Vrel. First, we evaluate several eigenvalue dispersion indices under various conditions, and show that only Vrel remains stable across samples size and the number of variables. We then demonstrate that Vrel accurately characterizes input patterns of covariation, so long as redundant dimensions are excluded from the calculations. However, we also show that the variance of the sampling distribution of Vrel depends on input levels of trait covariation, making Vrel unsuitable for direct comparisons. As a solution, we propose transforming Vrel to a standardized effect size (Z‐score) for representing the magnitude of integration for a set of traits. We also propose a two‐sample test for comparing the strength of integration between taxa, and show that this test displays appropriate statistical properties. We provide software for implementing the procedure, and an empirical example illustrates its use.Understanding how and why phenotypic traits covary is a major interest in evolutionary biology. Biologists have long sought to characterize the extent of morphological integration in organisms, but comparing levels of integration for a set of traits across taxa has been hampered by the lack of a reliable summary measure and testing procedure. Here, we propose a standardized effect size for this purpose, calculated from the relative eigenvalue variance,  Organismal phenotypes are the outcomes of complex developmental and evolutionary processes, where selective forces influence multiple traits simultaneously. When biological factors such as genic, ontogenetic, developmental, or functional linkages elicit concomitant changes in more than one trait, phenotypic correlations will ultimately arise. “Morphological integration” , describing the manner in which those traits are integrated , and identifying the causes of trait covariation , are questions that have garnered considerable attention in evolutionary biology. Olson and Miller's pioneering (1958) work espoused a research paradigm to interrogate these questions, which contemporary quantitative morphologists have adapted and utilized in subsequent investigations. However, as with all studies in quantitative morphology, such inquiries should not be performed on a haphazard collection of measurements, as little biological insight may be gleaned from summarizing a set of arbitrary traits with no known connection with one another. Rather, an understanding of the patterns of phenotypic integration, and their underlying causes, can only be attained through evaluating traits selected on biological grounds based on their biomechanical, functional, or developmental properties. In the work that follows, we advocate this philosophy, and assume that the researcher has first scrutinized their choices of phenotypic attributes prior to any evaluation of integration between them.p×p trait covariance matrix in some manner  and variable number (p). Indeed, this is an important property for integration indices, as it suggests that estimates of integration are not conflated with other attributes of the data that do not characterize the underlying covariance structure of the variables . This was equivalent to generating datasets of independent traits , or for the case of geometric morphometric data, generating shapes around the mean based on an isotropic model of landmark variation  and the number of variables (p). Here, we examined six eigenvalue dispersion indices, which are enumerated in Table Vrel: Pavlicev et al. T1: Van Valen T2: Van Valen Dr: O'Keefe et al. To understand the basic properties of eigenvalue dispersion indices, we used a series of computer simulations. Our initial experiments were performed using a set of random observations drawn from a multivariate normal distribution defined as: N independent observations from a multivariate normal distribution, N, where Σ was a p×p identity matrix . Likewise, in a second set of simulations we obtained 1000 datasets with p=30 variables at each of 17 levels of sample size: N=. For each dataset, the trait covariance matrix was calculated, from which we obtained the corresponding set of eigenvalues. These were then used to calculate each of the six eigenvalue dispersion indices described above (Table λ>0). This was in accord with recent work  increased , though several first increased until N=p, and then decreased at higher N. Taken together, these results indicated that under scenarios where input levels of trait covariation were minimal, most eigenvalue dispersion indices did not remain stable. Instead, most varied with both N and p, implying these indices did not purely measure patterns of integration. Rather, they confounded integration with trends generated by both sample size and the number of variables. As a consequence, treating values based on these indices as estimates of the magnitude of integration is ill‐advised, and direct statistical comparisons of them across datasets are uninformative.As is clear from Figure Vrel . Therefore, in contrast to all other eigenvalue dispersion indices, Vrel did accurately characterize input levels of covariation under these conditions.The one exception to this pattern was the relative eigenvalue variance, rel Fig. . Here, arel Fig. , it was Vrel to determine whether it was a suitable candidate metric for estimating levels of integration, and whether it may used to statistically compare the magnitude of integration across datasets. Similar to Watanabe , and for p=32 across levels of N=, 1000 datasets were generated by drawing N independent observations from a multivariate normal distribution, N. Here, Σ was a p×p trait covariance matrix which represented differing input levels of covariation: Vrel=. To accomplish this, input covariance matrices were obtained following the computational procedures of Watanabe . Next, for each simulated dataset, eigenvalues were obtained from the trait covariance matrix, and the nontrivial eigenvalues were used to calculate Vrel. For each N and p combination, the mean and variance of Vrel estimates were then obtained.The first set of simulations were performed using the procedure described in the previous section. Briefly, for Watanabe . SpecifiN=128 and p=32, at differing input levels of covariation: Vrel=. For each dataset, Vrel was obtained for the set of nontrivial eigenvalues obtained from the trait covariance matrix, and the sampling distributions at each input level of integration were generated. From these, the skewness and variance were observed. Additionally, to evaluate how the variance of Vrel changed across its range, we repeated the simulation procedure above (with N=128 and p=32) at various input levels of covariation that spanned the range of possible input values (Vrel=0.00→1.0). At each input level, the variance of Vrel was obtained from the corresponding sampling distribution. Simulation code for both procedures is found in the For the second set of simulations, we generated 2500 datasets of Vrel remained constant across differing levels of sample size and across differing numbers of variables , the sampling distributions of Vrel became more skewed , and was highest at intermediate values (Vrel≈0.5). Together, these observations complicate direct statistical comparisons of Vrel, as they indicate that the sampling distribution of Vrel can be non‐normal, and that the standard error of Vrel does not remain constant across its range. As such, an alternative testing procedure for evaluating levels of covariation, and comparing its magnitude across taxa, is required.When the sampling distributions of ion Fig. , we founwed Fig. . More imVrel must be converted to a standardized effect size for subsequent statistical evaluation. The logic underlying this suggestion is motivated by the properties of the bivariate correlation coefficient, ρ. As with Vrel, the sampling distribution of ρ becomes more non‐normal and skewed towards its extreme values  is transformed to −1→+1; matching that of ρ:Next, a linear transformation is applied to Vrel∗ is converted to a standardized effect size using Fisher's Z‐transformation ZVVrel to ZVrel for the simulated datasets examined previously. First, it can be seen that ZVrel remained constant across both N and p, implying that this desirable property of Vrel was retained in its corresponding effect size  have been estimated, one may test explicit hypotheses regarding the strength of morphological integration between datasets. For eigenvalue dispersion indices, such evaluations have previously been accomplished via bootstrapping procedures that establish confidence intervals for subsequent statistical hypothesis testing  for two samples can be directly compared in a hypothesis test by finding the two‐sample test statistic:Once standardized effect sizes /(p∗(N−1)+2). Vrel0 is then converted to an effect size using equations 2 & 3 above. With this conversion, ZR ranges from zero (no trait covariation) to positive values (increasing levels of trait covariation), and thus expresses the magnitude of integration in a manner analogous to that of Vrel. As such, ZR serves as a useful heuristic to aid in the interpretation of the magnitude of integration in phenotypic datasets.Additionally, because, the range of Watanabe , eq. 24)Z^12, we performed two sets of simulation experiments. The first set of simulations evaluated the type I and type II error, as well as model misspecification rates. Here, we generated 1000 datasets of N=128 and p=16 for each of five different “populations,” which contained differing levels of trait covariation . As in the previous simulations, N independent observations were drawn from a multivariate normal distribution, N, where Σ was a p×p trait covariance matrix representing the input level of covariation for that population: Vrel=. As above, input covariance matrices were obtained following the computational procedures of Watanabe , model misspecification rate (Vrel=0.6 vs. Vrel=0.6), or statistical power (1 ‐ type II error: Vrel=0.3 vs. Vrel=0.6 & Vrel=0.0 vs. Vrel=0.6). This procedure was repeated 100 times, and the mean outcome obtained.To evaluate the statistical performance of Watanabe . Next, VZ^12. Here, 1000 datasets were generated for p=10 traits for multiple levels of sample size N=, using the same procedure. Datasets were generated at several input levels of trait covariation: Vrel=. As above, input covariance matrices were obtained following the computational procedures of Watanabe  were compared the rest using Z^12. The proportion of times the hypothesis of equal covariation was rejected was treated as an estimate of type I error rate or statistical power, depending upon initial conditions. Simulation code for both procedures is found in the The second set of simulations evaluated type I error and statistical power of Watanabe . Next, VZ^12 displayed appropriate statistical properties. First, the type I error of tests based on Z^12 were at nominal levels for “no effect” comparisons  across six primate species. In primates, variation in pelvic morphology is largely driven by selection on locomotor performance and function Lewton, . For exaHomo sapiens: N=60, Hylobates lar: N=57, Chlorocebus pygerythrus: N=60, Macaca fascicularis: N=56, Gorilla gorilla: N=59, and Pan troglodytes: N=60). Next, from each surface scan, the locations of eight three‐dimensional landmarks . Based on the biomechanical considerations described above, we predicted that levels of integration would be significantly greater in the two cercopithecoid taxa (Chlorocebus and Macaca) as compared with the hominoid taxa. Finally and for comparison, we calculated Vrel for the size‐standardized data with both the trivial and information‐deficient dimensions removed, where the latter were determined by information‐theory . All analyses were performed in R, using functions written by the authors , and provided in the R‐package geomorph  and estimated levels of integration as Vrel=0.77→0.83). By contrast, when differences in size were accounted for, levels of integration in the size‐adjusted traits dropped substantially (Vrel=0.11→0.38), although the rank‐order of values across species was consistent in both datasets. The comparison of size‐adjusted and size‐unadjusted values indicated that size effects and intraspecific allometry contributed substantially to overall levels of integration in these taxa. Given the body size differences among species, this result was not surprising. Subsequent comparisons of size‐standardized measurements revealed that the strength of integration in pelvic dimensions was not consistent across species, and our two‐sample test comparing effect sizes (Z‐scores) revealed that some species displayed significantly greater levels of morphological integration as compared with others  displayed higher levels of integration as compared with several homonoid taxa (Homo and Gorilla); in accord with previous hypotheses. Somewhat surprisingly, one hominoid taxon, Hylobates, displayed the greatest levels of integration in their size‐adjusted traits, and was found to be significantly greater than patterns exhibited in the other hominoids  between the set of Vrel obtained using the nontrivial dimensions and those obtained when the information‐deficient dimensions removed, indicating that for this example, the small, but non‐zero