When stem diameter exceeds maxD, then the stem internode should be treated as a conic frustrum requiring two additional measures: a second, node‐adjacent diameter and a length. If the second diameter is less than maxD, then the power function allometry can be applied to the aerial tip; otherwise an additional internode is measured. This “two‐component” allometry—internodes as frustra and aerial tips as shrubs—can reduce estimated biomass error propagated to the plot‐level by as much as 50% or more where very large shrubs are present maxD is any diameter such that the ratio of single‐component to two‐component uncertainty exceeds the ratio of two‐component to single‐component measurement time. Guidelines for estimating maxD based on pilot field data are provided. Tall shrubs are increasing in abundance and distribution across Arctic, alpine, boreal, and dryland ecosystems. Estimating their biomass is important for both ecological studies and carbon accounting. Reducing field‐sample prediction error increases precision in multi‐stage modeling because additional measures efficiently improve plot‐level biomass precision, reducing uncertainty for shrub biomass estimates.We show that aerial tips are self‐similar fractals of whole shrubs and present a field method that applies this fact to improves accuracy and precision of biomass estimates of tall‐shrubs, defined here as those with diameter at root collar (DRC) ≥ 2.5 cm. Power function allometry of biomass to stem diameter generates a disproportionate prediction error that increases rapidly with diameter. Thus, biomass should be modeled as a single measure of stem diameter only if stem diameter is less than a threshold  Shrub biomass is an increasingly important measure in changing ecosystems. We present a simple method of reducing errors in its estimation. Foresters, for example, quantify forest stand wood volume using diameter at breast height (DBH) and ecologists increasingly apply allometry to calculate shrub biomass  = b ln(iD) + ln(a) + εi, where ln(a) is the constant of integration, and 2 < b < 3 captures the self‐similar, fractal geometry of woody vegetation.Expressed as a power function, biomass‐diameter allometry is theoretically reasonable if a relative growth increment in a volume‐related measurement, such as wet field‐mass nls with weighted regression  and uncertainty  implies that fitting power functions with log‐log plots can help control for heteroscedastic residuals  in southcentral Alaska. The individual uncertainties were then propagated across the wet field‐mass estimate for each sample‐plot , while increasing the precision and the accuracy of plot‐level biomass estimates. In particular, and by example, we show a substantial increase in precision over the sample plot estimates shown in Figure Applications of allometry that scale‐up from the individual to the plot‐level and beyond require methods that reduce propagated error. As an example, whole‐shrub biomass‐DRC (“one‐component”) allometry Figure  construct Figure  by Montet Figure . Assumin2L with D > maxD and terminal aerial shoots (tips) with minD ≤ D ≤ maxD, where maxD is a threshold diameter for treating stem internodes as conic frustra and minD defines the minimum shrub size . We initially identify maxD subjectively in plots of back‐transformed residuals versus arithmetic values of D as the diameter where biomass variability rapidly expands, then adjust maxD if the temporal cost of multiple measurements exceeds the benefit of increased precision. A sketch of how this can be accomplished is presented in the Discussion.Our goal was minimizing uncertainty in biomass estimates given power function allometry. Our protocol considers shrubs as “two‐component” structures Figure : stem inDRC > 2.5 cm = minD and includes 134 Alnus and Salix individuals  that we destructively sampled in southcentral Alaska to derive biomass‐diameter allometry  as internodes and terminal aerial tips, each individually weighed and measured.Our allometry example considers only shrub individuals with y Figure , shown p2.1D, single‐component allometric equations describe woody biomass M as a power function pM = aD, with p > 2; uncertainty also scales as a power function of D, with p > 2. The following two‐component field‐sampling algorithm reduces uncertainty in estimated biomass of large (maxDRC > D) shrubs, where diameter maxD offers the greatest acceptable uncertainty for pM = aD.Given diameter Step 1: Identify root collar.1D there.Step 2: Record diameter 1D ≤ maxD, stop; aerial tips with D ≤ maxD have acceptably low uncertainty. Else if 1D > maxD, identify stem internode above 1D as a conic frustrum. Record its length L and end diameters 1D > maxD and 2D (where 2D is measured just below the upper node swelling).Step 3: If 1D.Step 4: Return to Step 2 for stems above the node, treating each stem diameter as The individual shrub biomass estimate is the sum of biomass estimates for frustra and aerial tips with associated uncertainties. The uncertainty in each sample‐plot is calculated using Monte Carlo sampling of internodes and tips from lognormal distributions with parameters estimated from log‐log allometry.Aerial tip allometry Inspection of the 134 shrubs indicated that variability in M increased substantially at DRC > 7.5 cm ; and those two samples taken together (n = 132). We also calculated the percent overlap between the aerial tip allometric estimates (including prediction error) and individual shrub allometric estimates. Allometry was established by regressing ln(M) on ln(D), then exponentiating the 95%PI iupr and ilwr bounds of ln(iM) found with 2.5 ≤ iD ≤ 7.5 cm at intervals of 0.01 cm to determine uncertainty.m Figure , suggestStem internode allometry Stem internodes with D > 7.5 cm were modeled as regular conic frustra with diameters 1D and 2D and length L /12. We cut, measured, and weighed in the field as wet field‐mass 40 internodes from eight individual alder shrubs of two species (n = 20 internodes each from A. viridus and A. incana). Internodes varied as 2.7 ≤ D ≤ 36.1 cm (mean = 9.6 cm), 16.5 ≤ L ≤ 224.2 cm, and 0.2 ≤ M ≤ 9.1 kg (mean = 8.2 kg). We compared these linear regression wet‐density estimates  to wet‐density directly measured in two field‐cut stem pieces with known weight and estimated volume as measured by submersion in water (A. incana: 0.74 kg/L = 3.49 kg/4.73 L and A. viridis: 0.83 kg/L = 2.35 kg/2.84 L). Besides regression of untransformed internode variables, we also regressed ln(M) on ln(V), inspecting both regressions for heteroscedasticity.L Figure , where f2.2εi . Because the sum of lognormal distributions have no closed‐formed distribution  and seln(M) using log‐log models fit in R using lm, and se.fit = TRUE as arguments to the predict.lm function.1. For each shrub or shrub piece in each sample‐plot, estimate the expectedrlnorm function in R, following Bolker .3. Find the middle 95% quantile of the 33.1D ≤ 7.5 cm, whether rooted or not  on ln(D) regression for combined aerial tips and whole plants with DRC ≤ 10.2 cm was equivalent to M = 0.07 D2.437 , similar to the whole shrub‐only allometry of M = 0.07 D2.414  for individuals with 2.5 ≤ DRC ≤ 40.5 cm  with a wet‐density estimate of 0.84 ± 0.03 g/cm3 (estimate ± se). The directly measured wet‐density (0.83 g/cm3 = wet field‐mass/volume) was less than one‐half a standard error of the regression coefficient from the slope estimate. The wet field‐mass of Alnus incana was also well fit by frustrum volume ; the slope of the regression 0.77 ± 0.04 g/cm3 compared favorably to the directly measured wet‐density (0.74 g/cm3); the latter was less than one standard error from the point estimate of the slope. Nevertheless, residuals in these regressions visually appeared heteroscedastic in diagnostic plots.Wet field‐mass of alder internodes was well described by internode size as a frustrum volume using simple linear regression Figure . For AlnM) against ln(V) ignoring species (Supporting Information). The slope of this log‐log linear regression was essentially equal to one , suggesting a linear relationship through the origin. Taking the intercept  and exponentiating produced a 95%CI for wet‐density of the stem wood as 0.81 to 1.02 kg/L. We used the log‐log allometric version of internodes in estimating wet field‐mass.Because log‐transformation of the variables brought about homoscedasticity in residuals, and the difference in wood wet‐density between the two alder species was not significant at the 0.05 level, we regressed ln. Individual plant wet field‐mass point estimates from the two models differed by only about 10% on average , as would be expected given that the individual plants were included in constructing both model approaches.Uncertainty—defined as range in 95%PI found with Monte Carlo sampling for individual shrub biomass using single‐component allometry (DRC only) was on average double that using two‐component (tip + internode) allometry when comparing the eight shrubs of known biomass with measured internodes : t = 4.07, p =.0004, df = 16). Point estimates of total shrub wet field‐mass in sample plots  measured in 17 plots (169m2), the uncertainty in total sample‐plot biomass estimation using the two‐component method was 40% less than the single‐component method; this difference depended on the count of shrubs with DRC > maxD.Overall, for individual shrubs with DRC > 4precisely estimate plot‐level biomass to inform landscape‐level models with error propagation. The propagation of error in remote‐sensing informed biomass estimates is an increasingly important consideration. That importance motivates this study.Increases in shrubs in arctic and dryland ecosystems are well‐documented but poorly quantified as measured change in biomass. Part of the reason is a lack of allometry. Because these landscapes of increasing tall shrubs are often vast and remote, quantifying abundance there relies on remote sensing informed by fieldwork at the plot‐level  project‐specific allometry  versus DRC as a diameter near where variability in biomass increases dramatically. It is convenient to choose an integer value to increase speed of measurement in the field . To justify taking more measurements, the increase in precision of allometric prediction must outweigh the cost of increased measurement time.The cutoff diameter measure D2.4 [as found by regressing log(prediction error) on log(diameter)], showing clearly that as maxD approaches zero, so does the uncertainty in biomass estimate. A more rigorous approach than visual inspection of the raw data, is to choose an initial value for maxD from the untransformed allometry and apply it in the field in pilot sampling, so as to estimate the time required to make single component measurements versus two‐component ones. The ratio of time taken for two component measures to single component measures can then be compared to the ratio of expected single‐component prediction error to two‐component prediction errors using maxD.Given the allometry shown in Figure PE]) across all diameters requires both allometry of prediction error  is the scaling factor) and a probability density function of stem diameters, pdf(D), such that the expected prediction error for a single‐component method of a single randomly sampled shrub from pdf(D) isTo calculate the expected prediction error  is greater than or equal to the ratio of expected time for two‐component measure to time for single‐component measure /t1), then applying a two‐component strategy with maxD improves precision by a factor more than or equal to its cost in time: E[PE1]/E[PE2]≥(E[t2]+t1)/t1.If the ratio of expected prediction error for a single‐component to prediction error for two‐component  sampled during one day on Alaska's Kenai Peninsula. Using measurements of 1,286 tips and 87 frustra over a total of 14.7 person‐hours spent in within field plots, we applied a multiple linear regression as total time per plot = 0t + 1t * (count of single‐component measures per plot) + 2t * (count of two‐component measure per plot) + ε, finding 2R = 0.96, ε ~ N, and 1t = 26 s as mean time of single component measure and 2t = 111 s as mean time of two‐component measure. To calculate the expected time given the stem diameter distribution for the two‐component measure, E[t2], requires knowing the proportion of stems with diameters >maxD =10 cm  to calculate E[t2] = t2D>DmaPx to which the single‐component value 1t must also be added, because given a stem it is first measured to determine if it must be sampled as a frustrum. Hence the addition of 1t to t2 weighted by the probability that its diameter is greater than maxD.We provide an example of calculating times per measurement from actual field data with density with its defaults in base R; the output of density includes the random variable as x (here diameter) and the density value as y. We approximated the pdf in Equation (n = 512 diameters for x (range: 0.85–18.5 cm) estimated by density, calculated the prediction error at each ix using the allometry PE1=exp‐2.02D2.41, then multiplied each prediction error PE1 by the density estimate iy at each ix, summed and multiplied by Δx = (18.5–0.85)/512 to arrive atmaxD = 10 cm = 266x and approximating Equation ,,2), we cEquation  using thD ≥ maxD = 10 cm  gives E[t2] = (111 s) × (0.09) = 10.0 s and adding 1t = 26 s gives 36 s. The ratio of expected time for a two‐component measure to a single‐component measure is 36/26 = 1.38 ≤ 1.4 = EPE1/EPE2, suggesting that maxD is well‐chosen, because the cost in time of a two‐component measure is less than or equal to its benefit of reduction in uncertainty.Weighting the time of sampling stems requiring the two‐component method (111 s) by the proportion of stems with maxD requires allometry with known prediction errors, some prior knowledge of the distribution of stem diameters, and an estimate of the time required for both single‐component and two‐component measurements.In summary, to rigorously calculate an optimal As authors, we declare no conflict of interest in the publication of this study.Roman Dial: Conceptualization ; Data curation (lead); Formal analysis (lead); Writing‐original draft (lead). Bethany Schulz: Conceptualization ; Investigation ; Methodology (supporting); Supervision (lead); Writing‐review & editing . Eric Lewis‐Clark: Data curation ; Formal analysis ; Investigation ; Methodology ; Writing‐review & editing . Kaili Martin: Investigation ; Methodology ; Writing‐review & editing . Eric Anderson: Funding acquisition ; Investigation ; Project administration ; Resources ; Supervision ; Writing‐review & editing .Supplementary MaterialClick here for additional data file.


