Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients. Excessive mitochondrial matrix Ca2+ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture, and cell death. Moderate Ca2+ within the organelle, on the other hand, can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. Here, we aimed to determine in a quantitative manner if extra- or intramitochondrial Ca2+ modulates oxidative phosphorylation in mouse liver mitochondria and intact hepatocyte cell lines. To do so, we monitored the effects of more modest versus supraphysiological increases in cytosolic and mitochondrial Ca2+ on oxygen consumption rates. Isolated mitochondria present increased respiratory control ratios (a measure of oxidative phosphorylation efficiency) when incubated with low (2.4 ± 0.6 μM) and medium (22.0 ± 2.4 μM) Ca2+ concentrations in the presence of complex I–linked substrates pyruvate plus malate and α-ketoglutarate, respectively, but not complex II–linked succinate. In intact cells, both low and high cytosolic Ca2+ led to decreased respiratory rates, while ideal rates were present under physiological conditions. High Ca2+ decreased mitochondrial respiration in a substrate-dependent manner, mediated by mPTP. Overall, our results uncover a Goldilocks effect of Ca2+ on liver mitochondria, with specific “just right” concentrations that activate oxidative phosphorylation.Calcium (Ca Mitochondria are one of the central intracellular regulators of Ca2+ homeostasis (2+ uptake is lower than for the endoplasmic reticulum (ER), mitochondria can absorb very large amounts of the ion from the cytosol  complex    and by axchanger , 13, loc2+ transients are believed to provide a link between cytosolic Ca2+ signaling and the control of cellular energy demand by regulating ATP production. Pyruvate dehydrogenase (PDH), isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase (α-KGDH) within the mitochondrial matrix have increased activities in the presence of Ca2+, as uncovered through in vitro studies , often culminating in loss of function and cell death , have not been extensively determined.In theory, and when present at lower concentrations than those that lead to mPTP opening, Ca by Ca2+ , 23, shox volume , 25 and x volume , 27. How2+ modulates oxygen consumption fluxes under controlled side-by-side comparison conditions using different substrates, respiratory states, and their quantitative responses to specific, calibrated, and distinct amounts of Ca2+. We used mitochondria isolated from the liver, since a rich metabolic regulation by Ca2+ occurs in this tissue. As different Ca2+ amounts can be present in distinct isolated mitochondrial preparations, in order to minimize possible confounding results, we performed daily calibrations, which allowed us to uncover activating Ca2+ effects with more accuracy, as these were often subtle and concentration specific. We also explore how mitochondrial Ca2+ transport influences oxidative phosphorylation in intact hepatocyte-derived cells. Our results demonstrate that Ca2+ concentrations greatly impact mitochondrial respiration, with a Goldilocks-type effect, in which both too much and too little Ca2+ limit oxidative phosphorylation but the “just right” concentration promotes significant activation.We explore this knowledge gap here and determine if extra- or intramitochondrial Ca2+ additions on mitochondrial respiration, we used isolated mouse liver mitochondria in the presence of different substrates and measured oxygen consumption over time using an Oroboros high-resolution oxygraph. Ca2+ was added at different concentrations, followed by additions of ADP , oligomycin , and CCCP (to induce maximum electron transport). We determined residual Ca2+ concentrations present after mitochondrial isolation daily and then adjusted the amount of Ca2+ added so it was equal between biological replicates. Based on the amount of Ca2+ added and excess EGTA present in the buffer, we calculated the amount of free Ca2+ to which mitochondria were exposed, classifying conditions as no added Ca2+, low (2.4 ± 0.6 μM), medium (22.0 ± 2.4 μM), and high (52.9 ± 2.5 μM) Ca2+ concentrations.To gain insight into the effects of extramitochondrial Ca2+ on electron transport and oxidative phosphorylation, due to the known enhancement of PDH affinity in response to Ca2+ signaling , and was thus not included in further experiments. O2 consumption traces were then measured using low and medium Ca2+ additions and are shown as representative OCRs in B–D and quantified results in panels E–H.Complex I substrates such as pyruvate plus malate were chosen initially to study the effects of Caignaling , 16, 17.ver time A, and wa2+ concentrations strongly increased state 3 respiration, when oxidative phosphorylation is active (E), and state 3u, after the uncoupler CCCP (G). State 4 respiration, which occurs in the absence of ATP synthase activity and is limited by the inner membrane impermeability to protons, was stimulated by both low and medium Ca2+ concentrations when supported by pyruvate plus malate (F). Respiratory control ratios  are the ratio between state 3 and state 4 oxygen consumption and used as a proxy for oxidative phosphorylation efficiency, as they show the relative stimulation of oxygen consumption by production of mitochondrial ATP (2+ concentrations (H). This shows that oxidative phosphorylation supported by pyruvate plus malate has a tight relationship with mitochondrial Ca2+ and is more effective within a very specific range.We found that the presence of low Cas active E, and stler CCCP G. State s malate F. Respiros RCRs, H are therial ATP . We fountrations H. This s2+ uptake or cycling, using pharmacological inhibitors of the MCU , which prevents Ca2+ uptake, and of the NCLX , which prevents Ca2+ extrusion from mitochondria (I). Both RuRed and CGP reversed the increase in RCR promoted by low Ca2+ concentrations; this