dimensions had little effect on the empirical results. Overall, when viewed in light of the biomechanical predictions above, our data provide partial support for the hypothesis that when size is accounted for, hominoids present lower levels of integration in their pelvic morphology, relative to the cercopithecoid taxa. Thus, our findings are consistent with the biomechanical prediction that changes in locomotory mode have contributed to shifts in levels of morphological integration in Homo and Gorilla , based on the relative eigenvalue variance, Vrel found from the trait covariance matrix. This effect size is capable of capturing the magnitude of integration in a sample, and may be used in a testing procedure for comparing the strength of integration between datasets. In developing these tools, our work has revealed several key insights that advance the quantitative study of morphological integration, and further our understanding of the evolution of phenotypic diversity.A major interest in evolutionary biology is determining whether taxa display similar or different levels of morphological integration, and deciphering why such differences evolve. Accomplishing this task requires summary test measures that accurately characterize the magnitude of morphological integration, and statistical tools that facilitate comparisons across sets of taxa or traits. Unfortunately, quantitative comparisons of the strength of integration in a set of traits across datasets have been limited, in part because statistical tools to facilitate these comparisons have remained underdeveloped. In this article, we propose a standardized effect size for this purpose  provisions a procedure for its implementation. As illustrated with our empirical example, this approach provides a reliable means of comparing the magnitude of integration among species (or sets of traits), thereby opening the door to future examinations of the strength of integration in other empirical systems. Therefore, our proposed effect size (ZVrel), and associated two‐sample test, contribute important new comparative tools to the evolutionary biologist's quantitative arsenal for the study of phenotypic diversity.As a measure of the strength of integration, we proposed a standardized effect size, nge Fig. . Thus, tA+B=C), deficient dimensions will arise. Second, when data‐wide standardizations are performed, such as translation, rotation, and scaling of configurations of landmarks in geometric morphometric analyses , there will be at least p−(N−1) redundant dimensions .An important consideration when characterizing patterns of morphological integration is how to treat redundant dimensions: those that contain no variation, or those that contribute no additional information in terms of phenotypic variation and covariation. From an empirical perspective, there are three common reasons why redundancies may be present in morphometric datasets which are unrelated to morphological integration, yet result in covariance matrices that are rank‐deficient. First, when one variable is a perfect linear combination of others , and demonstrated by simulation that when those dimensions were included, Vrel displayed biased estimates . Similarly, recent work by O'Keefe et al. (2022) suggested that all rank‐deficient data dimensions—both those that contribute no variation, as well as those that contain small amounts of variation but do not provide additional information  contribute nothing to trait covariation and should be removed prior to estimating levels of integration, whether or not one should concomitantly exclude dimensions with small variation is less clear. Excluding the latter based on an entropy‐based definition of rank‐deficiency implicitly assumes that variation in dimensions with small eigenvalues are effectively noise. This need not be the case, although we are unaware of any study that has examined this issue. In our empirical example, removing dimensions with small variation had little effect on the biological conclusions when compared with removing only the trivial dimensions, though in another empirical example integration estimates varied considerably more . Further, one may compare the manner in which traits covary between taxa by an assessment of the extent to which the direction of their principal axes coincide  that are not independent of one another, but rather whose values are expected to be correlated in a manner as described by their phylogenetic relationships . R‐scripts for simulation tests are found in the Supplemental Information.Data for empirical example is available on DRYAD  Emprical variance in ZV rel as estimated across simulated datasets for differing levels of sample size (N).Figure 2: Simulation demonstrating an increase in estimates of Vrel with an increasing number of redundant dimensions, when using the trait covariance matrix as input.Figure 3: Simulation demonstrating a decrease in estimates of Vrel with an increasing number of redundant dimensions, when using the trait correlation matrix as input.Click here for additional data file.Data S1Click here for additional data file.Data S1Click here for additional data file.


Dictyostelium discoideum, the cytosolic calcium level oscillates synchronously, and calcium waves propagate through the cell population during the early stages of development, including aggregation. In the unicellular phase, the calcium response through Piezo channels also functions in mechanosensing. However, calcium dynamics during multicellular morphogenesis are still unclear. Here, live imaging of cytosolic calcium revealed that calcium wave propagation, depending on cAMP relay, disappeared at the onset of multicellular body (slug) formation. Later, other forms of occasional calcium bursts and their propagation were observed in both anterior and posterior regions of migrating slugs. This calcium signaling also occurred in response to mechanical stimuli. Two pathways—calcium release from the endoplasmic reticulum via IP3 receptor and calcium influx from outside the cell—were involved in calcium signals induced by mechanical stimuli. These data suggest that calcium signaling is involved in mechanosensing in both the unicellular and multicellular phases of Dictyostelium development using different molecular mechanisms.Calcium acts as a second messenger to regulate many cellular functions, including cell motility. In  In multicellular organisms, synchronized elevations of intracellular Ca2+ levels ([Ca2+]i), or bursts, occur in cell populations, which then propagate as waves among cells4. This phenomenon has been reported in multiple cell types and biological activities, including fertilization in eggs and endothelial wound repair6. Both [Ca2+]i bursts and wave propagation play key roles in orchestrating the activities of multiple cells in vivo and in vitro2. Cell–cell communication via Ca2+ signaling has been well investigated in animals in which Ca2+ waves are propagated by gap-junction communication or paracrine signaling4. A common factor evoking [Ca2+]i bursts is a mechanical stimulus; transduction of these stimuli into Ca2+ signals is mediated by various Ca2+ channels including inositol trisphosphate (IP3) receptors, transient receptor potential (TRP) channels, and the stretch-activated ion channel, Piezo12. These channels are broadly conserved in eukaryotes, including animals, plants, and amoebae12. In mammalian cells, the Piezo channel family members, Piezo1 and Piezo2, regulate mechanosensing, in which diverse cations, including Ca2+, flow in response to mechanical stimuli, and are responsible for various biological functions, including touch sensation and cell development11. The genome of the social amoeba, Dictyostelium discoideum, encodes a single Piezo homolog, pzoA13. Hence, there is a possibility that Ca2+ signaling is universally employed for mechanosensing.Ca2+ wave propagation in populations of eukaryotic cells is cell–cell communication in the aggregation of Dictyostelium discoideum. Following starvation, Dictyostelium cells aggregate and form cell masses called mounds. Cells in mounds differentiate into either prestalk or prespore cells and form migrating multicellular bodies, or slugs. During aggregation, cAMP signaling, known as cAMP relay, organizes the directed migration of cells16, and Ca2+ waves are propagated simultaneously among starved cells17. It has been assumed that cAMP relay is essential for the coordination of collective cell migration throughout Dictyostelium development19; however, recent studies have shown that the dynamics of cAMP signaling are altered after multicellularity begins20. In addition to Ca2+ wave propagation during aggregation17, transient [Ca2+]i elevation has been observed in mounds and slugs21. These results suggest that synchronous [Ca2+]i bursts and wave propagation occur not only during aggregation but also in later Dictyostelium development, including in the formation of mounds and slugs. Calcium signaling has been suggested to be involved in chemotaxis and stalk cell differentiation26. However, the dynamics of, and molecular mechanisms underlying [Ca2+]i signaling during the morphogenesis of Dictyostelium remain unclear.One example of Ca2+]i signaling during Dictyostelium development was investigated using fluorescent Ca2+ probes. This approach revealed the transition of [Ca2+]I signaling dynamics during cell aggregation and the formation of slugs of Dictyostelium, both of which have robust calcium signaling mechanisms in response to mechanical stimuli.In this study, i dynamics during development with genetically encoded calcium indicators (GECI). As previously reported17, cells expressing the Förster resonance energy transfer (FRET) sensor YC-Nano15 (Kd = 15 nM) showed clear oscillations and wave propagation in aggregation streams 27 is functional in Dictyostelium cells and is appropriate for visualizing [Ca2+]i dynamics. Oscillations of fluorescence signals and wave propagation were observed during both early aggregation (before streaming) and mound stages of cells expressing GCaMP6s    Fig. . The posels Fig. b. In conrly Fig. . A similted Fig. . These iDictyostelium, the IP3 receptor, IplA, which is localized in the endoplasmic reticulum (ER), is responsible for [Ca2+]i elevation in response to mechanical stimuli30. Expression of iplA mRNA is low during growth, peaks at about 9 h after starvation, and then decreases29. To confirm whether IplA is involved in [Ca2+]i bursts induced by mechanical stimuli in slugs, we measured [Ca2+]i responses to mechanical stimuli in slugs lacking iplA. When iplA− slugs were stimulated with a plastic rod, [Ca2+]i bursts occurred; however, even within the same slug, the incidence of responses dropped to 46% , and the response at the anterior region peaked at 15.7 ± 7.9 s (n = 18), earlier than in wild-type controls -N,N,N′,N′-tetraacetic acid (EGTA) to chelate Ca2+. All slugs overlayed with agar containing 1 mM EGTA showed an increase in [Ca2+]i in response to mechanical stimuli (n = 13)    Fig. a, b. How01) Fig. b, e. Add13) Fig. c. These es Figs.  and 5c. Dictyostelium, [Ca2+]i transients have been observed during both the mound and slug stages21; however, the actual dynamics of Ca2+ signaling have not been clarified, because most previous studies have focused on stages before aggregation32. In this study, [Ca2+]i imaging during Dictyostelium development with highly sensitive GECIs revealed that synchronized [Ca2+]i elevations and their propagation in cell populations occur continuously during the aggregation and mound stages; however, they disappear in the late stage of multicellular development. Ca2+ wave propagation depends on cAMP relay during the early aggregation and mound stages, and cAMP wave propagation disappears during tip elongation in the late mound20. This cAMP signal has been shown to induce transient [Ca2+]i elevation33. Therefore, changes in Ca2+ dynamics of during development follow the transition of cAMP relay. We found that [Ca2+]i bursts and their propagation occasionally occurred in slugs , two pore channels (tpc), a transient receptor potential (trp) channel, and an Msc-like channel (mscS)49. In higher eukaryotes, the stretch-activated Ca2+ permeable ion channel Piezo is involved in mechanical stimulus responses9. Recently, it has been reported that D. discoideum has a homolog of Piezo, PzoA. Disruption of the pzoA gene causes defects in the [Ca2+]i response to mechanical stimuli in amoebae31. Additionally, cells lacking PzoA develop normally under laboratory conditions; however, a defect is present in chemotactic migration under confined conditions31. Slugs lacking pzoA did not show a substantial difference in calcium response from that of wild-type control slugs. Notably, the Piezo channel in higher multicellular organisms functions only in the unicellular phase in Dictyostelium. Even within Dictyostelium, there are interesting changes, with Piezo acting as the main pathway during unicellularity, and pathways from the extracellular and ER during multicellularity. In iplA− cells, the [Ca2+]i response was faster than in the wild type. This indicates that the apparent single-peak [Ca2+]i burst is a mixture of a fast extracellular Ca2+ influx with a slower, yet more efficient, response from the ER. This delayed ER