Feeding an antibiotic-free diet is an inevitable trend in animal husbandry in China. In this study, high-throughput sequencing was used to analyze the gene expression differences in various intestinal segments of rabbits. Our analysis identified 168, 593, 2069, 334, 321, and 1423 DEGs in the comparison groups S_Z  vs. S_B (diarrhea in the duodenum of rabbits), K_Z  vs. K_B (rabbits with diarrhea in the jejunum), H_Z  vs. H_B (rabbits with diarrhea in the ileum), M_Z  vs. M_B (rabbit with diarrhea in the cecum), J_Z  vs. J_B (colon of rabbits with diarrhea), and Z_Z  vs. Z_B (rectum of rabbits with diarrhea), respectively. The reproducibility and repeatability of the results were validated by RT-qPCR. Enrichment analyses of GO annotations and KEGG pathways revealed the host DEGs that are potentially related to acute inflammation, stress response, tissue dehydration, adaptive immune response, protein binding, activation of related enzymes, migration of immune cells, and so on. In this descriptive study, our findings revealed the changes in the host transcriptome expression profile while feeding an antibiotic-free diet and suggested that feeding an antibiotic-free diet alters the host’s metabolic network and the expression of antiviral proteins.China officially promulgated the announcement of banning the use of antibiotics in the animal industry in 2020. However, the prohibition of antibiotics in the animal industry would reduce the feed conversion rate and increase the mortality of animals. In order to obtain information about the pathogenesis and host immune response of rabbits with diarrhea after being fed an antibiotic-free diet, we first analyzed the intestinal tissue sections of rabbits. Secondly, the gene expression differences of rabbit intestinal segments were analyzed by high-throughput sequencing. Our analysis identified 168, 593, 2069, 334, 321, and 1423 DEGs in the comparison groups S_Z  vs. S_B (diarrhea in the duodenum of rabbits), K_Z  vs. K_B (rabbits with diarrhea in the jejunum), H_Z  vs. H_B (rabbits with diarrhea in the ileum), M_Z  vs. M_B (rabbits with diarrhea in the cecum), J_Z  vs. J_B (colon of rabbits with diarrhea), and Z_Z  vs. Z_B (rectum of rabbits with diarrhea), respectively. The reproducibility and repeatability of the results were validated by RT-qPCR. Enrichment analyses of GO annotations and KEGG pathways revealed the host DEGs that are potentially related to acute inflammation, stress response, tissue dehydration, adaptive immune response, protein binding, activation of related enzymes, migration of immune cells, and so on. In this descriptive study, our findings revealed the changes in the host transcriptome expression profile after feeding an antibiotic-free diet and suggested that feeding an antibiotic-free diet alters the host’s metabolic network and the expression of antiviral proteins, which provides a theoretical basis for further study on the immune response of animals fed an antibiotic-free diet. Since the first use of antibiotics in piglets and chickens in 1949, it has been found that feeding diets containing antibiotics could maintain animal intestinal health and reduce livestock mortality . HoweverAfter the animal breeding industry banned the use of antibiotics, it caused a series of economic losses, such as increased animal mortality, increased breeding costs, and decreased quality of animal products ,7. In paIn this study, rabbits were fed an antibiotic-free diet, and a large number of rabbits had diarrhea. However, the immune mechanism of diarrhea in rabbits was not clear. Nowadays, high-throughput transcriptome technology can conduct very sensitive experiments in a cost-effective way. In this study, the differential genes of the rabbit intestinal transcriptome were analyzed by high-throughput technology so as to explore the pathogenesis of diarrhea in rabbits fed an antibiotic-free diet in order to provide a theoretical basis for the development of animal intestinal disease treatment.Experimental procedures in this study were approved by the Institutional Animal Care and Use Committee of the College of Animal Science and Technology, Sichuan Agricultural University, Sichuan, 611130, China.Two hundred Hyplus female rabbits at 35 days of age were selected from the Zhongtian rabbit farm  in three farm districts. Ten days before weaning, they were fed an antibiotic-containing diet together with female rabbits, and for 10 days after weaning, they were freely fed an antibiotic-free diet. All rabbits were raised under standard farm conditions and received routine vaccinations. At the end of the experiment, six rabbits were selected from the group in a healthy state and treated as the normal group (CON), and six rabbits were selected from the diseased rabbits according to the disease criteria and treated as the diseased group (DIA). The selection of the DIA standard was based on phenotypic differences, weight differences, stool differences, urine differences, and intestinal histopathological diagnosis.Feed ingredients and additives were prepared according to the nutritional requirements of INRA in France. Each rabbit was placed in a clean cage (60 × 60 × 50 cm) and placed in an environmentally controlled room ) .After fasting for 24 h, rabbits were blood let with electroshock. After that, we collected duodenum, jejunum, ileum, cecum, colon, and rectum intestinal tissue samples. Then the contents of the intestines of the rabbits were washed with normal saline. Samples placed in 3 mL cryotubes were stored in liquid nitrogen at −80 ℃. Finally, the collected intestinal tissue samples were used for transcription analysis.We selected seven rabbits with diarrhea and three healthy rabbits for intestinal morphological section analysis. The intestinal tissue specimens were fixed with 4% paraformaldehyde, trimmed, dehydrated, embedded in paraffin, sectioned, dewaxed with xylene, stained with hematoxylin and eosin (HE), dehydrated with alcohol, and sealed with resin. The histological changes of the whole tissue sections were observed under a microscope , and normal areas and obvious lesion areas were recorded with a microscope imaging system.® Ultra™ RNA Library Prep Kit for Illumina®  [The duodenum, jejunum, ileum, cecum, colon, and rectum of the DIA and CON were selected for RNA-seq analysis (n = 3). Total RNA was extracted from intestinal tissues of rabbits with diarrhea and healthy control rabbits by standard extraction method. The effective RNA was screened by Agilent 2100 biological analyzer (Agilent RNA 6000 nano). The screening criteria were RNA 7.0 Rin value and 28s/18S > 1.8. The purified double-stranded cDNA was repaired at the end, an A tail was added, and the sequencing connector was connected. The 370–420 bp cDNA was screened, amplified by PCR, and purified again, and the library was finally obtained. The NEBNextCA, USA)  was usedp-value ≤ 0.05, FDR ≤ 0.01, and genes with |log2 (foldchange)| > 1 were considered to be significantly differentially expressed genes. In order to predict the main biological and molecular functions of these DEGs, we used gene ontology (GO) classification and functional description. GO includes three aspects: molecular function, cellular components, and biological processes . Rabbit genes were annotated by KEGG (Kyoto Encyclopedia of Genes and Genomes) and the differential gene sets were enriched and analyzed by KEGG pathway. Significance levels for all GO and KEGG terms were corrected by controlling for the false discovery rate (FDR) of multiple pairing comparisons. Statistical software R (R version 4.0.5) and python (Python version 3.9.0) were used for statistical analysis.Image data measured by high-throughput sequencer were converted into sequence data (reads) by CASAVA base recognition and clean reads were obtained. Q20, q30, and GC contents of clean reads were calculated (S 1). The transcripts were reconstructed and annotated using StringTie (1.3.3b) . In addiThe HE-stained intestinal tissue samples  showed tThe correlation of gene expression level between samples is an important index to test the reliability of the experiment and the rationality of the sample selection. The higher the correlation coefficient between samples, the closer the expression pattern is. It can be observed in Among all the samples generated from these libraries, rabbits with diarrhea had an average of 45,800,180 double-ended raw reads and 44,413,253 clean reads. Healthy rabbits had an average of 46,213,220 double-ended raw reads and 44,918,133 clean reads. The GC content of the clean readings of all samples was between 44.73–57.14% . HISAT2 To identify the broad array of genes involved in response to rabbits with diarrhea, we analyzed the genes that responded to the rabbits with diarrhea A–F,H. Cop-value of ≤0.05 were considered significantly enriched. To classify and characterize DEG functions and pathways, we performed a gene ontology (GO) classification and functional annotation of molecular biological function, cellular components, and biological process , protein digestion and absorption (ocu04974), IL-17 signaling pathway (ocu04657), PI3K-Akt signaling pathway (ocu04151), MAPK signaling pathway (ocu04010), NF-kappa B signaling pathway (ocu04064), TNF signaling pathway (ocu04668), NOD-like receptor signaling pathway(ocu04621), HIF-1 signaling pathway(ocu00480), etc. . CompareComplement and coagulation are the main blood-derived proteolytic enzyme cascade reactions that limit bleeding and pathogen invasion ,32,33, aStudies have shown that an imbalance of cytokine secretion could cause intestinal epithelial barrier dysfunction and diarrhea . IL-17 aIt has been reported that activation of the NF-κB pathway in intestinal epithelial cells can cause severe inflammation . NF-κ B We also analyzed the interactions among the pathways involved in the PI3K-Akt signaling pathway. It is currently considered that the phosphatidylinositol-3-kinase (PI3K) pathway is an important signal transduction pathway for cell-surface receptors to control intracellular activities, and participates in all major leukocyte regulatory activities, such as receptors for antigen recognition and inflammation stimulation . In addiIn this study, we characterized the transcriptional profiles of rabbits with diarrhea and healthy rabbits after feeding them an antibiotic-free diet. The dynamic changes in differentially expressed genes between rabbits with diarrhea and healthy rabbits are helpful to understand the immune regulation mechanism of rabbits. Our results showed that there were significant differences in the metabolism and the expression of antiviral proteins and genes in the complement system in rabbits fed an antibiotic-free diet. It may be the cause of diarrhea in rabbits. These findings provide a unique insight into the regulatory mechanism of diarrhea in rabbits fed an antibiotic-free diet, and provide some new ideas for future research.