indicates that Ca2+ must enter the mitochondrial matrix to exert this effect, as RuRed prevents this uptake. The effect of CGP could also indicate that increased RCRs are dependent on Ca2+ cycling, as it inhibits Ca2+ extrusion from mitochondria through Ca2+ exchange with Na+  using pyruvate plus malate with CGP in the presence or absence of the mPTP inhibitor cyclosporin A (CsA). We found that the addition of CGP decreases CRC to 11.8 μM, in comparison with 24.5 μM under control conditions (J). In addition, incubation with CsA, as expected, greatly amplified CRC (43.9 μM with CsA under control conditions and 32.4 μM in the presence of CGP plus CsA) (K). CsA also decreased the effect of CGP (K), suggesting that the decrease in RCR promoted by NCLX inhibition by CGP is related to the induction of mPTP due to higher intramitochondrial Ca2+ accumulation.We investigated next if the increase in oxidative phosphorylation efficiency observed was dependent on mitochondrial Cachondria I. Both Rwith Na+ . Alternanditions J. In addlus CsA) K. CsA alt of CGP K, sugges2+ on oxidative phosphorylation, we performed similar experiments using α-ketoglutarate (α-KG) . Using this substrate, medium Ca2+ concentrations (22.0 ± 2.4 μM Ca2+) increased states 3 and the RCR, without changes in maximal respiration (B–H). Low Ca2+ concentrations did not present a significant effect when α-KG was used as the substrate. The beneficial effect of Ca2+ on mitochondrial oxidative phosphorylation supported by α-KG was partially attenuated by inhibition of Ca2+ release by the NCLX using CGP and totally prevented by inhibition of Ca2+ entry with RuRed (I), demonstrating again the need for Ca2+ entry into the matrix for the stimulatory effect. These results show that the specific “sweet spot” for enhanced electron transport capacity in mitochondria induced by Ca2+ varies with the substrate used. When using α-KG as substrate, the CRC was smaller than when using pyruvate plus malate (both in the absence and presence of CGP) (J): 7.2 μM under control conditions and 6.7 μM with CGP. CsA again increased all CRCs (K): 28.4 μM with CsA and 22.5 μM with CsA plus CGP.To further investigate the role of Cae (α-KG) , anotherin vitro , 16, 17. on α-KG A. Using piration , B–H. Loth RuRed I, demons of CGP) J: 7.2 μMall CRCs K: 28.4 μ2+, we performed similar experiments using the complex II substrate succinate in the presence of complex I inhibition with rotenone, to ensure no effect of contaminating or downstream NADH-reducing substrates. With succinate, mitochondria were able to retain a wider range of Ca2+ loads (A), so low, medium, and high concentrations were tested for their respiratory effects. Interestingly, using succinate, none of these Ca2+ concentrations led to a significant increase in state 3, maximal respiration, or RCR (2+ concentration (G), suggesting enhanced proton leak, possibly due to Ca2+ cycling or mPTP induction in a subpopulation of the mitochondrial suspension.To further investigate substrate-specific effects of Ca2+ loads A, so low, or RCR . A smallntration G, sugges2+ with two substrates metabolized by matrix NADH-linked dehydrogenases, we questioned whether Ca2+ influenced the formation of NAD(P)H. NAD(P)H autofluorescence was measured with pyruvate plus malate in the absence and presence of low Ca2+ concentrations (A) and with α-KG with and without medium Ca2+ concentrations (B). We saw no changes promoted by Ca2+ in NAD(P)H quantities under basal conditions, nor in the presence of any of the respiratory modulators used. We then wondered if the effects of Ca2+ could involve changes in mitochondrial membrane potentials (ΔΨm) and measured ΔΨm under our experimental conditions . ΔΨm using pyruvate plus malate as substrates is sustained (with or without Ca2+) throughout the measurements (C), with no changes between control conditions (no Ca2+) and low Ca2+ in the different respiratory states analyzed (D): basal state, after 10 min of Ca2+ addition, state 3 (after ADP), state 4 (after oligomycin), and state 3u (after CCCP). When using α-KG as the substrate, we noticed a gradual loss of ΔΨm with time (E). This could be due to mPTP formation, possibly in mitochondrial subpopulations, since we do not see significant Ca2+ efflux under these conditions. Despite this, ΔΨm quantifications were not significantly affected by Ca2+ (F), indicating changes in proton motive force cannot be mechanistically ascribed to the Ca2+ effects on electron transport.Since we measured an increase in oxidative phosphorylation efficiency by Catrations A and wittrations B. We sawurements C, with nanalyzed D: basal ith time E. This c by Ca2+ F, indica2+ enhances oxidative phosphorylation capacity and efficiency. This positive effect depends on the substrate used and requires Ca2+ entry into the mitochondrial matrix.Taken together, results using isolated mouse liver mitochondria and various substrates show that a low increase in mitochondrial Ca2+ modulate electron transport and oxidative phosphorylation in intact cells  (A–C). BAPTA-AM reduced maximal OCRs (C), but no changes were observed in basal respiration (B), which in intact cells corresponds to the respiration necessary to maintain normal levels of oxidative phosphorylation for typical cell function, in addition to the proton leak and nonmitochondrial oxygen consumption . Thus, decreasing physiological intracellular Ca2+ levels hamper maximal electron transfer capacity in intact cells but not sufficiently to decrease ATP-linked respiration under normal  conditions.To measure the respiratory effects of physiological Cad 20 μM) , A–C. BAmal OCRs C, but nopiration B, which 2+ on OCRs in intact cells required Ca2+ entry into the matrix, as we found in isolated mitochondria. Inhibition of the MCU or NCLX was promoted in AML12 cells both pharmacologically with RuRed or CGP and by silencing with specific siRNAs. We