response may be due to the fact that the signal from mechanoreceptors in the plasma membrane is transmitted via IP3. Moreover, in the unicellular phase of iplA− cells, no calcium response to mechanical stimuli was detected, even though IplA is not involved in blebbing, which is regulated by calcium signaling related to mechanical stimulation31. In a human colorectal carcinoma cell line, membrane blebbing is regulated by store-operated Ca2+ entry, which is controlled by ER proteins50. Alternatively, although no homolog of stromal interaction molecule (STIM) has been found in Dictyostelium51, unknown store-operated calcium channels (SOCs) may be transducing mechanical stimuli.ns Movie . Howevers show Ca+i elevat2+]i bursts at both the tip and posterior regions in slugs indicate the ability for rapid [Ca2+]i elevation in both prestalk and prespore cells. Previous studies have shown that [Ca2+]i in the anterior part of the slug is higher than that in the posterior22. However, in our study, this difference was not observed in slugs in a steady state, not receiving any external stimuli. On the contrary, if these previous data contain dynamic information, they are consistent with those of our study  following the manufacturer’s instructions57. The 5′- and 3′-flanking sequences were generated using polymerase chain reaction (PCR) and cloned into pKOSG-IBA-dicty1  in culture dishes at 21 °C. Transformants were maintained in 20 μg mL−1 G418 or 10 μg mL−1 blasticidinS .D. discoideum for efficient protein expression. The wild-type and mutant cells were transformed with ~ 1.5 μg plasmid via electroporation58, and transformants were selected using G418 or blasticidinS.Plasmids are listed in Supplementary Table 59 and R software. GCaMP6s oscillations were calculated by averaging differences between peaks. Data with at least three peaks in the oscillation were used for the analysis. In general, fluorescence intensities of GCaMP6s, Flamindo2, and the ratio of YFP/CFP channels of YCNano15 were normalized to their values at t = 0.In all experiments, cells were observed at 22 °C. Confocal images were taken using an A1 confocal laser microscope  with an oil immersion lens , or using an inverted microscope  equipped with a CSU-W1 confocal scanner unit (Yokogawa), two sCMOS cameras , and oil immersion lenses . GCaMP6s and YC-Nano15 were excited using 488 and 440 nm solid-state CW lasers, respectively. Epifluorescence micrographs were acquired using an inverted epifluorescence microscope  equipped with a 130 W mercury lamp system , sCMOS cameras , and objective lenses . Cells expressing GCaMP6s or Flamindo2 were observed using fluorescence mirror units U-FGFP . All images were processed and analyzed using the Fiji16. To induce development upon starvation, exponentially growing cells (1.5–3 × 106 cells ml−1) were harvested and washed three times in KK2 phosphate buffer . To monitor development, cells were plated on the entire surface of 2% water agar (2% w/v Difco Bacto-agar in ultrapure water) in 35 mm plastic dishes at a density of 5–7 × 105 cells cm−2  and incubated at 21 °C. Thereafter, plates were filled with liquid paraffin  to attenuate light scattering and for microscopy. Additionally, the “2D slug” method61 was applied for observing slug migration without three-dimensional scroll movement  was dropped on 2% water agar plates with 2 μL liquid paraffin. A coverslip was placed over the suspension and incubated at 21 °C for a minimum of 15 h.Imaging was performed as described previouslyDictyostelium cells expressing GCaMP6s were suspended in 1 mL developmental buffer  at a density of 5 × 105 cells mL−1 and incubated for 1 h. Thereafter, cells were exposed to 100 nM cAMP pulses at 6 min intervals for the next 5 h. Following starvation with cAMP pulses, cells were washed three times with 1 mL DB and resuspended in DB at a density of 106 per mL. Cell suspension (40 µL) was deposited onto a glass bottom dish. Cells were stimulated by adding 160 μL of 12.5 μM cAMP  to the cell droplet . During stimulation, confocal fluorescent micrographs of starved cells were acquired at 5 s intervals. Averaged fluorescence intensities of GCaMP6s in 5 μm2 regions positioned within the cytosol were estimated for each time point.7 cells mL−1, was deposited on 2% water agar with or without 1 mM EGTA and incubated at 21 °C for 12–15 h. Following slug formation, a piece of agar containing slugs was excised and placed upside down on a spacer attached to a 35 mm glass bottom dish . The spacer was filled with liquid paraffin to prevent desiccation during observation and to avoid light scattering. Slugs covered with agar were pushed with a 5 mm diameter plastic rod using a micromanipulator system  (Supplementary Fig. Five microliter of cell suspension, at a density of 4 × 10Supplementary Figures.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Video 4.Supplementary Video 5.Supplementary Video 6.Supplementary Video 7.Supplementary Video 8.Supplementary Video 9.Supplementary Video 10.Supplementary Video 11.Supplementary Video 12.Supplementary Video 13.Supplementary Video 14.Supplementary Video 15.


Acute myeloid leukemia is an aggressive cancer in children and novel therapeutic tools are warranted to improve outcomes and reduce late effects in these patients. In this study, we isolate and explore the protein profiles of leukemic stem cells and normal hematopoietic stem cells from hematologically healthy children. Differences in protein profiles between leukemic and normal hematopoietic stem cells were identified. These results provide an insight into the disrupted biological pathways in childhood acute myeloid leukemia. Moreover, differences in protein profiles may serve as potential targets for future therapies specifically aiming at the disease-propagating leukemic stem cells while omitting the normal hematopoietic stem cells.Novel therapeutic tools are warranted to improve outcomes for children with acute myeloid leukemia (AML). Differences in the proteome of leukemic blasts and stem cells (AML-SCs) in AML compared with normal hematopoietic stem cells (HSCs) may facilitate the identification of potential targets for future treatment strategies. In this explorative study, we used mass spectrometry to compare the proteome of AML-SCs and CLEC12A+ blasts from five pediatric AML patients with HSCs and hematopoietic progenitor cells from hematologically healthy, age-matched controls. A total of 456 shared proteins were identified in both leukemic and control samples. Varying protein expression profiles were observed in AML-SCs and leukemic blasts, none having any overall resemblance to healthy counterpart cell populations. Thirty-four proteins were differentially expressed between AML-SCs and HSCs, including the upregulation of HSPE1, SRSF1, and NUP210, and the enrichment of proteins suggestive of protein synthesis perturbations through the downregulation of EIF2 signaling was found. Among others, NUP210 and calreticulin were upregulated in CLEC12A+ blasts compared with HSCs. In conclusion, the observed differences in protein expression between pediatric patients with AML and pediatric controls, in particular when comparing stem cell subsets, encourages the extended exploration of leukemia and AML-SC-specific biomarkers of potential relevance in the development of future therapeutic options in pediatric AML. Conventional chemotherapy remains the mainstay in the treatment of acute myeloid leukemia (AML) in children ,2. Over Acute myeloid leukemia is a highly heterogeneous disease at both immunophenotypic and genotypic levels ,22. SomeIn many CD34 positive AML subtypes, the AML-SC population residing within the CD34+CD38− compartment is conceived as the cellular foundation for leukemogenesis ,31,32. ICryopreserved mononuclear cells (MNCs) from diagnostic BM samples from five pediatric patients with AML were included in the study based on the availability of sufficient pretherapeutic material (Supplementary methods). The samples from the leukemic patients were collected as part of the routine diagnostic workup. Clinical data were obtained from the AML database administered by the Nordic Society of Pediatric Oncology and Hematology (NOPHO) . Flow cyCells were sorted by FACS using a BD FACSAria III  to obtain proteomic profiles on isolated cell populations as described in detail in www.uniprot.org (accessed on 9 February 2020)). After filtering, a total of 456 proteins were identified in 49 eligible samples . Graph Pad Prism 9.4.3  was used for creating graphs. Principal component analysis (PCA) was performed in RStudio . Since the present analysis was a proteomic discovery-based study, we abstained from performing correction for multiple hypothesis testing since although this provides a wanted decrease in type 1 errors, it also increases type 2 errors with the risk of overlooking putative markers.Median and range were reported for the cell population frequencies unless otherwise stated. Student’s t-test was used for comparison of expression levels in the cell populations to identify significantly differentially expressed proteins  were expressed at lower levels in the AML-SCs and 18 (53%) were present at higher levels , the latSimilar to the AML-SCs from pAML23 and pAML29, we observed that the leukemic blasts from these two samples mapped together and at a different site than the remaining three patient samples and the HSCs and HPCs a. In conNext, to explore how protein expression patterns specifically distinguished PC1 and BC1 from HSCs, the following analyses were performed: First, when comparing PC1 to HSCs, 48 (77%) proteins were identified as upregulated and 14 (23%) proteins downregulated . Next, aIn summary, 17 proteins were differently expressed in the AML-SCs, PC1, and BC1 in the AML samples compared with the HSCs from the controls . Of thesIn this explorative study, we presented leukemia-associated protein profiles in stem cells and blast subsets from children with AML and compared them with HSCs/HPCs from age-matched hematologically healthy controls. Interestingly, based on the protein expression patterns, PCA analyses showed that the AML-SCs from pAML23 and pAML29 samples clustered with their CD34+ and CD34− blasts and in a different position than the healthy cell subsets. By contrast, the AML-SCs from pAML17, pAML20, and pAML21 displayed protein profiles that were, in part, similar to the control samples. Of particular interest was that the AML-SCs from pAML17 and pAML21 were positioned near the HSCs, suggestive of a more HSC-like protein profile in these AML-SCs. Still, the present study is a hypothesis-generating study and to obtain biological proof of these profiles and their putative functional impact, the data must be investigated in independent and ideally larger pediatric AML cohorts.Using IPA, we explored whether the proteins that were differentially expressed between the AML-SCs and HSCs were suggestive of pathway perturbations that could be of potential leukemogenic importance. Here, we found that signaling through the EIF2 pathway was significantly downregulated in AML-SCs. Physiologically, EIF2 signaling is important for protein translation initiation, which is inhibited in relation to cellular stress, resulting in reduced global protein synthesis . RecentlWe explored the protein profiles discriminating the AML-SCs, PC1s, and BC1s from HSCs. Overexpression of NUP210 was found in all analyses. In the context of both healthy hematopoiesis and leukemia, the function of this particular nucleoporin remains to be described. However, other nucleoporins, such as NUP98 and NUP214, are recurrently involved in gene rearrangements in AML , making Our data show that proteomic profiling of highly purified cell populations is technically feasible even with numbers as low as 1500 purified cells. A previous study, using reverse phase protein array (RPPA) of 121 selected protein targets, identified differences in the protein profile between AML-SCs and the bulk leukemia and leukemic CD34+ cells from adult AML samples . Due to One major strength of our data is that we de facto compared pediatric AML samples with the most biologically relevant control group of hematologically healthy, age-matched controls because age-related biological differences in HSCs have been established . BecauseLimited literature exists on proteomic profiling of pediatric AML. Prior studies have employed RPPA and 2D gel electrophoresis together with matrix-assisted laser desorption ionization time-of-flight MS for proteomic analysis of unfractionated BM samples ,58,59, aIn conclusion, we demonstrate altered protein expression patterns in purified leukemic cell subsets in pediatric AML. Importantly, the comparison of AML subsets to normal stem and progenitor cells is a way of obtaining a specific insight into the altered biological processes of potential therapeutic interest. In theory, severe toxicities such as myeloablation might be avoided by targeting proteins or pathways that spare, at least in part, HSCs. Our data pave the way for future investigations and the identification of such perturbed pathways that may when further investigated provide information on presumed molecular pathogenetic mechanisms and/or give rise to potential new candidates for targeted therapy in children with AML.