In 96-, 384-, or 1,536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+]i increase when stimulated with the GPVI agonist collagen-related peptide or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five parameters describing the Ca2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results present proof of principle of distinct receptor-type-dependent Ca2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM-linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra-high-throughput (UHT) method based on intracellular [Ca  •Tools are needed to screen for the anti-thrombotic potential of small molecules•Ultra-high-throughput assay to discriminate between GPVI and GPCR pathways•Calcium signaling as a profiling method to follow real-time platelet activation•Platelet calcium assays are highly sensitive to evaluate inhibitory drug effects Cell biology; Functional aspects of cell biology; Methodology in biological sciences Howeverxane A2) . In devexane A2) . This urxane A2) .In addition to GPCR, also the ITAM-linked receptor (ILR) for collagen, glycoprotein (GP)VI, regulates platelet activation . In mous2+ concentration [Ca2+]i acts as a common second messenger in response to both GPCR and ILR agonists. Agonist-induced increases in [Ca2+]i precede essentially all functional platelet responses, such as cytoskeletal reorganization, platelet adhesion, aggregation, secretion, and procoagulant and proinflammatory activity, and clotting  isoforms, resulting in cytosolic Ca2+ mobilization (via inositol triphosphate formation and intracellular Ca2+ release from the endoplasmic reticulum) and PKCα/β activation. The agonist CRP for ITAM-linked GPVI acts in a tyrosine-kinase dependent way, which results in activation of the PLCγ2 isoform and again causes inositol trisphosphate-mediated Ca2+ mobilization and PKCα/β activation. A common subsequent event is store-operated Ca2+ entry  and the ILR agonist, collagen-related peptide (CRP). After establishing time-dependent outcome parameters, we validated these using a robustness compound library and by a reference set of clinically relevant antiplatelet agents. Our results provide proof-of-principle evidence that new receptor-type-dependent antiplatelet drugs can be identified by this UHT screening.In the present study, we developed a UHT-based method for platelet i (9platelets/L) using a FlexStation 3 robot, which allowed the simultaneous measurement of one column at a time. The agonists were added by high-speed injection in order to obtain reproducible and highest responses. This resulted in relatively rapid and partially transient [Ca2+]i traces in response to the GPCR agonist thrombin, but in slower and more persistently high [Ca2+]i traces with the ILR agonist CRP  of Calcium-6 for Ca2+ in comparison with Fura-2 (224 nM) may be responsible for the small differences in response curves between probes. The clearly distinct shapes obtained with thrombin and CRP motivated us to further explore the use of Calcium-6 as an intracellular Ca2+ dye.To overcome this limitation, we determined again in 96-well format the high-wavelength dye Calcium-6, also loaded as acetoxymethyl ester with low leakage rate after de-esterification and commonly used in high-throughput assays with other cell types . Platelewith CRP D–1F. Pse [Ca2+]i . The hig2+]i increases over 10 min as an output parameter. For Fura-2 and Calcium-6, EC50 concentrations were highly similar for both agonists, i.e., 2–3 μg/mL for CRP , TRAP6 (for PAR1), 2-MeS-ADP (for P2Y1 receptor), and U46619 (for TP receptor). Agonist addition again was by optimized high-speed injection with the robot. Markedly, all GPCR agonists showed faster and more transient [Ca2+]i increases, when compared with the slower-onset and prolonged [Ca2+]i increases obtained with CRP  thr, PPACK B, and  was used, and a minimal volume of 2 μL of agonist solution was injection pipetted for achieving maximal responses. For analysis again pseudo-ratioing was applied, which in this case resulted in a sudden drop in fluorescence intensity due to the 33% dilution with agonist. A direct comparison of the overall assays' performances in 384- and 1,536- well formats is shown in Aiming to further downscale the assay, we moved to the 1,536-well format, which was possible by using the FLIPR-Tetra machine. Per well, 6 μL of Calcium-6-loaded platelets  maximal curve slope after agonist addition, (2) maximal fluorescence increase over time (maximum - minimum), and (3) area under the curve (AUC), the latter as an approximation of the net integrated [Ca2+]i increase over 10 min. Quantification showed that curve slopes were 16- and 9-fold higher with thrombin than with CRP, when measured in 384-  after agonist addition. We furthermore separated the biphasic curves into two parts, resulting in an early RoC (over 50–200 s) and a late RoC (over 200–450 s). The late RoC was considered as a best estimate of the later second-phase i increase measurements. Effects of the agonists on functional platelet responses were confirmed by flow cytometry (not shown). For all the 263 investigated compounds, effect values in quadruplicate were obtained of the three curve parameters  upon stimulation with thrombin or CRP. For mean effects of these three parameters, the Z score was calculated (number of standard deviations) in order to identify actively interfering compounds. Presentation of the data as a univariate scaled heatmap showed that the majority of compounds were without relevant effect, i.e., with ǀZǀ <4  interference of compounds with high absorbance at >480 nm  and (2) compounds affecting the agonist activity. Accordingly, using an appropriate hit triage cascade, an UHT-based screening procedure for novel antiplatelet drugs has now become possible.Evaluating the Z scores for all 263 compounds per class indicated some differences between CRP- and thrombin-induced i increases as evoked by GPCR or ILR agonists. In 96- and 384-well plates, we detected comparable, short-term [Ca2+]i transients induced by a range of physiologically relevant GPCR agonists: the PAR1/4 agonist thrombin, the PAR1 agonist TRAP6, the TP thromboxane receptor agonist U46619, and the P2Y1 receptor agonist 2-MeS-ADP. With all these agonists, the [Ca2+]i increases were not suppressed but slightly increased by the Syk tyrosine kinase inhibitor PRT060318. Because of this response similarity, we chose to perform further UHT assay development with the GPCR agonist thrombin only. On the other hand, the commonly used ILR agonist CRP for GPVI evoked a more prolonged Ca2+ signal that was completely blocked by PRT060318. The ILR C-type lectin-like receptor 2 (CLEC2) was not investigated, since the known, physiological ligands such a podoplanin are no more than weak platelet agonists.In this paper, we describe a UHT assay for platelet signaling responses, operating as 96-, 384-, or 1,536-well plate formats, based on the different profiles of [Ca2+]i increases comprise an essential signaling step, preceding almost all functional platelet responses . However, the 384-well measurements could be carried out at 50 μL per well (200 × 109/L), whereas the 1,536-well assay only required 6 μL per well (400 × 109/L). In the 384-well format, agonist-induced traces appeared to be highly reproducible, as apparent from the excellent, high Z′ factors. In the 1,536-well format, Z′ factors were slightly lower but within the acceptable range . With small adaptations, the current UHT Ca2+ signaling method can also be applied to cultured human cell lines and differentiated iPSCs.In future, the present UHT assays in 384- or 1,536-well plate formats can be employed for large small molecule screening approaches. The 1,536-well format is the gold standard in the academic drug discovery community and the pharmaceutical industry so as to reduce costs in reagents, consumables, and biological materials and make it time efficient owing to the high number of samples . So far,agonists . Other Ug Centre . For plag Centre  uses plaIn general, UHT screening assays, such as the one described here, render many positive hits, which may result not only in lead compounds but also in false-positive leads due to each particular assay . After i2+]i increases in platelets in 384- and 1,536-well plate formats. Herein, we were able to distinguish between GPVI- and PAR1/4-induced Ca2+ signaling and characterize inhibitory effects of compounds in early and late phases of the [Ca2+]i curves. Limitations of the study are the focus on only one ILR (CRP) and GPCR (thrombin) platelet agonist in both UHT formats, absence of discrimination between individual receptor isoforms (PAR1 and 4 for thrombin), and absence of discrimination between intracellular Ca2+ mobilization and Ca2+ entry. Another limitation of such UHT assays is the absence of continuous stirring, resulting in slightly delayed responses, although mixing of platelet suspensions with agonists was optimized by high-speed injection of relatively high volumes (10%–33%) of agonist solution. On the other hand, the absence of stirring suppressed the system heterogeneity, i.e., prevented the formation of large platelet aggregates (with CRP) or fibrin clots (with thrombin). Prolonged, biphasic Ca2+ curves of GPVI-stimulated platelets and aberrations in these with patients carrying genetic mutations in ORAI1 or STIM1 were previously also obtained in stirred, single-cuvette measurements .Further information and request for resources and reagents should be directed to and will be fulfilled by the lead contact, Delia I. Fernández . All subjects had normal platelet counts, as measured with a Sysmex XN-9000 analyzer . Where indicated, platelet concentrates from unknown healthy subjects were obtained from Sanquin , after written permission from Sanquin and full informed consent. A concentrate unit contained >2.5 x10% plasma . Concent2, 5.5 mM glucose and 0.1% bovine serum albumin). After addition of apyrase (1 U/mL) and ACD (1:15 vol./vol.), another centrifugation step was performed to obtain washed platelets  (Platelet-rich plasma (PRP) was separated from citrated blood by centrifugation at 240 g for 15 min . Alternalatelets . Platelealbumin) .9/L) were loaded with Fura-2-AM (3 μM) with pluronic (0.4 μg/mL) for 40 min at room temperature. After another wash step and resuspension of the platelets at 200 × 109/L, changes in [Ca2+]i were measured in the presence of 1 mM CaCl2 in 96-well plates using a FlexStation 3 (Molecular Devices) at 37°C during 10 min  were incubated at 1:1 vol./vol. with Calcium-6 dye solution for 2 h at room temperature, according to the manufacturer's instructions. After another wash, the loaded platelets in buffer containing 1 mM CaCl2 were resuspended at 200-400 × 109 platelets/L and distributed over 384-well or 1536-well plates. Dispersion of the platelet suspension over the 384-well plates was using a multi-pipette; dispersion over the 1536-well plates was with a Certus device . In each setting, a camera-equipped FLIPR-Tetra machine (Molecular Devices) was used to measure fluorescence increases, representing rises in [Ca2+]i, in all wells simultaneously (9/L) per well were injection pipetted with 5 μL agonist or vehicle solution. In the 1536-well format, 6 μL of dye-loaded platelets (400 × 109/L) per well were injection pipetted with 2 μL of agonist or vehicle solution. As a default procedure for this machine, mixing of the platelets with agonist was achieved by high-speed injection of a relatively large volume of agonist solution. In a pilot test, injection speed and volume were optimized per well-plate format to obtain highest and most reproducible platelet responses. Using a suitable optical filter set, the increases in Calcium-6 fluorescence were continuously measured at excitation and emission wavelengths of 485 nm and 525 nm, respectively (room temperature). All conditions were repeated in duplicate to quadruplicate wells (16 wells for Z' assessment). Off-line, the time traces per well were semi-ratioed by comparison in comparison to the baseline fluorescence (F/Fo) of the agonist medium-diluted signal , and area-under-the-curve (over 5–10 min). For each of these parameters, the assay's suitability was determined from the statistical effect size as Z' factor. This coefficient integrates the signal dynamic range and the test variation associated with the measured signal ; and n is a negative, maximum signal (stimulated platelets).Raw traces of changes in [Cad signal . An assa2+]i, traces were separated into early and late slopes . Traces of F/F0 over time were regressed to a smoothed curve . Early RoC was determined as the maximum differential between 50–200 s; late RoC as the mean change over 200–450 s.For the modeling of i rises, aggregation of platelets in platelet-rich plasma or in wash platelets was determined by light transmission aggregometry. In brief, washed platelets or PRP (250 × 109 platelets/L) were pre-incubated with indomethacin (10 μM) or DMSO (vehicle) for 10 min at 37°C. Maximal aggregation was induced by CRP (1 μg/mL) or thrombin (1 nM) while stirring at 1200 rpm at 37°C. Platelet aggregation was monitored using a Chronolog optical aggregometer . Maximal amplitude was quantified at 10 min after the addition of the agonist. In other platelet subsamples, washed platelets (100 × 109/L) were stimulated for 15 minutes with CRP (1 μg/mL), thrombin (1 nM), or left unstimulated, in the presence of 1 mM CaCl2. After stimulation, platelets were stained for Alexa Fluor-647 anti-CD62P mAb (2.5 μg/mL) and fluorescein isothiocyanate (FITC)-PAC1 mAb (1.25 μg/mL) to measure α-granule secretion and integrin αIIbβ3 activation, respectively. Measurements were in duplicates with an Accuri C6 flow cytometer  and data were analyzed with FlowJo software.For comparative measurements of platelet functions and [CaData were expressed as means ± SEM. GraphPad Prism 8  was used for the statistical analyses. Data normality was verified using a Shapiro-Wilk test, after which one tailed Student’s t-test or non-parametric Mann-Whitney test was used. For more than one group comparisons, a one-way repeated ANOVA test was used. Statistical significance was defined as P<0.05. Data sets were compared using Pearson or Spearman correlation analysis. The software R version 3.2.5 was used to calculate rates of changes, correlation analyses and for heatmap visualization. Statistical details per experiment are indicated in the figure legends.


We found that both CS, and CSE, rapidly elevated cytosolic Ca2+ in hASMC through stimulation of plasmalemmal Ca2+ influx, but excluded store-operated and L-type Ca2+ channels as mediators of this effect. Using a specific pharmacological inhibitor, or shRNA-driven knockdown, we established that both CS and CSE stimulated Ca2+ influx in hASMC through the neurogenic pain receptor channel, transient receptor potential ankyrin 1 (TRPA1). CS/CSE-dependent, TRPA1-mediated Ca2+ influx led to myosin light-chain phosphorylation, a key process regulating ASM contractility. We conclude that TRPA1 is likely an important link between CS/CSE exposure and airway hyperresponsiveness, and speculate that acute CS/CSE-induced Ca2+ influx could lead to exacerbated ASM contraction and potentially initiate further chronic pathological effects of tobacco smoke.Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with hyperresponsiveness of airway smooth muscle (ASM). Chronic exposure to cigarette smoke (CS) leads to airway inflammation and remodelling. However, the direct effect of gaseous CS or CS extract (CSE) on human airway smooth muscle cell (hASMC) function remains poorly understood. This study investigated the acute effect of CS/CSE on calcium homeostasis, a key regulator of ASM physiology and pathophysiology. Primary hASMC were isolated from non-smoking donor lungs, and subjected to Ca Airway smooth muscle (ASM) is at the centre of AHR and is more sensitive to contractile stimuli in the diseased state. This may be exacerbated by airway inflammation and remodelling, which are extensively linked to tobacco smoking9.Tobacco smoking is the largest risk factor for developing COPD, which is associated with airway hyperresponsiveness (AHR), amongst other lung conditions such as emphysema, chronic bronchitis and infections10. Experimental analysis of CS utilises one or both of its two distinct phases: the gaseous phase and the particulate phase. The gaseous phase entails the smoke that passes through a glass fibre filter with 0.3 µm pores, while the particulate phase, also known as the tar phase, consists of the relatively larger aerosol particles trapped in these pores11. Delivery of CS can be performed by direct aerosolisation into a closed chamber, commonly used for animal smoking studies, but have also been applied in in vitro experiments. Another delivery model is known as CS extract (CSE), produced by bubbling CS into a buffer solution, which can then be applied to in vitro or ex vivo samples, or collected for chemical analysis. The two delivery models are often reported in the literature as though to be synonymous, but certain volatile constituents potentially lost through vigorous bubbling during the production of CSE are retained in the closed chamber in the CS model.Cigarette smoke (CS) is a complex chemical mixture, containing over 4000 identified compounds. The most prominent of these are nicotine, acrolein, N-nitrosamines, various volatile compounds and heavy metals18. However, CSE, or CS, may also have a direct effect on the intrinsic contractility of the ASM6. ASM contractility, and hence the tone and calibre of the airway, is tightly regulated by intracellular Ca2+ signalling. Chronic CSE exposure enhances agonist-induced Ca2+ release and SOCE in hASMC, which correlates with upregulation of several components of Ca2+ signalling, including Ca2+ permeable channels, potentially leading to long-term disruption of Ca2+ homeostasis19. In relation to ASM contractility, there are varying effects reported of chronic CS or CSE exposure. Chronic in vivo CS exposure sensitises the responsiveness of rat bronchial SM to acetylcholine ex vivo20. Interestingly, rat bronchial segments cultured with CSE chronically are found to be hyper-contractile21, but bovine tracheal strips and hASMC cultured with CSE exhibit attenuated contractile responses14. This makes it difficult to extrapolate to what may occur in these circumstances in hASMCs.In vitro experiments have shown that chronic exposure of human ASM cells (hASMC) to CSE augments ROS generation, chemokine secretion, collagen deposition, proliferation, migration, apoptosis and inhibits wound-healing, all of which are hallmarks of the inflammatory profile and airway remodelling in CS-related airway diseases12 reported that transient exposure to CSE increased [Ca2+]i in hASMC, but the mechanism of action was not explored. Acute CSE treatment also leads to ex vivo constriction of guinea pig bronchi, but rapid relaxation of mice lung slices23. Otherwise, the direct effects of acute gaseous CS or CSE exposure on hASMC Ca2+ signalling, and subsequent downstream effects, remain poorly understood. In the present study, we therefore explored the effects of acute exposure to CS, or CSE, on Ca2+ homeostasis in primary hASMC. Importantly, we applied both the aqueous extract model as well as direct exposure to gaseous CS in order to tease out potential differences between the two delivery methods. Using a variety of pharmacological and molecular interventions, we established that both CS and CSE stimulate Ca2+ influx in hASMC through the TRPA1 channel, and lead to downstream activation of contractile signalling mechanisms.On the other hand, Yoon, et al.2+ concentration in isolated primary hASMC, the cells were exposed to one whole Kentucky 3R4F reference cigarette as described above. Real-time changes in [Ca2+]i was reported by changes in fura-2 fluorescence ratio, and the amplitude (peak [Ca2+]i minus baseline [Ca2+]i) and rate (change in [Ca2+]i per minute fitted with linear regression) of the calcium response were calculated from each individual experiment. In the presence of 1 mM extracellular Ca2+, exposure to the gaseous phase of one whole cigarette led to a rise in [Ca2+]i that was significantly higher, in both amplitude and rate, than the change in [Ca2+]i elicited by room air puffed with the same volume/frequency . This Ca2+ response typically started to rise after 4–6 puffs, and reached an initial peak 4.5–8 min after the first puff. More importantly, the CS-induced [Ca2+]i increase required extracellular Ca2+, as the response was abolished when CS was administered in a nominally Ca2+-free solution . This points to Ca2+ influx from the extracellular space as