found that an acute inhibition of the transporters  did not influence basal respiration (D and E), but MCU inhibition with RuRed led to significantly decreased maximal respiration (D and F). Cells in which NCLX was inhibited by CGP had similar profiles to untreated control cells (D–F).We then questioned if the effects of physiological levels of intracellular Capiration , D and Epiration , D and Fol cells , D–F.G–J). Interestingly, we found that overall cytosolic Ca2+ levels were similar in these cells, despite the lack of these major mitochondrial Ca2+ regulatory pathways, both under basal conditions and after ER Ca2+ depletion due to thapsigargin (TG) addition (K–M). Chronic inhibition of both channels did not significantly alter basal respiration (N and O), while MCU knockdown led to a significant decrease in maximal respiration (N and P). This shows that both short- and long-term MCU inhibition impact on mitochondrial activity in intact hepatocyte-derived cells.We then checked how prolonged inhibition of those channels impacted respiration by silencing MCU or NCLX with siRNAs , G–J. Inaddition , K–M. Chpiration , N and Opiration , N and P2+ levels and study the effects on mitochondrial oxidative phosphorylation, we used two approaches: a physiological stimulus with adrenaline (ADR) and a sarco-/endoplasmic reticulum Ca2+ ATPase (SERCA) pump inhibitor, TG. The effects of ADR and other Ca2+-mobilizing hormones on mitochondrial metabolic responses have been previously investigated in the liver . Although both strategies led to increased intramitochondrial Ca2+ levels, the kinetics of this effect were very different. TG induced a much higher increase in intramitochondrial Ca2+ than ADR. In addition, ADR induced a transient and moderate increase in intramitochondrial Ca2+, while intramitochondrial Ca2+ levels remained elevated and did not return to basal levels after TG (A).We first checked for intramitochondrial CaG or ADR A. Althouafter TG A.Figure B), which was transient and rapidly went back to control levels. This ADR effect resulted in a significant increase in basal respiration (C), with no changes in maximal respiration (D). We observed a similar effect with TG: an early increase in OCRs a few minutes after TG injection (E), reflecting a significant increase in basal respiration (F) but with no changes in maximal respiration (G). As with ADR, the effect of TG was also transient, but instead of returning to basal conditions, OCR with TG started to decrease between 4 and 8 min after its injection (E). We then designed a long-term evaluation of OCRs after TG injection and found that the decreased OCR is permanently maintained and not reversed over time , showing that the modulation of respiration by Ca2+ in intact cells is dependent on pyruvate oxidation, corroborating data obtained with isolated mitochondria , as well as chronic MCU inhibition with MCU knockdown (E–H), protects mitochondrial respiration against TG-induced decreases, indicating that Ca2+ from the ER released by TG is taken up by mitochondria and affects electron transport.Having uncovered the effects of mitochondrial Caed by TG . Acute Med by TG , A–D, asnockdown , E–H, pr2+ levels in AML12 cells transfected with a specific mitochondrial Ca2+ sensor (mito-GEM-GECO1) , which was prevented by preincubation with the intracellular Ca2+ chelator BAPTA-AM (C). In line with this, BAPTA-AM also protected against the TG-induced decrease in mitochondrial respiration  . This seaddition , A–C, whBAPTA-AM C. In linpiration .Figure 92+ overload in mitochondria is known to promote opening of the mPTP  handling and energy metabolism  additions in the presence of the complex I substrates pyruvate plus malate (2+ (22.0 ± 2.4 μM) in the presence of α-KG (2+ within the same concentration range (0.1–10 μM) in rat mitochondria from heart  but have decreased RCRs when Ca2+ was further augmented. In contrast, Ca2+ addition to isolated brain mitochondria energized by pyruvate plus malate leads to reduced RCRs, in a concentration-dependent manner, with no effect on PDH activity  than NADH-linked substrates (A and 2A), thus allowing us to test more Ca2+ concentrations on respiration supported by succinate (with negative results).With the exception of some careful studies showing intracellular Cancreases , a majoruccinate A than NAbstrates A and 2A,2+ levels in the nontumor AML12 hepatocyte-derived cell line (2+ concentrations are in the 100- to 150-nM range (K−L), which is much lower than concentrations that activate oxidative phosphorylation in isolated mitochondria, but does not reflect differences in local ion concentrations, such as those in the Ca2+-rich mitochondrial–ER contact sites, where Ca2+ concentrations may be up to 10 times higher than in the bulk cytosol , without affecting mitochondrial membrane integrity, as indicated by the lack of change in oligomycin-insensitive respiration (A). Second, inhibition of mitochondrial Ca2+ uptake with ruthenium red  or by silencing the MCU (N and P) also decreased maximal electron transport capacity in intact cells, indicating that ideal respiratory chain function requires both the presence of physiological levels of cytosolic Ca2+ and its uptake by mitochondria. Our results are compatible with data showing that genetic manipulation of MCU and consequent impairment of mitochondrial Ca2+ uptake disrupts oxidative phosphorylation and lowers cellular ATP in HeLa cells  induces a moderate (with ADR) or sustained (with TG) increase in intramitochondrial Ca2+ levels, leading to an early and transient stimulation of respiratory rates, followed by a decrease to control levels (with ADR) and an even further decrease with TG  was from Addgene; Bradford was from Bio-Rad Laboratories; siRNAs against MCU (ID s103465), NCLX (ID s100747), or negative control (#4390844) were from Ambion Inc; anti-MCU (#14997S) was from Cell