Furthermore, increased spontaneous Ca2+ release from intracellular stores can activate forward mode NCX. The resulting transient inward current causes delayed afterdepolarization (DAD)-dependent arrhythmias. Moreover, recently, NCX has been associated with impaired relaxation and reduced cardiac function in heart failure with preserved ejection fraction (HFpEF). Since NCX is upregulated in human chronic atrial fibrillation (AF) as well as heart failure (HF), specific inhibition may have therapeutic potential. Objective: We tested the antiarrhythmic, lusitropic and inotropic effects of a novel selective NCX-inhibitor (SAR296968) in human atrial myocardium. Methods and Results: Right atrial appendage biopsies of 46 patients undergoing elective cardiac surgery in a predominant HFpEF cohort (n = 24/46) were investigated. In isolated human atrial cardiomyocytes, SAR296968 reduced the frequency of spontaneous SR Ca2+ release events and increased caffeine transient amplitude. In accordance, in isolated atrial trabeculae, SAR296968 enhanced the developed tension after a 30 s pause of electrical stimulation consistent with reduced diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Moreover, compared to vehicle, SAR296968 decreased steady-state diastolic tension (at 1 Hz) without impairing developed systolic tension. Importantly, SAR296968 did not affect the safety parameters, such as resting membrane potential or action potential duration as measured by patch clamp. Conclusion: The novel selective NCX-inhibitor SAR296968 inhibits atrial pro-arrhythmic activity and improves diastolic and contractile function in human atrial myocardium, which may have therapeutic implications, especially for treatment of HFpEF.Background: In reverse-mode, cardiac sodium-calcium exchanger (NCX) can increase the cytoplasmic Ca Despite advances in heart failure therapy, morbidity and mortality in HF patients remain high, with an overall five-year mortality of 50%, which is even higher in end-stage HF ,2. HF ismodeling . Severalan heart ,5.+/Ca2+ exchanger (NCX1) have been found to be increased in HF, making it even more effective in competing with decreased SERCA activity for cytosolic Ca2+ degradation [2+ loading, resulting in decreased intracellular Ca2+ transients, increased diastolic Ca2+ levels on the cellular level, and decreased contractile force with diastolic dysfunction in vivo [In contrast, protein expression and activity of the cardiac sarcolemmal Naradation ,7. In adradation . In bothradation ,10, and radation . The und in vivo . Importa in vivo .Moreover, NCX seems to play a key role in impaired cardiac function in HFpEF ,15,16. B2+ homeostasis, pro-arrhythmic activity and contractile function in human atrial myocardium.The present study investigates the efficacy of a novel NCX inhibitor, SAR296968, developed for use in humans on intracellular CaA detailed description of the methods utilized is provided in the All procedures were performed according to the Declaration of Helsinki and were approved by the local ethics committee. All patients gave written informed consent prior to study inclusion. Biopsies of human right atrial appendages of patients undergoing aorto-coronary bypass grafting were used for chunk isolation of individual myocytes. Only elongated cells with cross-striations and without granulation were selected for experiments.SAR296968 was solubilized in DMSO to a 3 mmol/L stock and kept at −20 °C. The addition of a working solution accomplished final dilution. For control experiments, the “vehicle” groups used equal amounts of DMSO. For all experiments, the maximum DMSO concentration during the experiments was 0.1%.w/v) pluronic acid (30 min incubation), mounted on the stage of a laser-scanning confocal microscope  and superfused with normal Tyrode (NT) solution at room temperature. Fluo-4 was excited at 488 nm, and emission was collected through a 505 nm long-pass filter. Ca2+ sparks were analyzed using the SparkMaster plugin for ImageJ (Version 1.52) [2+ release, and the resulting transients were analyzed. Myocytes on laminin-coated recording chambers were loaded with 10 µmol/L fluo-4 acetoxymethylester in the presence of 0.02% (on 1.52) . For somMyocytes were mounted on the stage of a microscope . A ruptured-patch whole-cell current-clamp was used to measure cardiac membrane potential using microelectrodes (~5–7 MΩ). Access resistance was typically <15 MΩ after patch rupture. Action potentials were continuously elicited in current clamp configuration by square current pulses of 1–2 nA amplitude and 1–5 ms duration at variable basic cycle lengths. Signals were filtered with a 2.9 kHz Bessel filter and recorded with an EPC10 amplifier . All experiments were conducted at room temperature.2.Trabeculae were dissected from right-atrial appendage biopsies. For isometric force recordings, trabeculae were mounted in a chamber and connected to a force transducer. As previously described, trabeculae were superfused with Krebs–Henseleit solution and were electrically field-stimulated (1 Hz) . Contracp-values are two-sided, and the threshold for significance was set at p < 0.05. All data in graphs are expressed as mean patient data ± SEM. Continuous variables are expressed as mean ± SD or median (IQR) for baseline patient characteristics, and categorical variables as frequencies and percentages, respectively. Statistical analysis was conducted on mean patient data using SPSS version 28  and GraphPad Prism 9 . Normality was assessed by Shapiro–Wilk normality test. For experiments comparing more than two groups of paired data, RM-mixed effects analysis with Bonferroni’s multiple comparisons test was performed. In selected experiments, tests for linear trends between groups were performed. For this, data was arranged with inhibitor concentrations in ascending order. All reported 2+ spark frequency measured in human cardiomyocytes isolated from the right atrial appendage biopsies are displayed in 2+ spark frequency (CaSpF) was significantly reduced in the presence of the NCX inhibitor SAR296968 from (in 100 µm−1 s−1) 2.679 ± 0.1949  to 1.952 ± 0.1859 (300 nM) and further to 1.802 ± 0.1491 (3 µM) . Subgroup analysis revealed that the reduction of CaSpF was present in myocytes of HFpEF and non-HFpEF patients  was significantly increased by SAR296968 at 3 µM from 8.926 ± 0.836 to 11.58 ± 1.302 . Of note, SAR296968 did not significantly alter Ca2+ spark width, amplitude, or total SR Ca2+ leak but significantly increased Ca2+ spark duration at 3 µM . Interestingly, in the presence of SAR296968, the diastolic tension was reduced from 2.432 ± 0.2194 mN/mm2 (vehicle) to 1.803 ± 0.2711 mN/mm2 (300 nM) and 1.670 ± 0.2552 mN/mm2 (3 µM) . Intriguingly, this reduction in passive tension by SAR296968 was significant in HFpEF patients upon subgroup analysis . The increased SR Cap = 0.035 vs. vehicle). Additionally, there was a positive linear trend with increasing SAR296968 concentration (p = 0.018 for linear trend).Additionally, the developed tension as a measure of systolic contractile function was assessed. Although raw values did not significantly differ between groups  due to l2+ content and reduction in spontaneous SR Ca2+ release (see above), the ratio of developed force after a 30 s pause of electrical stimulation to steady-state developed force, i.e., the “post 30 s rest ratio”, was significantly increased by exposure to SAR296968 from 0.79 ± 0.09895 (vehicle) to 1.192 ± 0.1682 . There was a positive linear trend with increasing SAR296968 concentration (p = 0.02 for linear trend). In contrast to diastolic tension and systolic developed force, SAR296968 did not significantly alter trabeculae relaxation kinetics, indicated by the time to reach 80% of baseline level (RT80)  .Patch clamp experiments were performed on isolated human atrial cardiomyocytes to assess the potential safety of pharmacological NCX inhibition by SAR296968. The resting membrane potential was similar between groups A. ImportTo date, pharmacological NCX inhibition has been broadly tested in animal models, including rodents and large species ,20,21. HNa or ICa in vitro nor prolongation of the QT interval in vivo while providing high pharmacological selectivity with an IC50 of 74 nM for human NCX1 [2-sensitive currents (upon the voltage ramps from −120 mV to +60 mV) were analyzed and showed that SAR296968 inhibited both the NCX forward mode (at −90 mV Em) and the reverse mode (at +45 mV Em) with an IC50 for the forward mode of 34.9 nM (95% CI: 29.0–42.0) and 38.9 nM (95%CI: 30.6–49.5) for reverse mode in guinea pig cardiomyocytes [2+ extrusion mechanism from the cell in cardiomyocytes and is therefore involved in repolarization [Previously, the safety of SAR296968 has been studied. In a recent study, SAR296968 did not cause relevant inhibition of Iman NCX1 . In addimyocytes . Additiorization , we did 2+ leak is a hallmark of heart failure (HF) [2+ sparks occur during the diastole, and increased Ca2+ spark frequency and total Ca2+ leak, therefore, serve as arrhythmogenic triggers [2+ in exchange for 3Na+, which may facilitate the triggering of arrhythmias. Additionally, during conditions of increased diastolic SR leak, enhanced Ca2+ extrusion via NCX may further enhance this effect.An increased SR Caure (HF) , and is ure (HF) . Ca2+ sptriggers . The NCX+ influx contributes to increased intracellular Na+ concentrations [+ concentration in HFpEF [+ disrupts NCX function and can facilitate Na+/Ca2+ exchange in reverse mode, which results in a Ca2+ influx into the cell [2+ that enters the cell via reverse mode NCX could bind to the cytoplasmic surface of cardiac RyR2, increasing its open probability (Po) [o during cardiac excitation-contraction coupling [2+ influx during the action potential [+/calmodulin-dependent protein kinase II (CAMKII) by reverse mode NCX has previously been described, also in human atrial cardiomyocytes and discussed in detail [o and the frequency of Ca2+ sparks [2+ spark frequency observed in our experiments. In HF, but also sleep-disordered-breathing and atrial fibrillation, enhanced Natrations ,23,27. Iin HFpEF ,15,16. Ithe cell ,29. The ity (Po) . In factcoupling ,32. Morecoupling . Especiaotential . Subsequn detail . CAMKII + sparks . Thus, to is also present after inhibition of cardiac voltage-gated Na channels [2+ spark frequency can never be completely ruled out.A similar mechanism of indirect modulation of cardiac RyR Pchannels . Other dchannels . Finally2+ release events occur more frequently in atrial myocytes [2+ spark frequency was also increased in myocytes in a murine HFpEF model [2+ leak was not significantly reduced by SAR296968  can increase RyR2 open probability [2+ spark duration we observed at 3 µM  and NCX compete for Ca2+ removal, and therefore SERCA function may be improved by NCX inhibition, especially under conditions of increased NCX activity such as HF and AF. The experimental evidence provided in our study underlines improved SERCA function, as