arteriole diameter changes behind the SD wavefront.Spreading depolarization (SD) is a wave of mass depolarization that causes profound perfusion changes in acute cerebrovascular diseases. Although the astrocyte response is secondary to the neuronal depolarization with SD, it remains to be explored how glial activity is altered after the passage of SD. Here, we describe post-SD high frequency astrocyte Ca The outcome of acute brain injury worsens with the occurrence of secondary pathophysiological events that compromise cerebral perfusion in the subacute and chronic phase of injury. Notably, spreading depolarization (SD) waves ,2, which2+ waves have been considered as reliable indicators of astrocyte activation implicated in the glutamate release from Schaffer collateral terminals that innervate CA1 pyramidal neurons [2+ dependent gliotransmission, a process during which the astrocyte derived glutamate evokes slow inward currents (SICs) in pyramidal neurons to support their synaptic synchronization [2+ transients contribute to the regulation of cerebrovascular tone [2+ waves have been linked to both cerebrovascular dilation during neurovascular coupling [2+ transients and resting Ca2+ levels [2+ waves that temporally coincided with arteriolar constrictions in mice [+ release through large-conductance Ca2+-activated potassium channels (BK channels) on astrocyte endfeet [Astrocyte Ca neurons ,11. Addinization . Furtherlar tone ,13. Impocoupling ,15, or tcoupling ,17. Thiscoupling , or the + levels . As in p+ levels ,21, astr+ levels ,23,24,25 in mice ,27,28,292+ events and the arteriolar diameter variations during SD. We describe a characteristic pattern of large frequency astrocyte Ca2+ oscillations emerging a couple of minutes behind the Ca2+ wave of SD. The oscillations were reminiscent of a series of repetitive astrocytic Ca2+ spikes, which have recently been described in live brain slice preparations [2+ oscillations occur in vivo, in the normally perfused, intact brain of anesthetized mice. We examined the characteristic pattern of these Ca2+ oscillations and their spatio-temporal coincidence with SD-related cerebrovascular tone adjustments.In this study, we set out to explore the spatio-temporal relationship between the astrocytic Caarations . Here, w2+ changes and the arteriolar diameter variations during SD. We followed the design of our previously reported anesthetized mouse model, in which SDs were evoked by topical 1 M KCl application [2+ wave [Here, we set out to explore the spatio-temporal relationship between the astrocytic Calication . SD occu[2+ wave , and , the peak fluorescence maximum was temporally synchronized in cells of the field of view . The Ca2+ wave was also temporally coincident with the SD-related arteriolar constriction . The oscillations in individual cells were random and repetitive. Temporal coincidence of the Ca2+ spikes between cells were not observed, but the typical pattern of Ca2+ oscillations occurred in association with the plateau of the SD-related arteriolar dilation   D. Additianeously D.2+ waves and oscillations each displayed two distinct, non-overlapping pools of data, both the Ca2+ waves and the oscillations were divided into two subgroups by their temporal dynamics (2+ wave was short (≤33 s) in most of the cases , when SD travelled with a wave front from the origin of the rostral craniotomy to the imaging field (2+ wave of SD appeared to emerge from a single astrocyte and spread to neighboring somata (1–3). The Ca2+ waves measured in individual astrocyte somata in this recording were significantly longer lasting  cannot be attributed to the image sampling artifact, since the second SD in the same recording was associated with the short Ca2+ wave (14.94 ± 4.94 s).As the duration of Cadynamics A,E. The ng field A1. Howevg somata C1–3. The2+ oscillations was not linked to the direction of SD origin, because both short (<25 s) and long (>25 s) Ca2+ oscillations were observed with both propagation patterns observed .The duration of Ca2+ events. The SD-related Ca2+ wave peak fluorescence maximum was associated with the maximum degree of vasoconstriction . In contrast, the smaller amplitude Ca2+ oscillation peaks occurred during the highest degree of vasodilation   C. Althouve of SD D. At theincrease D insertsdilation E.2+ changes and vascular responses associated with SD in the anesthetized mouse somatosensory cortex. This is the first in vivo study to describe the delayed astrocyte Ca2+ oscillations following SD, and to investigate their temporal relationship to arteriolar diameter changes.In the present study, we visualized astrocyte Ca2+ waves indicate glial activity in response to mechanical, electrical, or pharmacological stimulation, which propagate from cell-to-cell with a relatively slow velocity of 10–30 µm/s [2+ waves (50-80 µm/s) [2+ waves measured in our model were typical of SD because they propagated at a speed of 77.5 ± 25.2 µm/s, delineating the direction of SD propagation  ,29. The pagation A-D. Addiur model .2+ events in individual astrocytes, we discriminated short (≤33 s) and long (>33 s) astrocyte Ca2+ waves during SD. A long Ca2+ wave was captured in one experiment, in which the SD-related Ca2+ elevation spatially radiated from a single astrocyte (1–3).Examining the duration of the SD related Castrocyte C1–3.2+ oscillations in astrocytes 2–4 min after the passage of the SD-related Ca2+ wave, occurring at a frequency of 0.66 ± 0.32 events/min/cell (0.01 Hz) in a chosen astrocyte soma. In agreement with the slice results of Wu et al., we observed that the frequency of these post-SD oscillations was three times higher than the spontaneous Ca2+ fluctuation of astrocytes during baseline (0.19 event/min/cell) [2+ oscillations in cell cultures and brain slices was shown in an earlier study [2+ oscillations among the cells [2+ oscillations have been shown to promote slow inward