Signaling; anti-β-actin (#ab8226) was from Abcam; fluorescent secondary antibodies (goat anti-mouse #926-68070 and goat anti-rabbit #926-68071) were from Licor.Culture medium (Dulbecco’s modified Eagle’s medium [DMEM]/F-12) (#11320), fetal bovine serum (FBS), insulin-transferrin-selenium (ITS), CaC57BL/6NTac male mice were bred and housed in the animal facility of the Faculty of Pharmaceutical Sciences and Chemistry Institute of the University of São Paulo, devoid of murine pathogens. Animals were maintained in collective cages (maximum four/cage) at controlled temperatures (23 °C) in a 12 to 12 h light/dark cycle with free access to food/water. We used 10- to 12-week-old mice. All procedures were conducted in accordance with the Ethical Principles of Animal Manipulation and approved by the Animal Ethics Committee (CEUA) of the Chemistry Institute (IQ) of the University of Sao Paulo (USP) under protocol CEUA-IQ/USP 196/2021.g, 4 °C, 4 min), and the pellet was discharged. The supernatant was further centrifuged at 9000g, 4 °C for 10 min. The pellet was resuspended in resuspension buffer  and centrifuged again . The pellet was resuspended in 125 μl of resuspension buffer, and total protein was quantified using the Bradford method.After deep anesthesia (4% isoflurane) followed by cervical dislocation, the abdomen was dissected and the liver was removed. Mitochondria were isolated by differential centrifugation. All steps were carried out at 4 °C, over ice. Briefly, the liver was chopped into small pieces, suspended in isolation buffer , and manually homogenized using a Potter–Elvehjem tissue grinder. To remove blood from the mixture, it was centrifuged twice  containing 100 μM EGTA and different substrates  and/or inhibitors (10 μM ruthenium red or 10 μM CGP-37157) at 37 °C with continuous stirring (700 rpm). After basal respiration was measured, CaCl2 was injected at different concentrations, as indicated. After reaching a steady state (as indicated by Ca2+ uptake measurements), state 3 respiration was measured by adding 1 mM ADP, followed by addition of 1 μM oligomycin (state 4) and 0.5 μM CCCP (state 3u). RCRs were calculated as state 3/state 4.Oxygen consumption was measured using a high-resolution Oxygraph-2k (O2k) respirometer (Oroboros Instruments). Mitochondria  were incubated in 2 ml of experimental buffer , medium Ca2+ , and high Ca2+ . According to the amount of Ca2+ added and the excess EGTA present in the buffer, we calculated the amount of free extramitochondrial Ca2+ to which mitochondria were exposed: low (2.4 ± 0.6 μM), medium (22.0 ± 2.4 μM), and high (52.9 ± 2.5 μM) concentrations, from now on called simply as low, medium, or high Ca2+. Fluorescence was monitored for 20 min. Each trace was followed by three 10 μM Ca2+, three 1 mM Ca2+, and three 3 mM EGTA additions, to allow the estimation of the Calcium Green 5N experimental Kd, and maximal (Fmax) and minimal (Fmin) fluorescence, respectively. Absolute fluorescence values (F) were calibrated into [Ca2+] through the formula [Ca2+] = Kd.(F − Fmin)/(Fmax − F). To determine CRC, several additions of 10 μM Ca2+ were made until mPTP opening occurred. These traces were performed with and without 5 μM CsA, as indicated.Ca done in , as wellex = 485 nm and λem = 586 nm, using an F4500 Hitachi Fluorimeter at 37 °C with continuous stirring. After a 100-s baseline interval, CaCl2 was added to achieve a low or medium Ca2+ concentration, and fluorescence was monitored for 20 min. Fluorescence values were calibrated to millivolts as described in (+ ionophore) and sequential additions of known KCl concentrations, and fluorescence was followed. A calibration curve was constructed based on fluorescence variations and estimated ΔΨm, calculated from the Nernst equation, ΔΨm = 60 × log(K+in/K+out), considering the intramitochondrial [K+]in = 120 mM and known extramitochondrial K+ concentrations (Kout).Mitochondrial membrane potentials (ΔΨm) were determined by measuring changes in the fluorescence of safranin-O, in quenching mode. Mitochondria (600 μg protein) were incubated in 2 ml of the same experimental buffer mentioned in the previous two sections, containing 5 μM safranin-O, 100 μM EGTA, and 1 mM pyruvate + 1 mM malate or 1 mM α-KG. Fluorescence was measured at λribed in . Brieflyex = 366 nm and λem = 450 nm, using an F2500 Hitachi Fluorimeter at 37 °C with continuous stirring. After a 100-s baseline interval, a CaCl2 solution was added to achieve the target Ca2+ concentrations, as indicated, followed by sequential additions of 1 mM ADP, 1 μM oligomycin, 0.5 μM CCCP, and 1 μM rotenone + 1 μM antimycin A. Fluorescence values were calibrated to nanomoles through a calibration curve constructed in experimental buffer, in absence of mitochondria, following additions of known NADH concentrations.NAD(P)H was determined by measuring changes in the NAD(P)H autofluorescence. Mitochondria (600 μg protein) were incubated in 2 ml of the same experimental buffer mentioned in the previous three sections, containing 100 μM EGTA and different substrates, as indicated. Fluorescence was measured at λ2.The hepatocyte-derived AML-12 cell line was maintained in DMEM/F-12 medium, which contains 17.5 mM glucose, 2.5 mM glutamine, and several other amino acids, supplemented with 10% (v/v) FBS, a mixture of insulin, transferrin, and selenium , 1% antibiotics , pH 7.4, at 37 °C in a humidified atmosphere of 5% COAML-12 cells were transfected with siRNAs against MCU, NCLX, or scramble RNA as a negative control (Ambion Inc), using Lipofectamine RNAiMAX and a reverse transfection protocol. Briefly, lipofectamine and siRNAs were diluted in Opti-MEM medium and were added to the cell suspension at a final concentration of 20 nM. Cells were kept overnight at 37 °C in DMEM/F-12 containing ITS, without antibiotics. After transfection, the medium was replaced by complete medium. Forty-eight hours after