SR Ca2+ content was increased by pharmacological NCX inhibition, as was the relative contractile force and post-rest contractile force, while simultaneously reducing pro-arrhythmic spontaneous Ca2+ release events. Moreover, at the doses investigated, SAR296968 did not negatively influence action potential characteristics. Prolongation of action potential duration could further increase diastolic Ca2+, but this is not the case with SAR296968. On the other hand, both the sarcoplasmic reticulum Ca2+ release events by activating Ca2+ -induced Ca2+ -release. In conclusion, NCX inhibition may be a potential novel therapeutic strategy to combat AF and improve contractility, especially in HFpEF patients. The enhanced NCX expression has been reported in different cardiac diseases, including heart failure and atrial fibrillation ,8. NCX u2+ concentration.Due to the limited availability of tissue, SAR296968 was the only NCX-inhibiting compound investigated in two different concentrations (300 nM and 3 µM) in this study. Although the detailed pharmacological profile regarding potency and selectivity of SAR296968 indicates a high selectivity for NCX , we cann2+ release events were suppressed by pharmacological NCX inhibition. Additionally, the SR Ca2+ content was increased accordingly. Importantly, these effects were reached while maintaining a high inhibitor selectivity, and no effects on cardiac action potential characteristics were detected. These results demonstrate the potential of this novel compound developed for use in humans as a novel therapeutic pharmacotherapeutic strategy, especially for HFpEF patients.In this study, we were able to show that SAR296968, a potent NCX inhibitor, significantly increased the developed force of human atrial trabeculae, while simultaneously significantly reducing diastolic tension. Moreover, the frequency of spontaneous SR Ca


Ca2+ signaling in the intact human artery intima has not been well characterized. Here, we employed confocal imaging and a custom analysis algorithm to assess the spatially and temporally dynamic Ca2+ signaling profiles of human peripheral arteries isolated from the amputated legs of patients with advanced CVD  or patients who had lost limbs due to non-cardiovascular trauma. In all tibial artery branches (0.5–5 mm diameter) assessed, the intima consistently elicited a broad range of basal Ca2+ signals ranging from isolated focal transients to broad waves. Arteries from patients with existing CVD displayed a restricted intimal Ca2+ signaling pattern characterized by diminished event amplitude and area. Stimulation of type-4 vanilloid transient receptor potential channels (TRPV4) amplified endothelial Ca2+ signals; however, these signals remained smaller and spatially confined in arteries from patients with CVD verses those without CVD. Our findings reveal a characteristic underlying basal Ca2+ signaling pattern within the intima of human peripheral arteries and suggest a distinct truncation of the inherent Ca2+ profile with CVD.Endothelial dysfunction is a key feature of cardiovascular disease (CVD) including atherosclerosis. Impaired endothelial signaling leads to plaque formation, vascular wall remodeling and widespread cardiovascular dysregulation. The specific changes along the vascular intima associated with atherosclerosis, including the vulnerable circulation downstream of the flow obstruction, remain poorly understood. Previous findings from animal models suggest that preservation of a distinct Ca The vascular endothelium is a pivotal determinant of cardiovascular health. Risk factors including diabetes, hypertension and obesity along with altered shear stress along the vascular wall promote endothelial dysfunction . Charact2+-dependent signaling along the endothelium is crucial for many processes that preserve vascular function, including nitric oxide signaling, secretion, and nuclear transcription  correspond to vascular function, including vasodilator and shear stress responses. Although effects of chronic pathologic signaling on endothelial Ca2+ signatures remain unknown, we previously reported that 2 weeks of reduced blood flow  substantially restricts the size and range of Ca2+ dynamics along the endothelial network . Indeed, TRPV4 channels have surfaced as particularly pivotal conduits for dynamic Ca2+ signaling in the endothelium . We describe a robust dynamic Ca2+ signature along the intima of human arteries and a distinctive restriction of Ca2+ dynamics associated with existing CVD.Dynamic Cas, two CaCl2, 10 HEPES, 10 glucose; pH 7.45) at 4°C and transported to the laboratory for study. Branches from the anterior and/or posterior tibial artery (three to five artery segments of 0.5–5 mm diameter) were dissected and employed in the experiments described within 8–18 h of amputation procedure. We have observed that extended tissue storage beyond this point can impact endothelial signaling and function. Notably, while most main tibial artery segments obtained from CVD patients contained extensive atheroma, the distal branches extracted for study did not have visible plaque and diameters were comparable among preparations employed. All arteries were treated identically, regardless of patient CVD status. Tissues were excluded from study if they were obtained beyond 18 h of the amputation surgery. All procedures were approved by the University of South Alabama Institutional Biosafety Committee.Neurovascular bundle segments were removed from the limbs of patients who had undergone amputation surgery due to advanced cardiovascular disease or severe limb trauma. All tissues ware acquired after complete processing and evaluation by the University of South Alabama College of Medicine Department of Pathology. Patient gender, age, and the presence or absence of diagnosed peripheral artery disease and/or diabetes were recorded, and the tissue was deidentified. Tissues were stored in HEPES-buffered physiological saline solution  at room temperature for 40 min in the dark. After washing and 30 min equilibration, inserts were inverted and placed in a glass-bottom chamber containing HBSS (tissue 100 μm from glass). The chamber was mounted on an Andor Revolution spinning disk confocal system , and Ca2+-dependent fluorescence was measured at eight frames/sec at 25°C using iQ software . For each tissue sample, recordings were obtained from two separate intimal fields and subjected to analysis offline. 16-bit image sequence data were processed using a custom variation of the algorithm LC_Pro (2) for each event. A nuclear stain  was used to assess the status of the endothelial layer following Ca2+ measurements. Any areas lacking endothelial nuclei  were circumscribed and assessed relative to the entire field using ImageJ; intimal fields with denuded areas >20% were not included in analysis.Artery segments were cut open longitudinally and mounted on sylgard inserts, intima side up, using tungsten micropins as previously described . When pim LC_Pro . This anArteries were embedded in OCT and then cut into 20 µm cross-sections. After fixation in cold acetone for 10 min at room temperature, tissues were blocked in the blocking buffer  including 0.2% Triton X-100. TRPV4 protein was labeled with rabbit primary antibody (Alomone Labs) and Alexa 555 secondary antibody . Vascular endothelial cells were labeled with conjugated lectin-specific antibody . Elastic lamina autofluorescence was assessed using 488 nm excitation and nuclei were labeled with DAPI . Digital image stacks were collected using a Nikon A1R confocal microscope  and NIS Elements software.Reagents were purchased from Sigma-Aldrich , Cal-520 AM from AAT Bioquest  and GSK1016790A Fisher Scientific  Tungsten wires (for making pins) were purchased from Scientific Instrument Services .via Dunns post-test. p values <0.05 were considered significant.Data are presented as means ± SE and/or as medians with full distributions where values are not normally distributed. All statistical analysis was performed with GraphPad Prism nine software. Due to limited tissue samples, data in respective groups were combined and analyzed as collective groups. Student’s t-test or Mann-Whitney was used for comparison of two independent Gaussian and non-Gaussian datasets, respectively. Kruskal–Wallis was employed for nonparametric analysis of multiple data sets with subsequent comparisons We obtained samples from 10 patients who had undergone elective amputation surgery due to either advanced clinical cardiovascular disease  or non-cardiovascular related trauma. Two samples were excluded from study because they were obtained more than 18 h after the amputation procedure. One additional sample was excluded because the endothelium was severely damaged, precluding subsequent imaging and analysis. In all, samples from seven patients were used for study; four from patients who had diagnosed severe cardiovascular disease (CVD) and three from patients with no previously diagnosed cardiovascular disease (nCVD). Patient data is summarized in 2+ signaling profile of the human peripheral artery intima, we performed confocal imaging on open artery segments loaded with the Ca2+ indicator Cal-520 AM  were widely distributed and positively skewed in all vessels  in nCVD arteries compared to 4.9 ± 1.1 s-1  in CVD. This suggests higher event frequency in CVD arterial intima but the apparent difference did not reach statistical significance. Mean amplitudes were 5.5 ± 0.2 vs. 3.1 ± 0.1 ΔF , mean durations were 7.9 ± 0.3 vs. 7.4 ± 0.3 (medians 4.4 vs. 4.0), and mean areas were 62.5 ± 4.1 vs. 33.0 ± 1.7 . 