the primary source of the CS-induced [Ca2+]i elevation, and that intracellular Ca2+ stores are not likely to contribute to this response.To investigate the effect of CS on cytosolic Ca2+]i was through a plasma membrane-bound Ca2+ channel, we tested the effect of gadolinium (Gd3+), a lanthanide ion that non-specifically blocks Ca2+-permeable pathways at the PM. Indeed, both the amplitude (52.6%) and rate (43.6%) of the CS-activated Ca2+ response were significantly inhibited by Gd3+ , suggesting that Ca2+ influx through PM-bound channels was the main source of this response.To ascertain if the CS-induced rise in [Ca2+]i, and this was concentration-dependent . This response typically reached a peak within 1–2.5 min of exposure to 50% CSE, and 3–6.5 min following exposure to 10% CSE. As for the CS-induced rise in [Ca2+]i, the CSE-induced change in [Ca2+]i was also abolished by the removal of extracellular Ca2+ . Since a 5-min exposure to CSE appeared to be sufficient to activate Ca2+ influx, we developed a calcium-addback protocol, whereby cells were first exposed to CSE in 0Ca2+ for 5 min to synchronise influx for the whole population of cells, then Ca2+ was added back in the absence of CSE. This reduced inter-experiment variability in response profiles.To directly compare the effect of CSE to that of gaseous CS, diluted CSE was delivered to hASMC through a perfusion system. Similarly to gaseous CS, exposure to diluted CSE also raised Ca+i, and t2+ addback protocol revealed that the continual presence of CSE was not required for maintained Ca2+ influx, and that the channels remained open following removing of extracellular Ca2+ (data not shown). More importantly, following an extended washout period (at least 25 min after removing CSE), both the amplitude and rate of the Ca2+ addback response was attenuated but not abolished (data not shown), suggesting that CSE’s activation of the Ca2+ influx pathway was likely to be reversible. Using the addback protocol, we examined the effect of Gd3+ on CSE-induced Ca2+ influx. Gd3+ also significantly attenuated both the amplitude (74.8%) and rate (76.0%) of CSE-activated Ca2+ influx in hASMC , providing further evidence that a PM-bound Ca2+ channel was responsible for this response.Experiments with this Ca2+ response elicited by the two tobacco product delivery methods were similar, the response profiles bore subtle differences. In the presence of 1 mM extracellular Ca2+, once cells were exposed to 10% and 50% CSE, [Ca2+]i started to increase within a minute; whereas in the CS model, there was a noticeable time lag of 2–3 min (4–6 puffs) before [Ca2+]i began to rise. Indeed, on average it took longer for CS-induced Ca2+ response to reach a peak than the CSE-induced response (4.5–8 min vs. 1–2.5 min for 50% CSE and 3–6.5 min for 10% CSE).Although the general Ca2+ influx pathways in hASMC, we considered the potential contribution of store-operated Ca2+ channels (SOCC) and the voltage-gated L-type Ca2+ channels (LTCC), both of which play an important role in refilling SR Ca2+ stores in ASMC24. The involvement of SOCC was studied using CPA (10 µM), a SERCA inhibitor, which depletes SR Ca2+ stores and activates SOCC. After store depletion and the subsequent SOC influx reaching a plateau, CS from one cigarette was puffed onto hASMC as previously described. CS was able to instigate an additional elevation in [Ca2+]i when SOCC were already active  did not significantly attenuate CS-induced Ca2+ influx  significantly inhibited the amplitude and rate of both CS- (71.2/94.7% inhibition) and CSE- (69.0/94.0% inhibition) induced Ca2+ influx in hASMC  against TRPA1. A shRNA sequence containing an empty vector (E) was used as a negative control. The relative expression of TRPA1 mRNA, normalised to two housekeeping genes, was significantly lower in cells transfected with the shRNA targeted towards TRPA1 than those transfected with the empty vector  was higher than reported in the initial screening studies (4.9–7.5 µM), albeit in different cell types and against different agonists40. We also used a genetic approach to ascertain the role of TRPA1, and used mRNA levels to quantify TRPA1 expression; studying TRPA1 protein levels, whenever a validated, specific, antibody emerges, could provide additional evidence for changes in TRPA1 expression in these cells. Our shRNA-driven knockdown of TRPA1 was not fully efficient (mean 2-ΔΔCT for TRPA1-shRNA transfected cells was 0.44–0.75) although we were able to detect a ~ 30–60% attenuation of CS/CSE-induced Ca2+ influx with this knockdown model. A complete knockout animal or cell model may provide more definitive evidence of this interaction. CSE and its components were reported to activate TRPA1-mediated Ca2+ influx in sensory neurons of WT, but not TRPA1-deficient, mice33; we hypothesise that ASMC from TRPA1-deficient rodents would not respond to CS/CSE challenge. Moreover, other Ca2+ channels may potentially be involved, since we observed that neither HC-030031 nor TRPA1 knockdown completely abolished CS/CSE-induced Ca2+ influx. For instance, although our data suggested that SOCC were unlikely to be the major facilitators of acute CS/CSE-induced Ca2+ influx, this interpretation assumed that CPA fully activated SOCE in hASMC, which may not be the case. Therefore, SOCC could play a minor role in mediating this influx, but several other candidates to consider include, TRPV141, TRPV442, TRPM843, and reverse-mode NCX44, as these proteins were reported to be activated by CSE or CSE constituents in other cell types. Furthermore, as CS-induced Ca2+ influx was blocked by Gd3+, the involvement of other Gd3+-sensitive channels, including stretch-activated cation channels45 and various TRPC, TRPM, and TRPV channels46, could be further explored.HC-030031 has been reported to be highly selective for TRPA1. At 10 µM, the compound did not inhibit other Ca2+ responses in hASMC  unless otherwise specified. HC-030031, felodipine, and CPA were purchased from Tocris . The primary antibody against p-MLC (3675S) and the secondary anti-mouse HRP-linked antibody (7076S) were purchased from Cell Signaling Technology , while the primary antibody against MLC (M4401) was from Sigma-Aldrich. 3R4F reference cigarettes were purchased from the Kentucky Tobacco Research & Development Center, University of Kentucky. Glycerol stocks of bacteria carrying the plasmid vector encoding shRNA targeted against TRPA1 (TRPA1-shRNA) or the empty control vector (EV) were obtained from Lenti-shRNA Core Facility, UNC Chapel Hill. PCR primers were purchased from Integrated DNA Technologies .56). These donors were all adults over the age of 18, and informed consent was obtained for procurement of lung tissues. All experiments presented in this study were performed in accordance with local and institutional guidelines and regulations.Human tracheal rings were procured from non-smoker excess donor lungs from Freeman Hospital, Newcastle upon Tyne and the Cystic Fibrosis Centre, UNC Chapel Hill. Ethical approval was obtained for the collection of non-smoking human donor lung tissues from Newcastle and North Tyneside Local Regional Ethics Committee (16/NE/0230) and the University of North Carolina Institutional Review Board , and cultured in DMEM/F-12 media . Media was replaced every 48–72 h. Cultured primary hASMC were grown in a humidified incubator at 37 °C with 5% CO2+ imaging experiments and shRNA transfection, hASMC were seeded onto 25 mm glass coverslips, and grown in 6-well plates until ~ 60–90% confluent (Ca2+ imaging) or ~ 40–60% confluent (transfection). For the MLC phosphorylation assay, hASMC were seeded directly onto 6-well plates until fully confluent. Cells were serum-starved for 24–48 h before Ca2+ imaging experiments or MLC phosphorylation assay.For CaCS generated from Kentucky 3R4F reference cigarettes was delivered through a Borgwaldt LM1 smoke engine , into a closed chamber in which hASMC were situated. A glass fibre Cambridge filter pad (0.3 µm pore size) was placed in the tubing to filter out the particulate tar phase. Exposure of hASMC to CS was carried out in 13 puffs with a 30 s interval in between; each puff was 35 ml, delivered over a duration of 2 s. CS persists in the closed chamber after delivery for the full duration of each experiment. Exposure to room air, with identical puff delivery conditions, was used as a control.2, 1 MgCl2, 10 NaHEPES, 10 D-glucose; for the nominally Ca2+-free solution, CaCl2 was omitted and replaced with 1 mM EGTA; solutions were adjusted to pH 7.4). The stock solution was made fresh at the beginning of each day of experiments, and kept on ice throughout the day. CSE was diluted into HEPES-buffered solution for use immediately prior to each experiment.The stock solution of 100% CSE was produced by bubbling one 3R4F reference cigarette, with the particulate phase filtered out, into 25 ml of HEPES-buffered solution  for 60 min at 37 °C, washed with HEPES-buffered solution, and left to de-esterify for 15 min at room temperature. Coverslips were mounted onto a Nikon epifluorescence microscope equipped with a 20 × lens and appropriate filters, and the 340/380 emission ratio (F340/380) of fura-2 fluorescence was recorded using a Princeton Instruments/Hamamatsu CCD camera. Single cells were circled as areas of interest , and images were collected every 5–15 s (5 s for the influx phase). For CS experiments, cells were left in a static bath (1 ml HEPES-buffered solution) and were covered with a customised chamber with tubing connected to the smoke machine for CS delivery. For CSE experiments, a perfusion system was assembled to allow for fluid exchange (2 ml/min). All experiments were performed at room temperature.Primary hASMC grown on glass coverslips were loaded with the cell-permeable i change.Summary data is presented as mean ± SEM. All experiments were performed using hASMC isolated from 2 to 4 donors . n denotes number of repeated independent experiments . Statistical analysis was performed using GraphPad Prism 8, with statistical significance indicated by an alpha value of p < 0.05. Non-parametric tests were performed for datasets that did not pass the D’Agostino & Pearson test. The type of analysis for each dataset is indicated in the respective figure legend. Linear regression was performed to estimate the rate of [CaSupplementary Figure S1.