transfection, cells were seeded for experiments and left for an additional 24 h.Mcu , Nclx , Hmbs , and B2m  genes. Amplification data were analyzed by the 2(−ΔΔCt) method using the mean of Hmbs and B2m Ct values as housekeeping. The housekeeping choice was based on the stability value of the gene or combination of genes calculated by the NormFinder Software , loaded onto SDS-PAGE gels, and electrotransferred to PVDF membranes. Membranes were blocked with 5% BSA in TTBS  for 1 h at room temperature before overnight incubation with MCU (1:1000) and β-actin (1:5000) primary antibodies at 4 °C. Fluorescent secondary antibodies  were incubated for 1 h at room temperature prior to fluorescence detection using a ChemiDoc Imaging System (Bio-Rad). Quantification of band densitometry was performed using the FIJI ImageJ software .6 cells in 4 ml complete medium in 60-mm cell culture dishes. After 24 h, cells were loaded with 5 μM Fura-2 AM for 1 h at 37 °C. After the loading period, cells were trypsinized and suspended in Krebs-Henseleit buffer without CaCl2 . About 1 × 106 cells  were checked for basal cytosolic Ca2+ levels and after ER Ca2+ depletion with TG using an F4500 Hitachi Fluorimeter at 37 °C with continuous stirring. Fluorescence was measured at excitation 340/380 nm and emission 505 nm. Each trace was followed by 4% Triton X-100 (50 μl) and 60 mM EGTA (150 μl) additions, to allow the estimation of maximal (Rmax) and minimal (Rmin) fluorescence ratio, respectively. Intracellular Ca2+ concentrations, [Ca2+]i, were calculated as described in (2+]i (nM) = Kd × [(R −Rmin)/(Rmax − R)] × Sfb, where the Kd for Ca2+ binding to Fura-2 at 37 °C = 225 nM and Sfb is the ratio of baseline fluorescence at 380 nm.After MCU or NCLX silencing, AML-12 cells were seeded at 1.5 × 10ribed in  through 2+ was measured in AML-12 cells expressing the mito-GEM-GECO1 sensor. Cells were seeded at 50,000 cells in 500 μl complete medium on 30-mm cell culture glass dishes, previously coated with poly-D-Lysine. After 24 h, cells were transfected overnight at 37 °C in DMEM/F-12 without antibiotics containing Lipofectamine 2000 in the presence of a plasmid encoding a mitochondrial-targeted sequence of the Ca2+ sensor GEM-GECO1 , as indicated. After incubation, plates were placed in the equipment and OCRs were measured under basal conditions, followed by different injections: a) TG  or same volume of dimethyl sulfoxide in control wells or ADR ; b) oligomycin ; c) CCCP ; d) antimycin and rotenone . Measurements were mostly done using the standard timepoints of the Seahorse equipment (3 min mixing + 2 min waiting + 3 min of measurements), except for A and D, where time points were 20 s mixing + 3 min of measurements (zero time waiting), to uncover the short ADR effects. Cell-free wells were incubated with the same medium for background correction, calculated by subtracting changes observed from the experiments in the presence of cells. OCR values obtained were normalized per amount of total protein in each well. For that, at the end of each experiment, the medium was removed, cells were PBS-washed and lysed with RIPA buffer, and total protein was quantified using the BCA Pierce protocol. Values were then normalized again by dividing the OCRs obtained by the average OCRs from the controls of each experiment.Cells were seeded at 30,000 cells per well in 100 μl DMEM/F-12 medium  on Agilent Seahorse XF24 cell culture microplates and allowed to adhere overnight. Oxygen consumption was measured using a Seahorse Extracellular Flux Analyzer (Agilent Technologies). For that, cells were washed thrice with 500 μl DMEM/F-12 containing 1% P/S and 5 mM Hepes. Media did not contain bicarbonate or FBS. Cells were kept for 1 h in a humidified incubator at 37 °C without CO6 cells were incubated in 2 ml of the same medium used in Seahorse experiments in the absence or presence of 2 μM TG and/or inhibitors, at 37 °C with continuous stirring (400 rpm). After basal respiration, 1 μM oligomycin was injected, followed by 5 μM CCCP, to calculate maximal respiration.Different substrate oxidation effects on respiration were evaluated using inhibitors for the three primary substrates that fuel mitochondria: glucose/pyruvate , glutamine , and long-chain fatty acids . Oxygen consumption was measured in AML-12 cells in suspension using a high-resolution O2k respirometer (Oroboros Instruments). About 1 × 10t test was used to compare differences between treated and untreated conditions, one-way ANOVA followed by Šidak was used to compare multiple conditions with one independent variable, and two-way ANOVA followed by Tukey was used to compare multiple conditions with two independent variables, with confidence levels set to p < 0.05. Data shown in scatter graphs are individual biological replicates, averages, and standard deviations.Statistical analysis was carried out using GraphPad Prism 8 Software for at least three independent experiments . Student’s All data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