2+ events in CVD artery intima, especially with respect to amplitude and spatial area. The general truncation of event size is visually evident in the x-y-t plots shown in 2+ signals are projected over time. The restricted stippled pattern in the CVD artery compared to the nCVD artery reflects the profile of smaller Ca2+ signals along the intima.Next, we compared the Ca2+ signaling in the arterial endothelium, and their capacity to generate and relay Ca2+ signals may be altered in cardiovascular disease. We extended our evaluations to assess the expression and relative effect of TRPV4 stimulation in CVD and nCVD arteries. Immunofluorescence staining (2+ dynamics in a subset of arteries using the agonist GSK1016790A (−1 to 3.5 ± 0.2 s−1 in nCVD and from 5.5 ± 0.6 s−1 to 4.1 ± 0.06 s−1 in CVD), possibly reflecting the general coalescing of many local transients into continuous events and waves (2 to 53.5 ± 4.7 µm2) and both the amplitude and area of CVD events ; Exposure to vehicle (0.01% DMSO) had no effect. Both event amplitude and area remained lower in GSK1016790A-stimulated CVD arteries compared to stimulated nCVD arteries. The truncation of events in CVD vs. nCVD arteries is evident in the time-projected plots shown in 2+ signal profile . This approach allows for detection and isolation of a wide range of events as well as the characterization of each event with space and time. Such continuous spatio-temporal tracking distinguishes events that might otherwise be coincidently sampled and misinterpreted by fixed ROIs, providing a valuable approach for future elucidation of signals within complex networks.Studies over the past decade have exposed the crucial impact of dynamic endothelial Cadilation . While edilation . For ins2+ profiles. This also ensured comparable readouts for CVD and nCVD vessels, allowing us to assess altered signaling in the diseased circulation, even where the intima remained intact. Indeed, we observed that while many arterial branches from CVD patients exhibited some degree of neointima , the endothelium was usually intact. For Ca2+ signal quantification, we have found that tracking frequency as well as maximal amplitude, duration and area of detected events provides a useful assessment of signal profile. We noted a trend of increased event frequencies in CVD arteries although additional sampling in future studies will be needed to determine if this is a common feature of CVD or whether it may be specific to a condition, disease, age or some other variable. Analysis of Ca2+ parameter profiles showed that CVD artery distributions for amplitude and area were consistently left-shifted compared to nCVD arteries, suggesting the arterial intima of CVD patients elicit truncated Ca2+ signals compared to those of non-CVD patients. Overall, analysis of distribution differences revealed that CVD arteries have a higher density of events with amplitudes of one to three ΔF, durations of 2.5–5 s, and areas of 4–16 μm2, and a lower density of events with amplitudes of 5–10 ΔF, durations of 10–40 s, and areas of 30–250 μm2 compared to nCVD. This truncation of Ca2+ event size is similar to the effect of chronic low flow on mouse carotid arteries wherein 2 weeks of reduced blood flow resulted in endothelial dysfunction and significant arterial wall remodeling, including neointima formation , allowing for localized signal targeting and providing a trigger for larger Ca2+-induced Ca2+ release events  . Extended investigation of the mechanism driving this shift and the implications for TRPV4 mediated signaling under conditions of shear stress in CVD is warranted.TRPV4 cation channels elicit focal Caticulum) . Growingticulum) . In the rtension . The imp2+ profiles and the importance of discerning specific Ca2+ signal distributions. While mean parameter values can provide basic information about the events, assessment of full distributions is needed to characterize the disparate nature and range of signals. This will be particularly important for distinguishing Ca2+ parameter shifts with specific perturbations  and determining distinct physiologic and pathologic impacts. Indeed, our data suggest TRPV4 channel stimulation has a complex effect on the endothelial Ca2+ signal profile. While spatial area of the signals expands, as evidenced by a general right shift in the event area distribution, the profile for event duration appears to flatten, essentially redistributing to the left and right. This may reflect the widespread triggering of focal Ca2+ entry events and subsequent spread of many of these events as internal Ca2+ stores are recruited. Notably, this expanded duration profile is not evident in CVD arteries. A key point is that shifts in a parameter distribution may provide important information not reflected by mean values. Overall, our current findings suggest CVD limits the capacity of TRPV4 signaling. Specific aspects of these changes, including the mechanism underlying redirection of Ca2+ signals and the direct functional consequences remain to be determined.The current findings underscore the complexity of biological Ca2+ imaging studies. These are likely smooth muscle cells just below the internal elastic lamina or part of the neointima of CVD arteries. As these cells were part of the continuous cell population and we could not definitively identify and remove specific cells from analysis, they were included. Overall, we estimate these cells contributed <5% of the signals analyzed and had little impact on the net assessment. Future iterations of the imaging and analysis applied here should allow for discrete discrimination of the vascular intima as well as the specific impact of the neointima itself. This should also include assessment of endothelial status and the relative impact of apoptotic or necrotic cells on the prevailing Ca2+ profile. While the current study was limited by the modest number of tissue samples, the findings serve as a valuable foundation for future mechanistic investigations and development of new approaches for preserving homeostatic endothelial signaling.It should be noted that cells other than endothelial cells were sometimes captured in our Ca


Several lines of evidence suggest that there are three possible positions of tropomyosin on actin commonly called Blocked, Closed/Calcium and Open or Myosin states. These states are thought to correlate with different functional states of the contractile system: inactive-Ca2+-free, inactive-Ca2+-bound and active. The inactive-Ca2+-free state is highly occupied at low free Ca2+ levels. However, saturating Ca2+ produces a mixture of inactive and active states making study of the individual states difficult. Disease causing mutations of troponin, as well as phosphomimetic mutations change the stabilities of the states of the regulatory complex thus providing tools for studying individual states. Mutants of troponin are available to stabilize each of three structural states. Particular attention is given to the hypertrophic cardiomyopathy causing mutation, Δ14 of TnT, that is missing the last 14 C-terminal residues of cardiac troponin T. Removal of the basic residues in this region eliminates the inactive-Ca2+-free state. The major state occupied with Δ14 TnT at inactivating Ca2+ levels resembles the inactive-Ca2+-bound state in function and in displacement of TnI from actin-tropomyosin. Addition of Ca2+, with Δ14TnT, shifts the equilibrium between the inactive-Ca2+-bound and the active state to favor that latter state. These mutants suggest a unique role for the C-terminal region of Troponin T as a brake to limit Ca2+ activation.Striated muscle contraction is inhibited by the actin associated proteins tropomyosin, troponin T, troponin I and troponin C. Binding of Ca Naturally occurring mutants of troponin have been identified that stabilize each of the states of regulated actin. Those states of actin have different abilities to stimulate myosin ATPase activity. In addition to giving insight into muscle disorders, these mutants have provided tools to study normal muscle function and regulation. The use of troponin mutants as tools will be apparent in the first part of this document as we review concepts of regulation, the states of regulated actin and quantitation of those states. These concepts are essential as the mutants of troponin that we studied function by changing the distribution of those actin states.via C protein (Muscle movement occurs as two filamentous protein complexes (myosin and actin) slide past each other in a process that is driven by the concentration gradient of ATP and the free energy of ATP hydrolysis. Actin binding to myosin greatly increases the rate of ATP hydrolysis by myosin; the conformational changes that occur during the cycle of ATP hydrolysis are coupled to force production. Cardiac and skeletal muscles are regulated primarily through modulation of the cofactor activity of actin through tropomyosin and troponin. This modulation may be fine-tuned by post translational modification such as phosphorylation of sites in TnI and TnT  and trop protein  or nebul protein . Phospho protein . Here we2+ binding to TnC opens a hydrophobic patch to which the switch region of TnI can bind . S1 that is extensively modified with NEM binds with high affinity to regulated actin even at saturating ATP levels and is able to activate regulated actin under those conditions  decreased activation at saturating Ca2+ but increased activation at low free Ca2+ or 3) increased activation at all Ca2+ levels. Several examples of these three classes have altered equilibrium constants among the inactive and active states of regulated actin  decreased activation at all Caed actin . Alterin2+ sensitivity. It is likely that these mutations stabilize the inactive states of regulated actin. Fifteen mutants of TnT and six mutants of TnI, associated with hypertrophic cardiomyopathy, have increased Ca2+ sensitivity; those mutants likely stabilize the active state. The association between the effect of mutations on Ca2+-sensitivity and the type of disorder has been previously noted  and 2 (active). State 1 had two substates: one without Caer model  describe binding . The ATPA, is then given by (vobs—vmin)/(vmax—vmin) where vobs is the measured ATPase rate, vmin is 0.67* the rate with zero calcium added and in the presence of EGTA, vmax is the rate measured at saturating Ca2+ and NEM-S1 and at the same actin concentration used in the other measurements. The term 0.67 comes from the rate of ATPase activity with a phosphomimetic mutant of TnI having Glu replacing the 3 phosphorylatable residues (max can be estimated by stabilizing the active state with both Ca2+ and activating or force-producing type crossbridges. The use of NEM-S1 to stabilize the active state (mentioned earlier) may involve rather large corrections  was set to zero and the rate of binding to actin in the presence of saturating Ca2+ (a mixture of the C and O states) was assumed to be at its maximum value. Although useful, such estimates are model dependent. Relaxing the restrictions led to different estimates of the actin distributions  reduces Caactivity . Actin fvity  in one case and that replaced the last 28 amino acids with a novel stretch of 7 amino acids in the other case . Muscle  binding . The acttrations  providedtrations .2+ activation of ATPase activity and sensitize fibers to Ca2+  was not formed.Wild type regulated actin filaments containing acrylodan-labeled tropomyosin show an increase in fluorescence when they progress to the inactive-Ca Δ14-TnT , or 289C Δ14-TnT  indicati2+-free state and gives a 30% enhancement of the acrylodan-tropomyosin signal did not rescue the total absence of signal with Δ14-TnT  permit faster binding  is faster than to actin in the inactive-Ca2+-bound state (C state)  . That diC state) .2+ (2+-bound) state even at low free Ca2+.The HAHA mutant of TnT had other similarities to Δ14 TnT. For example, the acrylodan-tropomyosin fluorescence assay failed to detect evidence of the B state in the virtual absence of Ca2+ . However2+ over values obtained with wild type actin filaments. Placing 289C-HAHA TnT into a cardiac fiber preparation increased the Ca2+ sensitivity by 0.24 pCa units. Overall, HAHA TnT behaved similarly to Δ14 TnT.HAHA TnT increased the ATPase activity at saturating Ca2+-bound state of wild type actin filaments to the inactive-Ca2+-free