currents (SICs) in neurons [2+ activity of astrocytes might contribute to the NMDA receptor driven hyperexcitability after SD [2+ oscillations had a smaller fluorescent peak amplitude when compared to the prior Ca2+ waves, which could be due to different mechanisms in the background of these two events. For instance, antagonism of GABAB receptors, depletion of intracellular Ca2+ stores, or blockade of IP3 receptors all attenuated the Ca2+ oscillations, but had no significant effect on the SD-related Ca2+ wave [2+ waves rely on multiple pathways mobilizing external Ca2+, post-SD oscillations are largely dependent on Ca2+ release from the endoplasmic reticulum (ER) or mitochondria.We confirmed the occurrence of delayed random Cain/cell) . Althouger study ,34, we d neurons ,34 the iafter SD . Astrocya2+ wave ,34. One 2+ wave and the arteriolar constriction in our present study also stands in good agreement with previous reports [2+ dependent arachidonic acid release in brain slice preparations [+ release through BK channels with KCl evoked SDs in anesthetized mice [2+ oscillations and arteriolar dilation after SD.The SD-linked arteriolar diameter changes consisted of a short constriction and a subsequent dilation, confirming our earlier findings . The spa reports ,29. The arations , or perized mice . Such me2+ oscillations and the vasodilation element of the vascular response is rather unlikely, because the onset of vasodilation preceded the Ca2+ oscillations, which clearly emerged with the peak of the arteriolar dilation. Furthermore, it has also been shown that SD-associated glial Ca2+ transients  do not necessarily coincide with arteriole diameter changes [The causality between the post-SD Ca changes . Therefo2+ oscillations following SD, and are complementary to the in vitro findings of Wu et al. [Taken together, our results provide the first in vivo characterization of delayed astrocyte Cau et al. . Althougu et al. . TherefoThe experimental procedures were approved by the National Food Chain Safety and Animal Health Directorate of Csongrád County, Hungary. The procedures were performed according to the guidelines of the Scientific Committee of Animal Experimentation of the Hungarian Academy of Sciences  Gov. of Hungary), following the EU Directive 2010/63/EU on the protection. The experiments were reported in compliance with the ARRIVE guidelines.n = 6) were anesthetized with 1% Avertin , and mounted on a stereotactic frame incorporating a heating pad [Male C57BL/6 mice  The baseline diameter of penetrating arterioles was above 5 μm in order to have reliable assessment of vasoconstriction with respect to pixel size ; and (iii) The penetrating arteriole optimally appeared in cross sectional view. Vascular diameters were either measured manually at baseline , maximum constriction, subsequent maximum dilation, and recovery or using the” Diameter” plugin in Fiji [2+ waves  by placing 4–10 µm ROIs on the soma of selected astrocytes. Cells expressing both Caanalysis . The vesanalysis . Additio in Fiji . Recordi2+ waves C.p < 0.05* or p < 0.01**. Distinct statistical methods are provided in each figure legend in detail.Quantitative data are given as mean ± standard deviation (st.dev.). Statistical analysis was conducted with the software SigmaPlot 12.5 . Datasets were evaluated by a two-tailed paired T-test or a Mann-Whitney Rank Sum Test. Levels of significance were set at


Autophagy is a highly conserved cellular degradation process that prevents cell damage and promotes cell survival, and clinical efforts have exploited autophagy inhibition as a therapeutic strategy in cancer. Chloroquine is a well-known antimalarial agent that inhibits late-stage autophagy. We evaluated the effects of chloroquine on cell viability and proliferation of acute myeloid leukemia acute myeloid leukemia (AML) cells derived from 81 AML patients. Our results show that chloroquine decreased AML cell viability and proliferation for the majority of patients. Furthermore, a subgroup of AML patients showed a greater susceptibility to chloroquine, and using hierarchical cluster analysis, we identified 99 genes upregulated in this patient subgroup, including several genes related to leukemogenesis. The combination of chloroquine with low-dose cytarabine had an additive inhibitory effect on AML cell proliferation. Finally, a minority of patients showed increased extracellular constitutive mediator release in the presence of chloroquine, which was associated with strong antiproliferative effects of chloroquine as well as cytarabine. We conclude that chloroquine has antileukemic activity and should be further explored as a therapeutic drug against AML in combination with other cytotoxic or metabolic drugs; however, due to the patient heterogeneity, chloroquine therapy will probably be effective only for selected patients. Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. It is an aggressive malignancy characterized by the accumulation of immature leukemic cells in the bone marrow ,2. CurreFLT3) and isocitrate dehydrogenase (IDH) mutations) are targets for new pharmacotherapy -thymidine incorporation from day six to seven of in vitro culture, i.e., the incorporation reflects characteristics of the minor cell subset that is able to survive and proliferate after seven days of culture . Our resBasal levels of autophagy vary among AML cell lines and there is also a variation between patients; a higher autophagic flux was described for AML patients with complex karyotype whereas no differences were detected in AML with recurrent genetic abnormalities with prognostic value . We alsoBafilomycin 1A is a V-ATPase inhibitor that blocks the fusion between autophagosomes and lysosomes ,74; chloSNX2, FLT3, PFKP, and CCL23) involved in AML leukemogenesis  biomarkers that are associated with chloroquine sensitivity. Personalized or precision therapy is now considered in AML . If chlo