In recent years, the Ca2+ mediated calcium signaling pathway has been studied systematically in various mammals and fungi, indicating that the pathway is conserved among organisms. The pathway consists mainly of complex Ca2+ channel proteins, calcium pumps, Ca2+ transporters and many related proteins. Crz1, a transcription factor downstream of the calcium signaling pathway, participates in regulating cell survival, ion homeostasis, infection structure development, cell wall integrity and virulence. This review briefly summarizes the Ca2+ mediated calcium signaling pathway and regulatory roles in plant pathogenic fungi. Based on discussing the structure and localization of transcription factor Crz1, we focus on the regulatory role of Crz1 on growth and development, stress response, pathogenicity of pathogenic fungi and its regulatory mechanisms. Furthermore, we explore the cross-talk between Crz1 and other signaling pathways. Combined with the important role and pathogenic mechanism of Crz1 in fungi, the new strategies in which Crz1 may be used as a target to explore disease control in practice are also discussed.Ca Unlike other second messengers, Ca2+ does not need to be synthesized but instead controls intracellular Ca2+ content through a series of complex regulatory mechanisms when responding to external signals. The CaN-Crz1 signaling cascade in fungal cells can be activated by different external stimuli, such as high temperature, low temperature, hypertonicity, alkalinity, oxidative stress, ethanol stress, light sources, antifungal drugs and others. The signal transduction mediated by Ca2+ can cause an instantaneous increase in intracellular Ca2+, which is generally considered to be the switch to turn on the signaling pathway [2+ content is caused by the entry of extracellular Ca2+ into cells through Ca2+ channel proteins Mid1 and Cch1 on the plasma membrane, or the release of Ca2+ from the intracellular calcium pool [2+ combines with calmodulin (CaM) to form a Ca2+/CaM complex and then activates calcineurin (CaN), which further dephosphorylates transcription factor Crz1 and allows it into the nucleus to regulate the expression of target genes [2+/calmodulin/Crz1 signaling pathway, also known as the CCS  pathway [2+ sensor proteins of the calcium pathway system in fungi, pointing out that the calcium homeostasis system is involved in a variety of life processes, such as cell growth, conidia production, stress response and maintenance of normal organelle function. We highlight recent findings on how transcription factor Crz1 regulates growth and development, stress responses, pathogenicity of pathogenic fungi and its regulatory mechanisms based on discussing the structure and localization of Crz1. In addition, cross-talk between Crz1 and other signaling pathways and how recent advances in our understanding of CaN-Crz1 signaling cascade might be used in practice to explore new strategies for disease control are also discussed.Ca pathway ,4,5,6,7.ium pool ,9,10. Inet genes . The pat pathway . At pres pathway ,16,17,18 pathway ,22,23,242+ in fungal cells is in the low range of 50 to 100 nM [2+ levels in cells is controlled by a complex Ca2+ homeostasis regulatory system  and low-affinity Ca2+ transport system (LACS). The HACS, composed of Mid1 and Cch1, is responsible for Ca2+ uptake at low calcium concentrations (about 100 nM) [2+-related protein in the Ca2+/calmodulin/calcineurin/Crz1 signaling pathway, plays a critical role in regulating a variety of physiological activities activated by the calcium signaling system in fungal cells [Arabidopsis thaliana, can be activated by hyperosmotic shock and, therefore, was named calcium-permeable stress-gated cation channel 1 (CSC1) [P. chrysogenum and CefP of A. chrysogenum [2+ from the vacuole into the cytoplasm [Two pathways have been reported to participate in extracellular Ca 100 nM) ,30,31. R 100 nM) ,33. Cch1al cells ,37,38,39al cells ,30,40. Tal cells ,42,43,44al cells . The TRPal cells ,47,48,49ysogenum . The Yvcytoplasm ,52. FLC ytoplasm . Some st2+ from the cytoplasm to various organelles against the concentration gradient. For fungal cells, vacuoles rather than endoplasmic reticulum are the most important calcium pools, where the concentration of Ca2+ is about 104 times that of cytoplasmic [2+ storage is maintained by the action of two transporter proteins, Ca2+-ATPase Pmc1 and Ca2+/H+ exchanger Vcx1 [2+ ions, thus allowing S. cerevisiae to grow in high concentrations of either calcium or manganese ions [2+-ATPase (SPCA) family, which mediates the transport of Ca2+ and Mn2+ in Golgi under normal physiological conditions [There are many kinds of calcium pools in fungal cells, such as endoplasmic reticulum, Golgi apparatus and vacuoles. Different calcium pumps are distributed in these calcium pools, and are responsible for transporting Caoplasmic ,55. Thisger Vcx1 ,58,59,60ger Vcx1 ,62,63. Wese ions . Pmr1 . Interestingly, exogenous Mg2+ addition could restore the growth phenotype at pH 9, but the ΔBcCrz1 growth defect phenotype did not recover at pH 3 [Crz1 is essential for tolerance to high pH conditions in yeast. Upon stimulation of alkaline conditions, Cand PHO12 ,120. The at pH 3 .Crz1 mutants in P. digitatum, M. oryzae and B. cinerea were seriously damaged in the medium containing cell wall inhibitors [ΔVpCrz1 was significantly increased on CM agar medium containing SDS, CR or CFW, which was inconsistent with previous reports. It was suggested that VpCrz1 acted as a negative regulator of cell wall stress in V. pyri [Crz1 mutant demonstrated resistance to SDS in human pathogenic fungus Candida lusitaniae, indicating that Crz1 negatively regulated cell membrane integrity, while Crz1 was found to respond to SDS by an unknown mechanism independent of CaN [The growth of hibitors ,90,102.  V. pyri . Similart of CaN . S. cerevisiae increases its sensitivity to azole drugs, while its overexpression reduces the sensitivity [P. digitorum as well as ΔPdCrz1 reduced imidazole and difenoconazole tolerance [C. neoformans, Crz1 homologous phospholipid binding protein Cts1 was identified as a CaN substrate for high-temperature stress [ΔCgCrz1 in C. glabrata could not grow as normally as the WT at 40 °C [Crz1 demonstrated poor adaptation to ethanol stress, while the multi-copy plasmid of Crz1 improved the tolerance to ethanol stress. Therefore, Crz1 was crucial for the survival of yeast cells under ethanol-induced stress [C. neoformans that Crz1 is involved in cell survival, biofilm formation and fluconazole sensitivity in the hypoxic environment [In addition, the involvement of Crz1 in fungal stress resistance was also reflected in the tolerance of antifungal drugs, temperature and ethanol. It has been reported that the damage