Particularly, fundamental aspects of neuronal function such as synaptic transmission and neuronal plasticity are regulated by Ca2+, and neuronal survival itself relies on Ca2+-dependent cascades. Indeed, impaired Ca2+ homeostasis has been reported in aging as well as in the onset and progression of neurodegeneration. Understanding the physiology of brain function and the key processes leading to its derangement is a core challenge for neuroscience. In this context, Ca2+ imaging represents a powerful tool, effectively fostered by the continuous amelioration of Ca2+ sensors in parallel with the improvement of imaging instrumentation. In this review, we explore the potentiality of the most used animal models employed for Ca2+ imaging, highlighting their application in brain research to explore the pathogenesis of neurodegenerative diseases.Calcium (Ca Intracellular Ca2+ concentration ([Ca2+]i) must be tightly controlled in terms of both space and time to generate regulated signals. This dynamic control is obtained through the orchestrated interplay of Ca2+ transport systems, localized in the plasma membrane (PM) and the membranes of the intracellular Ca2+ stores and the Ca2+ buffering systems.Ca2+ generates versatile signals in the nervous system i. The investigation of Ca2+ signals through fluorescence imaging is now of utmost importance in order to gain information regarding not only single-cell, activity-dependent [Ca2+]i variations, but to also map neuronal population activity and functional connectivity i not only in the cytosol, but also in the subcellular compartments i with no interference from focal plane shifts, photobleaching, or dye leakage.An interesting alternative to single-protein sensors consist of using ratiometric indicators, in which variations of the ratio of the fluorescence emitted by two fluorescent proteins are correlated to [Caf probes , in whic2+ imaging experiments in live tissues/animals were performed with epifluorescence microscopy equipped with charged coupled detector (CCD)-based cameras using synthetic dyes. However, the application of wide-field microscopy is limited by light scattering across the z-axis and resolution and contrast fall as the imaging plane goes deeper into the tissues. As a result, this technique can only be successful with small and transparent invertebrates [2+ dynamics within cells localized deeper in tissues. Importantly, this permitted to move from cell cultures and isolated tissues to the imaging of the intact brain of an entire transgenic animal, exploring neuronal circuitry in vivo [Efforts for the amelioration of existing GECIs and for the development of new ones are continuously ongoing. This path precedes in parallel with the improvement of imaging instrumentation . Indeed,tebrates . These ltebrates , thus ontebrates ,53. The tebrates  allowed  in vivo ,53,54,55 in vivo ,57,58,59 in vivo . Miniatu in vivo . 2+ imaging has developed into a powerful tool for the imaging of the brain in living animals. As technology advances, upgraded systems could be designed to map less accessible brain areas in order to obtain a larger view of the nervous system with increased spatio-temporal resolution.Thanks to this parallel development of fluorescent indicators and dedicated instrumentation, Cahttp://www.alzforum.org/research-models (accessed on 17 August 2021)), are now available. Among species, mice are the most common animal models used in research. This is mainly due to their similarity to humans at the genetic and physiology level as well as their relatively short generation time and lifespan, which make them useful for aging studies ,63. More2+ imaging in mice. GECIs can be expressed through in utero electroporation [Since their development, GECIs are the elective probes for ex vivo/in vivo Caporation  or by inporation . Howeverporation . Moreoveporation . 2+ probes, GECI-expressing transgenic mice have been developed [2+ imaging studies, especially those investigating processes related to aging and neurodegeneration in old mice, to a rapid improvement.To overcome these limitations and to reach a more stable, long-term expression of Caeveloped ,68,69,702+ dynamics in cellular and subcellular compartments. Improved versions of GCaMP6 have been recently generated, i.e., the enhanced version GCaMP7 [2+ dynamics in vivo [The most employed GECIs for live imaging in mice are the GCaMPs. In particular, GCaMP6 ,39 has bn GCaMP7  and the  in vivo ,71 (Tabl2+ channels (CaV1). To overcome this drawback, a new GCaMP-X variant was generated by protecting Ca2+-free CaM to avoid CaV1 perturbation [2+ indicators can be used, e.g., via Cre/LoxP-mediated recombination [2+ imaging [Although commonly used, GCaMPs could present side effects when expressed chronically. Cytotoxicity and abnormalities in brain activity have been reported  due to turbation . Alternabination  or by thbination . Modern bination  improvedbination  and also imaging .2+ sensor iGECI [2+ imaging experiments. Another limitation of GCaMPs is represented by their excitation emission spectra. Indeed, traditional GCaMPs cannot be used in mice that concomitantly express other GFP-based proteins. Similarly, their simultaneous use with optogenetic tools is prevented, due to the overlap of a GCaMP excitation spectrum with those of light-sentitive ion channels, such as channelrhodopsin (ChR2). To overcome this weakness, improved red-shifted GECIs with a sensitivity comparable to GcaMP6 were generated: mRuby-based jRCaMP1a and jRCaMP1b, mApple-based jRGECO1a , and a nor iGECI . The new2+ signals. Given their importance in physiology and their involvement in the pathogenesis of neurodegenerative diseases [2+ probes have been developed to target them [2+ at the cellular level are available and are used routinely. However, this type of measurement is still poorly exploited in living animals. Organelles play a fundamental role in shaping cellular Cadiseases ,78,79,80get them ,81. Nowa2+ dynamics in cultured cells. Until recently, however, measurements of mitochondrial Ca2+ dynamics in vivo were limited to the use of the virus-mediated delivery of GECIs (see above). Mice lines have only been generated for the expression of mitochondria-targeted bioluminescence-based sensors [2+ sensor 4mtD3cpv [2+ handling in vivo.As far as the mitochondria are concerned, a large number of probes, both chemical  and gene sensors ,87 which4mtD3cpv , which w2+ sensor expression in mice and imaging techniques, Ca2+ signalling alterations in a variety of diseases have been described [2+ dysregulation and AD [APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) genes. In the late 1980s, Ca2+ homeostasis alterations were proposed to be responsible for AD-linked neurodegeneration [2+ handling impairment is an early event in AD pathogenesis [Thanks to the tremendous improvement in Caescribed ,88,89. Fn and AD . AD is tneration . Indeed,ogenesis ,90.https://www.alzforum.org/research-models (accessed on 17 August 2021)), along with new tools for Ca2+ imaging, allowed the investigation of Ca2+ dynamics at different levels, ranging from in vivo multiphoton imaging of Ca2+ dynamics within organelles [2+ imaging in awake mice [2+ imaging techniques have recently been applied to explore hippocampal activity in a knock-in AD mouse model carrying mutated APP. These mice were crossed with a transgenic mouse line that co-expresses the Ca2+ indicator GCaMP7 and a Ca2+-insensitive red fluorescent protein (DsRed2) in neurons [2+ transients monitoring GCaMP7 fluorescence, while the red fluorescent protein, showing stable fluorescence independent on neuronal activity, was used to precisely align the imaging field during subsequent acquisitions. Ca2+ signals were followed together with Aβ deposition for several months while the animals were subjected to specific tasks that allowed the estimation spatial and temporal representations in the hippocampus separately, revealing that place cells were mostly impaired in AD mice, especially in the proximity of Aβ deposition [In this context, the development of several transgenic AD mouse models  channel superfamily [2+ entry (SOCE) molecular players stromal interacting molecule 1 (STIM1) and ORAi1 [2+ signaling toolkit are conserved between mammals and flies [Drosophila, such as voltage- and ligand-gated PM Ca2+ channels [2+ release channels the inositol 1,4,5-trisphosphate receptor  [2+ ATPases located on PM (PMCA) [+/Ca2+ exchanger NCX  [2+ interacting proteins, e.g., Calcineurin (CaN), Calmodulin (Cam), Ca2+/Calmodulin-dependent protein kinase II (CaMKII), the and Ca2+/Calmodulin-dependent serine protein kinase (CaSK).The lack of genetic redundancy and the easy genetic manipulation via well-consolidated knock-down and knock-out strategies make the characterization of protein function in erfamily  and the nd ORAi1 ,100. Indnd flies . Single channels , the Ca2ed itpr)  and the ed itpr) ,105, theM (PMCA) , Sarco/EM (PMCA) , the secM (PMCA) , the Na+X  , Ca2+ inDrosophila allowed the generation of several lines for the expression of GECIs. Most frequently, the expression of indicators relies on the Gal4-UAS binary expression system [2+ sensor under the control of an upstream activating sequence (UAS) so that the gene is silent in the absence of the transcription factor Gal4. A second fly strain expresses Gal4 under the control of a cell specific promoter. Upon mating, the resulting progeny will express the probe in the specific cell type determined by the Gal4 line promoter. UAS lines for the controlled expression of both cytosolic and organelle-targeted GECIs have now been developed in flies and exploited to unravel