state was accompanied by movement of TnT-289 toward tropomyosin-190  to monitor changes in the CB region of TnT and to map the changes that the CB region of TnT produces in other key regions of troponin.Regulation of mammalian striated muscle contraction involves alterations in protein-protein contacts among TnC, TnI, TnT, tropomyosin and actin. Because the CB region of TnT causes large changes in the response to CaReaders may wish to consult more detailed descriptions of troponin structure than are present in this manuscript. A detailed description of the structure of a large part of the troponin complex is available . Most re2+-free state from the inactive-Ca2+-bound state, CB residues 275 and 289 approach tropomyosin-190 . Other data suggests that a site in the C-terminal region of TnT binds to tropomyosin near Cys 190 under conditions of very low free Ca2+ . That se2+ , or with2+ , or with2+ .2+ binding to TnC caused the CB region of TnT to move away from actin as well as from tropomyosin (2+ binding. The extent of movement of the CB region away from actin-tropomyosin increased when Ala replaced the basic residues within the CB region of TnT. This further movement reflects the shift from about 30% occupancy of the active state to 70% occupancy under these conditions.Further FRET studies showed that Capomyosin . That is2+ binding to the regulatory site(s) of TnC opens a hydrophobic pocket to which the switch region of TnI can bind  increased the resting force and increased the Ca2+ sensitivity of fibers  all result in cardiomyopathies. This observation complicates treatment of cardiomyopathies as it is critical to establish the natural balance. For example, the Ca2+-sensitizer Bepridil does increase activity at low free Ca2+ levels but it inhibits activity at high Ca2+ levels and seems to stabilize the inactive intermediate state of the actin filament  operate together. Fortunately, troponin mutants may contribute to answering these and other questions.The CB region of TnT reduces basal activity of regulated actin and limits Ca


Research into the complex signaling pathways is motivated by cardiac pathologies including arrhythmia and maladaptive myocyte remodeling, which result from Ca2+ dysregulation. Of interest to this investigation are two types of Ca2+ currents in cardiomyocytes: 1) background Ca2+ entry, i.e., Ca2+ transport across the sarcolemma from the extracellular space into the cytosol, and 2) Ca2+ leak from the sarcoplasmic reticulum (SR) across the SR membrane into the cytosol. Candidates for the ion channels underlying background Ca2+ entry and SR Ca2+ leak channels include members of the mechano-modulated transient receptor potential (TRP) family. We used a mathematical model of a human ventricular myocyte to analyze the individual contributions of background Ca2+ entry and SR Ca2+ leak to the modulation of Ca2+ transients and SR Ca2+ load at rest and during action potentials. Background Ca2+ entry exhibited a positive relationship with both [Ca2+]i and [Ca2+]SR. Modulating SR Ca2+ leak had opposite effects of background Ca2+ entry. Effects of SR Ca2+ leak on Ca2+ were particularly pronounced at lower pacing frequency. In contrast to the pronounced effects of background and leak Ca2+ currents on Ca2+ concentrations, the effects on cellular electrophysiology were marginal. Our studies provide quantitative insights into the differential modulation of compartmental Ca2+ concentrations by the background and leak Ca2+ currents. Furthermore, our studies support the hypothesis that TRP channels play a role in strain-modulation of cardiac contractility. In summary, our investigations shed light on the physiological effects of the background and leak Ca2+ currents and their contribution to the development of disease caused by Ca2+ dysregulation.The intricate regulation of the compartmental Ca Intricately regulated levels of Ca2+ concentrations are critical for electrical activity, excitation-contraction coupling, and other signaling pathways. In many cardiac diseases, the delicate balance of Ca2+ cycling is perturbed. Ca2+ dysregulation underlies maladaptive cardiac remodeling. A complete understanding of all components of Ca2+ handling is essential for the development of therapeutic strategies to attenuate cardiac pathologies.Ca2+ signaling in cardiomyocytes are well characterized. Ca2+ signaling related to excitation-contraction coupling primarily relies on sarcolemmal Ca2+ entry through voltage-gated L-type Ca2+ channels (LTCC) to trigger Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyR), and, subsequently, Ca2+ extrusion via the sodium-calcium exchanger (NCX) and reuptake into the SR through sarco/endoplasmic Ca2+-ATPase (SERCA). Other Ca2+ currents contribute to the modulation of Ca2+ concentrations in cardiomyocytes as well. Of interest to this study are background Ca2+ entry through the sarcolemma and SR Ca2+ leak. Understanding of the physiological role of these Ca2+ currents is still incomplete.Many ion channels underlying the Ca2+ transport across the sarcolemma from the extracellular space into the cytosol through LTCC and NCX, sarcolemmal Ca2+ transport comprises background Ca2+ entry. In resting cardiomyocytes, [Ca2+]i is of the order of 100 nM, which indicates the existence of a background Ca2+ entry pathway to balance the Ca2+ efflux of NCX  at normal heart rates   channels, which are also sensitive to Gd3+. The mdx mouse model of muscular dystrophy exhibits [Ca2+]i and [Na+]i overload that can be blocked by Gd3+, and the increase in cation entry has been suggested to involve TRPC channels (mdx mice exhibit increased expression of a putative stretch-activated channel (SAC), TRPC1. Elevated [Ca2+]i levels can also be reduced to [Ca2+]i levels of WT myocytes when exposed to SAC blockers streptomycin or GsMTx-4  . Connexi by Gd3+ . While c by Gd3+  and may,channels . Myocyte GsMTx-4 . Upregulnjection . Backgro2+ entry . Further2+ entry .2+ transport across the SR membrane from the intracellular store into the cytosol is known as SR Ca2+ leak. During an action potential, activation of RyR clusters triggered by Ca2+ entry through LTCC results in a synchronized release of a large amount of Ca2+ from the SR that forms the Ca2+ transient and leads to cardiomyocyte contraction. Activation of RyR clusters causes Ca2+ sparks. These sparks are an important pathway for SR Ca2+ leak. RyR Ca2+ leak can occur also through a mechanism independent of sparks (2+ leak includes a component separate from RyRs (3) receptor (IP3R), which is a Ca2+ release channel expressed at lower densities than RyRs in cardiomyocytes and found upregulated in heart failure (HF)   and ther or stress-induced slow increase in contractility (SSC), describing the gradual increase in twitch force corresponding to an increase in [Ca2+]i transients that develops over several minutes when stretch is sustained. Members of the TRP family are likely candidates for mechanotransduction of the SFR.TRP channels constitute primary candidates for explaining background and leak Ca stretch . Cardiom stretch . The rap2+ currents are still poorly defined, they may play important roles in regulating Ca2+ homeostasis and contractility and altered Ca2+ dynamics in cardiac disease. The lack of complete understanding of the identity and mechanics of these channels, in addition to their relatively small amplitude compared to the voltage-gated Ca2+ channels, make them difficult to study in vivo. A computational model provides the advantage to study the currents and their roles in isolation from other cellular mechanisms. In this study, we use a mathematical model of a human ventricular myocyte to analyze the individual contributions of background Ca2+ entry and SR Ca2+ leak to the modulation of Ca2+ transients and SR Ca2+ load. We also assess the effects of the Ca2+ currents on cellular electrophysiology.Though background and leak Ca2+ current (ICabk) through the sarcolemma is defined as a summation of junctional  and subsarcolemmal  components:junc = 0.11 and Fsl = 0.89 are constants that determine the fraction of total background current corresponding to the junctional and subsarcolemmal spaces, respectively, Gbkg is the maximum conductance (5.513e-4 A/F) of the channels, Vm is the transmembrane voltage, and ECa,junc and ECa,sl are Nernst potentials corresponding to the junctional and subsarcolemmal spaces, respectively. SR Ca2+ leak (Jleak) describes the Ca2+ flux out of the SR:leak is the leak constant (5.348e-6/ms), and [Ca2+]SR and [Ca2+]j describe the Ca2+ concentrations in the SR and junctional space, respectively. In this study, we modulated Gbkg and Kleak independently and in conjunction to investigate the effects of altered sarcolemmal Ca2+ entry and altered SR Ca2+ leak, respectively.We applied a mathematical model of a human ventricular myocyte . The mod2+ currents in the cell model is shown in 2+ current into the junctional  and subsarcolemmal  compartments are determined by Ca describes the L-type Ca2+ current, IpCa describes the sarcolemmal Ca2+ pump current, and INCX describes the NCX current.A graphical summary of the Ca2+ concentrations in the junctional compartment [Ca2+]j, subsarcolemmal compartment [Ca2+]sl, bulk cytosol [Ca2+]i, and SR [Ca2+]SR are given by:mem is the membrane capacitance, F is Faraday’s constant, Vjunc is the volume of the junctional compartment, Vsl is the volume of the subsarcolemmal compartment, Vmyo is the volume of the bulk cytosol, and Vsr is the volume of the SR. JCa,juncsl is the rate of Ca2+ flux from junctional to subsarcolemmal compartments and JCa,slmyo is the rate of Ca2+ flux from the subsarcolemmal compartment to bulk cytosol. JRYR describes the SR Ca2+ release and JSERCA describes the SR Ca2+ pump. Ca2+ buffering is described by JCa,B,junction in the junctional compartment, JCa,B,sl in the subsarcolemmal compartment, JCa,B,cytosol in the bulk cytosol, and dCsqnb/dt in the SR. We refer to , diastolic [Ca2+]i, systolic [Ca2+]i, [Ca2+]i amplitude, diastolic [Ca2+]SR, systolic [Ca2+]SR, and [Ca2+]SR amplitude. Sensitivity analyses of each measured parameter were then normalized to the measurement of the default model values and fit to the quadratic equation:bkg or Kleak. We evaluated the factor of the quadratic term, A, the linear term, B, and the constant term, C, of this fit for relative comparisons of the effects of modulated background Ca2+ current or modulated SR Ca2+ leak. This analysis was repeated for the model ran at 1, 2, and 3 Hz electrical excitation.To investigate the effects of altered background Cabkg and Kleak to understand how the two Ca2+ currents interact and influence Ca2+ handling in the cardiomyocyte together.We subsequently performed a dual-sensitivity analysis for the modulations of Gm, [Ca2+]i, and [Ca2+]SR following modulation of the background Ca2+ entry current, ICabk, at pacing rates of 1, 2 and 3 Hz  display increases in both diastolic and systolic [Ca2+]i (2+ handling between HFpEF and HF with