questions of physiological and pathological relevance.The ease of transgenesis in n system ,111 as fDrosophila nervous system proved particularly helpful in the study of neuronal function in physiology and pathology. In the adult fly, both a central and a peripheral nervous system can be recognized. The central brain and the optic lobes are lodged in the head capsule. Neurons from the brain descend in the ventral nerve cord, and motor neurons project towards muscles . The adrefer to . In the refer to . Similarrefer to ,116. Imprefer to . MoreoveDrosophila as a model organism is the high accessibility of its nervous system at all developmental stages. Ca2+ imaging procedures have been adapted or specifically developed for the study of Ca2+ dynamics in vivo in the nervous system in embryos [Drosophila larvae are partially transparent, and the GECIs expressed in superficial tissues, such as central nervous system (CNS) or sensory neurons, can be excited through the intact animal’s cuticle, provided that the fluorescence is strong enough. The main obstacle to larval Ca2+ imaging is the sample movement due to continuous body wall muscle contraction, which can be partially avoided using sticky tape [2+ imaging with other genetically encoded tools, e.g., optogenetics [2+ imaging experiments, e.g., larval neuromuscular preparation [From the experimental point of view, the great advantage of using  embryos ,119, lar embryos ,122,123, embryos . A criticky tape  or chambcky tape  and corrcky tape  or, mostcky tape ,128. Singenetics ,130,131 genetics ,133. GECgenetics ,27,134, paration  or the iparation . 2+ imaging techniques can be applied to explore a wide range of biologically relevant questions in Drosophila [2+ imaging techniques to investigate the response of specific brain regions to various sensory stimuli. These studies mainly investigated olfaction [2+ imaging using GECIs has been successfully applied to the Drosophila brain to investigate the neuronal plasticity underlying associative learning and memory formation [2+ imaging to be performed in freely walking flies during sensorial and social behaviors [Optical Caosophila . Mechanilfaction ,141,142,lfaction , auditorlfaction , taste [lfaction , mechanolfaction , and thelfaction . Moreoveormation  and the ormation . Whole-behaviors . Long-teehaviors .Drosophila a powerful model system to elucidate the Ca2+ dynamics involved in pathogenic processes in neurodegenerative diseases. Remarkably, over 60% of known human disease-causing genes have a fly homolog [The potential of the described approaches makes  homolog , and a wDrosophila AD models have been generated, and neuronal Ca2+ homeostasis has been evaluated using different approaches, e.g., loading the chemical indicator fura-2 in dissociated neurons [2+ levels have been measured in dissected third instar larval brains [As an example,  neurons ,151 or i neurons  or in vi neurons . AD fly l brains . Danio rerio) is a small freshwater teleost native of Southeast Asia belonging to the cyprinid family. In recent years, it has become a prominent vertebrate model in neuroscience as well as in other fields of biological research. The reasons for its increasing popularity stem from characteristics that allow it to overcome many limitations that emerge when working with other vertebrates, especially tissue opacity and the large size and complexity of the brain. Given its small size, rapid development  and the short generation time , zebrafish are easy to manipulate and to raise in the laboratory [Zebrafish , it maintains the basic vertebrate brain organization, although at a lower-level complexity. Moreover, the transparency of the embryos and larvae makes the entire nervous system optically accessible, allowing the brain-wide monitoring of neuronal activity throughout the whole CNS in real time and at single-cell resolution ,162,163.Zebrafish disease models can be generated in order to explore the progression of pathologies and the alterations of related signaling pathways in vivo ,170,171.2+ imaging, electrophysiological recordings, and optogenetics, allowing the investigation of neuronal activity in vivo at single cell resolution as well as full circuit reconstruction. One of the great advantages of zebrafish is the ease of application of techniques ranging from advanced Ca2+ in zebrafish. However, they require injection procedures and do not allow the selective loading of specific cell types. For these reasons, the GECIs represent the elective choice for Ca2+ imaging experiments in zebrafish. Transgenic lines can be generated by microinjecting [Dextran-conjugated and membrane-permeant chemical indicators have been employed to image Canjecting  the GECInjecting . Transge2+ imaging experiments to be performed in intact, free moving animals [2+ sensors in specific cell types, in combination with appropriate imaging techniques , is that it allows to image neuron functional dynamics at early stages of zebrafish development as well as during neurodegeneration. As a result, a morphological and functional reconstruction of the pathways underlying brain function and dysfunction can be obtained with a high spatial and temporal resolution, a task that is currently only achievable in transparent zebrafish larva. GECIs, such as aequorin, cameleons, and GCaMPs, have been exploited in zebrafish, allowing Ca animals ,178. Neu animals ,160,161 Caenorhabditis elegans is a nonparasitic nematode firstly described as a model organism in 1963. Only a decade later, it was clear that worms could be used as a genetic tool to study aging and neurodegeneration [C. elegans genome was completely sequenced in 1998 [neration . The sucneration . Moreove in 1998 , and pos in 1998 ,183. C. elegans nervous system is extremely simple when compared to mammals yet complex enough to consider this model suitable to study neurodegeneration. Notably, the full connectome of the 302 neurons comprising the hermaphrodite worm nervous system has been mapped [C. elegans presents standard glutamatergic [The n mapped ,185,186.matergic , GABAergmatergic , cholinematergic , dopaminmatergic , and sermatergic  transmisC. elegans led to the identification of homologs of Ca2+ signalling components, including PM [2+ channels, such as IP3R [2+ pumps SERCA [2+ signalling modulators are present in C. elegans, including CaM, kinases, and phosphatases [The availability of the full genomic sequence of uding PM ,193 and  as IP3R  and RyR  as IP3R ,196; theps SERCA  and PMCAps SERCA ; transpops SERCA ; and SOCps SERCA . Furtherphatases ,202,203.C. elegans an advantageous animal model is its genetic manipulability. Forward and reverse genetic approaches have been largely exploited in worms, and a considerable number of transgenic lines have been generated over time [C. elegans models of neurodegenerative diseases to search for small molecules as possible therapeutic treatments [One of the features that make ver time ,205. Morver time  allows rver time ,207. Phaeatments ,209.C. elegans models carrying gain/loss of function mutations of genes involved in neurodegenerative diseases have been generated, recapitulating many of the characteristics of the disease at the cellular level [Consistently, ar level ,211. The2+ sensors can be expressed in C. elegans in a tissue specific manner. A fundamental feature for Ca2+ imaging experiments is the transparency of the worms throughout their lives. It is thus possible to record Ca2+ sensor fluorescence in vivo in the intact animal. The two most broadly used Ca2+ sensors in C. elegans are GCaMPs and FRET-based cameleons, the most frequently expressed in neurons or muscular cells [C. elegans have been performed using untargeted probes expressed in the cytosol, relevant examples of Ca2+ sensors expressed in organelles exist, e.g., mitochondria [2+ imaging, in which about 100 neurons were simultaneously monitored in moving worms [Caar cells ,213,214,ar cells . Althougchondria ,217,218.ng worms ,220.C. elegans models have provided critical information on the role of Ca2+ signaling in aging [2+ during aging have been recorded in live, non-anesthetized worms [The application of these powerful techniques to in aging . As an eed worms ,222.2+ imaging. The combination of genetic accessibility and methodological advances has enabled the exploration of Ca2+ dynamics not only inside single cells but also among a population of neurons. As a result, cellular and circuit neurophysiology can be explored in physio-pathological conditions.Here, we depicted four commonly used animal models used to study neurodegeneration, highlighting their characteristics and potentiality in the field of CaDrosophila models of neurodegenerative diseases exist where Ca2+ dynamics have been successfully explored. Additionally, Ca2+ probes can be expressed in C. elegans and zebrafish, two models that have great potential in terms of genetic manipulation. We foresee that these organisms will be profitably used to explore Ca2+ dynamics in models of neurodegenerative disorders [Many relevant examples of mouse and isorders ,224.2+ sensors and imaging instrumentation, the study of Ca2+ dynamics is progressively moving from in vitro samples to animals in vivo, providing an ever-growing number of strategies for investigating neural function and dysfunction. It is however worth to mention that in recent years, three-dimensional (3D) brain organoids from patient-derived, inducible pluripotent stem cells (iPSCs) have been developed, showing themselves to be useful models for studying different aspects of aging and neurodegeneration [Thanks to the improvement of both Caneration ,226,227.


Myomaker. In parallel, while Piezo1 knockdown also lowers Ca2+ influx in response to stretch, Piezo1 activation increases Ca2+ influx in response to stretch and enhances myoblasts fusion. These findings may help understand molecular defects present in some muscle diseases. Our study shows that Piezo1 is essential for terminal muscle differentiation acting on myoblast fusion, suggesting that Piezo1 deregulation may have implications in muscle aging and degenerative diseases, including muscular dystrophies and neuromuscular disorders.Mechanical stimuli, such as stretch and resistance training, are essential in regulating the growth and functioning of skeletal muscles. However, the molecular mechanisms involved in sensing mechanical stress during muscle formation remain unclear. Here, we investigated the role of the mechanosensitive ion channel Piezo1 during myogenic progression of both fast and slow muscle satellite cells. We found that Piezo1 level increases during myogenic differentiation and direct manipulation of Piezo1 in muscle stem cells alters the myogenic progression. Indeed, Piezo1 knockdown suppresses myoblast fusion, leading to smaller myotubes. Such an event is accompanied by significant downregulation of the fusogenic protein  Skeletal muscles are highly specialised tissues composed of multi-nucleated, post-mitotic myofibres. Since nuclei within a myofibre (myonuclei) do not divide after development, the production of new myonuclei, to sustain muscle maturation and repair, is entrusted to muscle stem cells (named satellite cells (SCs)). SCs are found on the surface/periphery of postnatal skeletal myofibres ,2,3 and +, K+, Ca2+, and Mg2+ with a preference for Ca2+ i were measured by ratiometric imaging with Fura 2-AM at 340 and 380 nm, and the emitted light signal was read at 510 nm. Images were then analysed on ImageJ imaging software. Three independent myotubes from each condition were selected and analysed. The changes in ratios were calculated by subtracting basal values from peak values. The values were then normalised to ionomycin data.Changes in intracellular calcium i) upon modulation of Piezo1 activity in cultured myotubes. Using the customised stretch silicon bio-chambers i (Piezo1 siRNA-mediated knockdown led to nearly 50% and 40% reduction on Piezo1 mRNA in EDL-derived myoblasts (2+]i increases significantly in both EDL-derived and SOL-derived myotubes compared to no-stretch (0%) controls (2+]i. Reduction of Piezo1 severely suppressed in [Ca2+]i in response to stretch compared to control cells (siRNA controls)  . Ca2+ isn/fusion ,19,20. Tchambers , we cult [Ca2+]i . Piezo1 yoblasts  and SOL-yoblasts  respectiyoblasts . Under ccontrols a–h. Of nontrols) a–d. Indemyotubes c with nees , Yoda1 administered myotubes already begin to show an enhanced i, which most likely translates in the depression of the influx of Ca2+ into cultured myotubes exposed to stretch. In contrast, activation of Piezo1 significantly increased [Ca2+]i, which means the enhancement of Ca2+ influx. Our results propose that Piezo1 is a novel intracellular Ca2+ regulatory protein in skeletal muscle function. Ca2+ plays a crucial role in skeletal muscle function, maintenance, and plasticity. All myofibres use Ca2+ as their main regulatory and signalling molecule i must be attained in order to elicit a response, in their case ATP efflux. Our data support the presence of a stretch-dependent increase in Ca2+ influx. Remarkably we found that activation of Piezo1 resulted in increased [Ca2+]i even without stretch, suggesting that the activation threshold of Piezo1 was lowered. Furthermore, the data showed that reduction of Piezo1 expression significantly blunted any significant increase of [Ca2+]i in response to stretch. These results, for the first time show the need for Piezo1 to respond to stretch and permeate Ca2+ into myotubes. The findings also propose the presence of a physical threshold that must be attained before Piezo1 mediated Ca2+ influx is significantly increased. Moreover, as with Miyamoto et al. i when Piezo1 is activated. Whether this leads to altered cellular/myotube viability in the form of ATP release remains a subject for future research.The silicon bio-chamber experiments revealed that the mechanical activation threshold of Piezo1 was severely blunted by Piezo1 downregulation. In a similar set of experiments , Miyamoto et al. also showed a distance dependent increase of Canditions . The reso et al. , we find2+ influx. Indeed, since the early 1990s, it has been postulated that defective mechanotransducers, also referred to stretch activated channels (SACs), contribute to the high influx of Ca2+ and, hence, maintain higher Ca2+ resting levels in Duchenne’s muscular dystrophy (DMD) [2+ at hazardously high levels. It is possible that the reduced fusion we observed from an over-active Piezo1 channel  led to decreased fusion in both EDL- and SOL-derived myotubes . This adhy (DMD) ,31. Howe channel  stems frPiezo1 siRNA was on average more efficient compared to the main siRNA from Qiagen used in this study  that are directly the result of mechanical stress should consider analysis of Piezo1. In the context of therapeutic strategies against muscular dystrophies such as DMD, not only must we unravel the spatiotemporal regulation of Piezo1 expression, but we must be aware of this channel’s ability to alter its Ca2+ influx threshold by adapting or inactivating its gating capacity in response to repetitive stimuli. Pharmaceutically, small activating molecules, such as Yoda1, may prove beneficial. However careful attention must be given to the half-life and pharmacokinetics of these agonists in vivo before even considering them as viable drugs for human consumption. Piezo1′s importance in skeletal muscle maintenance and function will undoubtedly grow as new research aims to explore the mechanisms and signalling pathways this remarkable mechanosensor employs. Moreover, given Piezo1’s pivotal role in myogenic progression and its expression in satellite cells, it is likely that altered Piezo1 activity may associate with pathogenesis of satellite cell-opathies [The data presented in this study showed that the Piezo1 channel is present in SC-derived myoblasts and myotubes but expressed at a higher proportion in the latter. Downregulation of Piezo1 significantly lowered the fusion capacity during myotube formation, growth and maturation. In contrast, Piezo1 activation increased fusion. Future research examining changes in myotube function (integrity, Caopathies .


NaL) increases arrhythmia propensity in the heart, whereas suppression of the current is antiarrhythmic. In the present study, we investigated INaL in canine ventricular cardiomyocytes under action potential voltage-clamp conditions using the selective Na+ channel inhibitors GS967 and tetrodotoxin. Both 1 µM GS967 and 10 µM tetrodotoxin dissected largely similar inward currents. The amplitude and integral of the GS967-sensitive current was significantly smaller after the reduction of intracellular Ca2+ concentration ([Ca2+]i) either by superfusion of the cells with 1 µM nisoldipine or by intracellular application of 10 mM BAPTA. Inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII) by KN-93 or the autocamtide-2-related inhibitor peptide similarly reduced the amplitude and integral of INaL. Action potential duration was shortened in a reverse rate-dependent manner and the plateau potential was depressed by GS967. This GS967-induced depression of plateau was reduced by pretreatment of the cells with BAPTA-AM. We conclude that (1) INaL depends on the magnitude of [Ca2+]i in canine ventricular cells, (2) this [Ca2+]i-dependence of INaL is mediated by the Ca2+-dependent activation of CaMKII, and (3) INaL is augmented by the baseline CaMKII activity.Enhancement of the late sodium current (I NaL) remains active throughout the entire AP. INaL contributes to plateau formation and is responsible for largely half of the transmembrane Na+ entry through voltage-dependent Na+ channels i-dependent augmentation of the current in rabbit i with either nisoldipine or BAPTA decreases INaL just like CaMKII inhibition with KN-93 or AIP does. Our results also suggest that in the presence of nisoldipine or BAPTA, the lower [Ca2+]i can reduce CaMKII activity so much that INaL will become significantly smaller than with normal intracellular calcium homeostasis.We hypothesized that the Cath KN-93 A and AIPth KN-93 B than unth KN-93 C. INaL i and AIP D, howeve2+-dependent behavior of the GS967-sensitive current could also be demonstrated under current clamp conditions when APs were elicited by electrical stimulation; 1 µM GS967 significantly shortened the AP duration (measured at 90% repolarization) in a reverse rate-dependent manner and significantly decreased the amplitude of the plateau phase  as presented in 2+-chelator  the intrinsic dependency of APD changes on baseline APD. These three effects seemed to compensate each other, causing similar GS967-induced APD shortening in the presence of BAPTA-AM throughout the investigated cycle lengths.Based on our results , INaL be lengths C. It is 2+]i-dependency of INaL because the GS967-induced plateau depression appeared only in cells with normal calcium homeostasis and not in the presence of BAPTA-AM.Our current clamp results indirectly indicate the [Ca2+]i modulate INaL in canine ventricular cells. Whenever [Ca2+]i was reduced—either by applying 10 mM intracellular BAPTA or by perfusing the cells with 1 µM nisoldipine—the density and the integral of INaL significantly decreased. The Ca2+-dependency of INaL was evident under both action potential voltage clamp and current clamp conditions. Furthermore, this Ca2+-dependent modulation of INaL was likely due to the contribution of CaMKII in regulating the current because inhibition of CaMKII with KN-93 or AIP decreased the density of the current to a similar level observed with nisoldipine or BAPTA. The CaMKII-related stimulation of INaL observed by us in healthy canine ventricular cells was like those reported in rabbit i i [NaL when CaMKII is maximally activated at high [Ca2+]i levels, so we could not estimate the total CaMKII-dependent fraction of INaL.Our results indicate indirectly that in paced canine ventricular cardiomyocytes with intact calcium homeostasis—just like in the physiologically active, working ventricle—CaMKII is partially activated. This is indicated by the marked reduction of I [Ca2+]i  at room  [Ca2+]i  carried 2+/CaM significantly increased both CaMKII and Camui activity in lysates of HEK cells expressing the Camui construct. Furthermore, the Camui sensor detected increasing CaMKII activation as either the pacing frequency or the bath Ca2+ concentration was increased in primarily isolated rabbit cardiomyocytes. Both in HEK cells and in adult cardiomyocytes, the addition of 1 μM KN-93 blocked the FRET change associated with activation of Camui. This suggests that KN-93 prevents CaMKII activation resulting from the physiological excitation-contraction coupling. This is in line with our results, where blocking CaMKII activation either by KN-93 or by AIP resulted in a smaller INaL than under control conditions.Erickson et al.  also conNaL to CaMKII activity i or inhibition of CaMKII in rabbit ventricular cells under control, normoxic conditions, but only in hypoxic myocytes i-dependent regulation of INaL, therefore, neither KN-93 nor BAPTA-AM could further reduce INaL under normoxic conditions. Fu and colleagues also showed that hypoxia increased diastolic [Ca2+]i that leads to CaMKII activation. Furthermore, Lu et al. found that hypoxia might also stimulate CaMKII directly i modulates INaL under physiological conditions, a process likely mediated by CaMKII.In conclusion, our results demonstrate that in canine ventricular cells i in canine ventricular cells.I2+]i-dependence of INaL is mediated by the Ca2+-dependent activation of CaMKII.The [CaNaL is augmented by the baseline CaMKII activity.I