reduced ejection fraction (HFrEF) is preserved [Ca2+]SR in HFpEF vs. reduced [Ca2+]SR in HFrEF  by around 10% for maximal Gbkg modulation. It has also been shown that APD prolongation leads to increased Ca2+ (2+ signaling. Another important note is that APD increase is known to be inotropic, e.g., in rat ventricular myocytes (2+ entry to contractility. Conversely, prolonged APD can induce torsades de pointes tachycardia, leading to life-threatening ventricular fibrillation (Modulating Krequency . For thetentials . While Gsed Ca2+ , suggestmyocytes . This inillation .in vivo. However, there are also caveats of mathematical modeling that should be considered. It should be noted that the definitions of ICabk and Jleak used in this model are general simplifications and meant to reproduce poorly defined currents. The equations lack specific gating conditions of the currents. The current equations were not parameterized to match experimental data which is only incompletely characterized in human ventricular myocytes. Instead, the current equations are adjusted such that the model reproduces overall physiological action potentials and calcium transients. This is an important consideration, since the magnitudes of these currents could be largely different in living cells. Thus, interpreting the results of this study should focus on the qualitative trends. As the specific ion channels that contribute to Ca2+ entry and leak are identified and characterized, future work can aim to refine the current definitions and provide detailed current models to replace the general simplifications of ICabk and Jleak.Mathematical modeling of cellular electrophysiology provides a valuable resource for studying how aspects of cellular physiology interact and affect one another. It provides a means to investigate questions that cannot be easily answered + channels and isoforms of the Na+/K+-ATPase are modulated by localized Ca2+ concentrations (2+-dependent terms in the definitions of these currents. The inclusion of these interactions may alter the effects we see on Vm in this study. Future work could address this limitation.In a similar way, other ion currents in the model are not fully defined. For example, some Ktrations . However


However, the cellular events leading to the rise in [Ca2+]cyt following L-asparaginase-induced ER Ca2+ release remain obscure. Here, we show that in acute lymphoblastic leukemia cells, L-asparaginase causes mitochondrial permeability transition pore (mPTP) formation that is dependent on IP3R-mediated ER Ca2+ release. This is substantiated by the lack of L-asparaginase-induced ER Ca2+ release and loss of mitochondrial permeability transition pore formation in cells depleted of HAP1, a key component of the functional IP3R/HAP1/Htt ER Ca2+ channel. L-asparaginase induces ER Ca2+ transfer into mitochondria, which evokes an increase in reactive oxygen species (ROS) level. L-asparaginase-induced rise in mitochondrial Ca2+ and reactive oxygen species production cause mitochondrial permeability transition pore formation that then leads to an increase in [Ca2+]cyt. Such rise in [Ca2+]cyt is inhibited by Ruthenium red (RuR), an inhibitor of the mitochondrial calcium uniporter (MCU) that is required for mitochondrial Ca2+ uptake, and cyclosporine A (CsA), an mitochondrial permeability transition pore inhibitor. Blocking ER-mitochondria Ca2+ transfer, mitochondrial ROS production, and/or mitochondrial permeability transition pore formation inhibit L-asparaginase-induced apoptosis. Taken together, these findings fill in the gaps in our understanding of the Ca2+-mediated mechanisms behind L-asparaginase-induced apoptosis in acute lymphoblastic leukemia cells.Acute lymphoblastic leukemia (aLL) is a malignant cancer in the blood and bone marrow characterized by rapid expansion of lymphoblasts. It is a common pediatric cancer and the principal basis of cancer death in children. Previously, we reported that L-asparaginase, a key component of acute lymphoblastic leukemia chemotherapy, causes IP3R-mediated ER Ca Acute lymphoblastic leukemia (aLL) is a devastating cancer of immature lymphocytes. It largely afflicts children, representing more than a quarter of all childhood cancers, and causing most of the fatalities from cancer in children . L-aspar2+ channel to form a functional HAP1-Htt-IP3R complex that regulates IP3-stimulated ER Ca2+ release. HAP1 loss inhibits HAP1-Htt-IP3R formation and thus L-asparaginase stimulation of ER Ca2+ release   as a nov release . Loss ofsistance . These fpoptosis . However2+ homeostasis and apoptosis. The outer mitochondrial membrane (OMM) is easily permeable to Ca2+ while the inner mitochondrial membrane (IMM) consists of the mitochondrial calcium uniporter (MCU) complex that mediates mitochondrial Ca2+ influx    , the ER-channels  and voltchannels . These cchondria . Once ERe occurs  via the  channel . Howevere (mPTP)  but alsoe (mPTP) , which ae (mPTP)  that alle (mPTP) . The mPTnditions , sustainnditions .2+]cyt is caused by mPTP formation that results from ER-mitochondria Ca2+ transfer and subsequent ROS production. Thus, our findings define the Ca2+-mediated mechanisms through which L-asparaginase perturbs intracellular Ca2+ homeostasis to cause apoptosis in aLL cells.In the current study, we utilized SEM patient aLL cells expressing or depleted of HAP1 by retroviral transfection, and demonstrate that L-asparaginase-induced aLL cell apoptosis triggered by a lethal rise in  for 30 min then stimulated with 100 mIU L-asparaginase were analyzed using a Shimadzu RF 5301 PC spectrofluorometer  at λex = 495nm and λem = 530nm.To measure ER Ca2+]mt, *+pRS and *+pRS-shHAP1 (0.5 × 106) loaded with 2 μM of Rhod-2 AM [in Ca2+-free KRH buffer containing 5 μM EGTA] for 1 h then pre-treated with 2 μM XeC or 3 μM RuR were stimulated with 100 mIU L-asparaginase and analyzed using a Shimadzu RF 5301 PC spectrofluorometer at λex = 550nm and λem = 588nm. Peak amplitudes were quantified as ratios of fluorescence (F/F0) after addition of L-asparaginase. F0 represents basal fluorescence or fluorescence before stimulation with L-asparaginase.To measure [Ca2+]cyt,*+pRS and *+pRS-shHAP1 cells (0.5 × 106) grown on poly-L-ornithine-coated glass coverslips were loaded with 5 μM Fluo-4 AM [in Ca2+-free KRH buffer] for 1 h then pre-treated with 2 μM XeC, 3 μM RuR or 1 μM CsA and stimulated with 100 mIU L-asparaginase. Ca2+ transients were analyzed by single-cell Ca2+ imaging using an Olympus X71 inverted microscope  at λex = 485 nm and λem = 530 nm. Fluorescence intensities were measured in individual cells (n = 10) every 2 s. Data were analyzed using ImageJ 1.4.1 . The integrated Ca2+ signals , CsA (1 μM) or Mito-Tempo (5 μM) then stimulated with 100 mIU L-asparaginase for 12 h were stained with MitoSOX red (5 μM) and MitoTracker green (200 nM) or DCFDA (5 μM) for 30 min at 37°C. MitoTracker green was used to label mitochondria in live cells. Cell images were acquired using an Olympus 1 × 71 inverted microscope  at 160 to ×360 magnification. Fluorescence intensity of captured images (from a field with at least 200 cells) were measured using the ImageJ software. Values from cells stimulated with L-asparaginase alone were normalized to 1.*+pRS and *+pRS-shHAP1 cells (1×104) seeded on 96-well plates coated with 0.2 mg/ml poly-L-ornithine and pre-treated with RuR (3 μM), CsA (1 μM) or Mito-Tempo (5 μM) for 30 min then stimulated with 100 mIU L-asparaginase for 12 h were stained with Hoechst 34580 and FITC-Annexin V. FITC-positive apoptotic cells were counted 12 h post-treatment at ×10 magnification using a I×71 Olympus inverted microscope attached to a 37°C incubator with 5% CO2. The percentage of FITC-positive apoptotic cells was determined from a field of ∼100 Hoechst 34580-stained cells using the Olympus CellSens software .*+pRS and *+pRS-shp < 0.05 for experiments involving two treatment groups. For experiments involving more than two treatment groups, one-way Analysis of Variance (ANOVA) with Tukey Honestly Significantly Different (HSD) post hoc tests were performed.Student’s t-test  was performed at 2+ release that is mediated by IP3R causes mPTP formation. To investigate if L-asparaginase-induced IP3R-mediated ER Ca2+ release , when mPTP is closed  stably depleted of HAP1 by infection with retrovirus carrying pRS-shHAP1 (*+pRS-shHAP1)   were use-shHAP1) , a key c complex , inhibit release . Treatmepulation , indicat2+ release, results from Ca2+ entry into mitochondria. Since mPTP formation is associated with Ca2+ overload in mitochondria (*+pRS-shHAP1 cells pre-treated with Ruthenium Red (RuR), a potent MCU inhibitor (*+pRS-shHAP1 cells stimulated with L-asparaginase (right panel). Cyclosporine A (CsA), an mPTP inhibitor . To do so, *+pRS and *+pRS-shHAP1 cells loaded with the cell permeable mitochondrial Ca2+ dye, Rhod-2 AM33, were treated with L-asparaginase, and analyzed for mitochondrial Ca2+ increase by spectrofluorometry. As shown in 2+ release in *+pRS cells , a potent inhibitor of IP3R  in aLL cells. Since a rise in [Ca2+]mt has been associated with the generation of mitochondrial ROS  in breast cancer cells or knocking out Cdk5 in primary mouse embryonic fibroblasts (MEFs) is associated with mPTP formation, ROS increase [Capoptosis . Our notβγ-mediated rise in [Ca2+]cyt through phospholipase C (PLC)   was shown to trigger G C (PLC) , we prop C (PLC)  whereby  purple) . In this purple) ) in aLL 2+ transfer, ROS production and mPTP formation. Thus, results from our studies not only fill in the gaps in our understanding of the Ca2+-mediated mechanisms by which L-asparaginase induces aLL cell apoptosis, but also offer a fresh perspective on targeting ER Ca2+ release, ER-mitochondria Ca2+ transport, ROS and/or mPTP in leukemic cells for aLL therapy.In conclusion, our findings indicate that L-asparaginase-induced aLL cell apoptosis requires ER-mitochondria Ca


These scoding’)  where thesponses . Great p signals , spurrin2+ signalling is central to diverse aspects of plant development as well as responses to environmental perturbations  and acts as an executor of the general stress response  family , which acts as an immune elicitor upon cellular damage in plants. Finally, while most signalling work has been performed using model plants such as Arabidopsis, Zhou et al have examined how Ca2+ signalling and transporters contribute to flagellin-induced immune responses in cotton (Gossypium hirsutum), identifying a Ca2+ efflux transporter as a potential regulator of defenses to Verticillium wilt.Many Ca) family . Sun et in planta Ca2+ dynamics continue to expand and improve (2+ signals, from organelles to single cells to whole plants. Recent insights have also expanded our understanding of the repertoire of Ca2+-permeable channels in plants and their potential functions in plant immunity (The tools available to researchers that allow for monitoring of  improve , allowinimmunity . Such brAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.