of Crz1 in sitivity . Similarolerance . In C. ne stress . The ΔCgat 40 °C . Ethanold stress . It has ironment .S. cerevisiae, the core consensus site for Crz1 binding is 5′-GNGGCKCA-3′ [Trichoderma reesei was identified as 5′-GDGGCKBNB-3′ [2+. PMC and PMR belong to the P-type ATPase superfamily, which can obtain energy by hydrolyzing ATP to drive Ca2+ transport from the cytoplasm to the vacuole and the Golgi, respectively, to maintain intracellular calcium homeostasis [PMC and PMR genes is significantly induced in response to Ca2+, but the expression levels are not highly activated in the Crz1 mutants [2+ from the cytoplasm to various organelles, resulting in a disruption of calcium homeostasis, which may account for the sensitivity of Crz1 mutants to Ca2+. ENA1, ENA2, and ENA3 belong to the encoding plasma membrane Na+/Li+-ATPase, which are necessary for yeast survival under high Na+ and Li+ concentrations, and their expression is also induced by CaN in a Crz1-dependent manner [MEP1, ENB1, PHO84, PHO89 and KHA1 are also regulated by CaN-Crz1 pathway [β-1,3 glucan synthase (FKS) and the chitin synthase (CHS) are essential for maintaining cell wall integrity. In the Crz1 mutant, both FKS and CHS expression are disrupted [CRH1, RHO1, SCW10 and KRE6 are also regulated by the CaN-Crz1 pathway [P. oxalicum, an industrial fungus, Crz1 plays a role in cellulase synthesis by regulating the expression of cellulose decomposition genes such as cbh1, eg1 and eg2 [SUR1, CSG2, YSR3, ERG26, HES1 and PLB3, as well as genes involved in vesicular transport such as GYP7, YPT53, YIP3, PEP12, RVS161, SHE4, CVT17, CVT19 and VPS36, all of which are regulated by Crz1, thus enables cells to maintain normal membrane function and complete the process of substance delivery to the cell surface [C. neoformans, Crz1 exhibits a specific CaN-independent response to different environmental stress stimuli [C. dubliniensis, Crz1 regulates haptotropic (surface-sensing) responses independently of CaN [The zinc finger domain of Crz1 specifically binds to the 24 bp CDREs sequence to initiate target gene expression ,124. In GCKCA-3′ , and theCKBNB-3′ . Therefoeostasis ,113. In  mutants ,91,102. t manner ,126. In  pathway . Under eisrupted ,92,112.  pathway . In P. o and eg2 . Express surface . However stimuli ,128, Fury of CaN .CHS and FKS biosynthesis [At present, it has been found that Crz1, a downstream transcription factor of the calcium signaling pathway, is not only related to calcium signaling but also participates in the transcriptional regulation of other signaling pathways. The cell wall integrity (CWI) pathway, one of the MAPK cascades pathways, maintains cell wall integrity by mediating cell wall biosynthesis. Since cell wall integrity is critical for cells to cope with environmental stress, CWI pathways need to cross-talk with other proteins or pathways to enhance their transduction ability ,131. Numynthesis ,132,133.Sho1 sensor and the other by a system consisting of Sln1, Ypd1 and Ssk1 [Sln1 branch. In addition, the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway is also antagonistic to the CaN-Crz1 signaling pathway. It was found that Crz1 is a substrate for PKA, which is functionally opposite to the CaN signaling pathway, and PKA can directly phosphorylate Crz1 to inhibit its nuclear localization and activity [The high-osmolarity glycerol (HOG) pathway is used to regulate various stress genes for osmotic protection, and activation of this pathway is regulated by two upstream branches, one mediated by the and Ssk1 ,137,138.and Ssk1 ,102,116.and Ssk1  found thactivity .S. cerevisiae, Neurospora crassa and mammals, it has been shown that external signals are sensed by G protein-coupled receptors (GPCRs) [2+, thereby activating calcium signaling pathway [GPR1 or GPA2 genes, the calcium influx induced by addition of high glucose was inhibited, which suggests the physiological process requires the Gpr1p/ Gpa2p receptor/G protein-coupled (GPCR) complex [In  (GPCRs) ,142. Aft (GPCRs) . Among t pathway ,145,146. pathway ,148. Fur complex ,150.S. cerevisiae, DNA microarray data indicated that a total of 150 genes responded to the alkaline pH environment, but the expression of many alkali-induced genes was inhibited in the CaN or Crz1 mutants, suggesting that calcium signaling is involved in the alkaline stress response [C. albicans to the alkaline environment [C. albicans activated the calcium influx system in response to alkaline stress, and both Rim101 and Crz1 were involved in the activation of PHO89 promoter induced by alkaline stress, indicating that Rim101 and Crz1 signaling pathways had potential chelating effects in C. albicans response to alkaline stress. In addition, the interaction between CaN-Crz1 and heat shock proteins (Haps) is involved in response to different environmental stress conditions [C. albicans [A. fumigatus, the MAPK, Hsp90, and calcineurin signaling pathways are linked and play a role in drug resistance and development [In response . The Rimironment . Wang etironment  confirmenditions . Hsp90 palbicans . In A. felopment . These d2+ concentration changes and then activating expressions of target genes. As an important transcription factor downstream of the calcium signaling pathway, Crz1 is highly conserved in fungi and plays a critical role in growth, development, tolerance to stress conditions and pathogenicity. Although our insight into Crz1 biological function has recently advanced with unprecedented speed, there are still some open research problems that urgently need to be addressed: (1) the specific molecular mechanism of Crz1 in transcriptional regulation of target genes in calcium homeostasis system still needs to be further elucidated, (2) It is necessary to further carry out genetic and biochemical analysis experiments combined with transcriptome sequencing technology to understand the metabolic pathway regulated by the transcription factor Crz1 in fungi, (3) new, environmentally safe, species-specific strategies for disease control, such as RNA interference (RNAi) technology, should be explored based on clarifying its regulatory mechanism of Crz1.In response to complex environmental stimuli, fungi regulate multiple cellular metabolic processes by sensing intracellular Ca


Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca2+ release-activated Ca2+ (CRAC) current and, in association with TRPC1, the store-operated Ca2+ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca2+ channel (ARC/LRC), store-independent Ca2+ influx activated by the secretory pathway Ca2+-ATPase (SPCA2) and the small conductance Ca2+-activated K+ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1β, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1β, the implications of the Ca2+ signals triggered by each variant, and their downstream modulatory effect within the cell.Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP Ca2+ is also heavily implicated at the cellular level, where it mediates gene expression, secretion, cell proliferation, and apoptosis. Due to its pleiotropic effects, altered intracellular Ca2+ homeostasis induces pathological conditions i), while only NFAT5 is sensitive to osmotic stress i promoted by opened PM Ca2+-channels activate the Ca2+-dependent calmodulin (CaM), which contains EF-hand motifs to sense [Ca2+]i, among other proteins. As a result, heavily phosphorylated resting NFAT proteins located in the cytoplasm become dephosphorylated by calcineurin, starting the calcineurin-NFAT pathway i microdomains within ER-PM junctions. The Orai1α region located within the amino acid residues 39–59 (AKAR region) interacts directly or indirectly with the scaffolding protein A-kinase anchoring protein 79 (AKAP79) [2+]i microdomains during SOCE.Since the identification of Orai1α and Orai1β variants has only recently been reported, previous studies mostly described Orai1α-NFAT interactions. The role of the Orai1α subunit in calmodulin and NFAT1 activation is not only restricted to the generation of local high i themselves are not sufficient and that it is mandatory for the formation of Orai1/AKAP-79/calmodulin/NFAT1 complexes to drive NFAT1 translocation.The Orai1α-AKAP79 interaction seems to be specific since the AKAR region is absent in Orai2 or Orai3 subunits, and they are less able to interact with AKAP79 . Interesr extent . In summr extent . Given t2+]i oscillatory frequencies modulate alternative NFAT1/NFAT4 patterns of translocation, which were previously observed using different agonist concentrations i microdomain formed near the channel, and a slow inactivation (SCDI) occurring over seconds triggered by overall rises in [Ca2+]i [2+]C rises, but this work also proposes that FCDI modulates the frequency of [Ca2+]C oscillations. The lesser sensitivity to CDI exhibited by Orai1β has been attributed to the absence of Ser34, which, in Orai1α, is phosphorylated by protein kinase A (PKA), leading to CDI initiation. The lack of CDI entails the promotion of different oscillatory [Ca2+]i frequencies by Orai1β than those generated by Orai1α. Thus, Orai1α leads to lower frequency [Ca2+]i oscillations than Orai1β, which might underlie the activation of NFAT isoforms at different degrees depending on the agonist concentration i are sufficient to activate NFATs in contrast to Kar and coworkers and that their oscillatory nature modulates NFATs function. Regarding other NFAT isoforms, there are no studies about the possible interactions between Orai1β and NFAT2 or NFAT3. Orai1α activity triggers NFAT2 translocation i . CDI wasntration ,117. Botd manner ,125,126.d manner ,16, exced manner . Althouglocation . Whether2+ [2+ and exhibits a low affinity for this ion. This isoform is able to form complexes with Orai1 channels [Mammalian adenylyl cyclases comprise nine (AC1–9) transmembrane enzymes and one cytosolic isoform (AC10) that catalyze the synthesis of cyclic AMP (cAMP) from ATP . AC isof2+ ,136,137.2+ . PKA, fo2+ . Target 2+ ,136,137.channels ,140, botchannels , and reschannels  directlychannels . The Orachannels . Thus, tc), that activates the complexed PKA, which, in turn, phosphorylates Orai1α at Ser34. The consequence of Ser34 phosphorylation is the activation of CDI since the mutation of this phosphorylation site (the exchange of a Ser34 by Arg34) abrogates CDI [2+ oscillations, establishing different NFATs translocation patterns [2+ signaling. It is known that cAMP modulate Ca2+ homeostasis . In sumIt has been reported that embryonic kidney HEK293 cells do not express AC8, but they can recruit AKAP79/PKA and phosphodiesterase 4 (PDE4) complexes nearby activated Orai1 channels in the absence of cAMP microdomains suggesting that there should be an alternative source of cAMP able to trigger PKA activity close to Orai1 .2+-signaling machinery in cancer [In cancer cells, the cAMP/PKA pathway promotes proliferation, migration, and invasive properties, as well as other aspects of their physiology . Moreoven cancer . Indeed,n cancer . In thesn cancer . Since On cancer ,152. Finn cancer ,158,159,n cancer ,161. Oven cancer . Therefon cancer .Icrac. In addition, Orai1 promotes store-dependent activation of the Isoc current involving TRPC1. Two variants of Orai1 are expressed at the protein level in mammalian cells. These variants, Orai1α and Orai1β, generated by alternative translation initiation, differ in the N-terminal 63 amino acids lacking in the short form, Orai1β. While these forms have been reported to support Icrac with similar efficiencies, their participation in Isoc depends on the cell type investigated, and Iarc activation is unique to Orai1α. Orai1 variants also differ in their sensitivity to fast Ca2+-dependent inactivation, which explains the different Ca2+ signals mediated by these forms when expressed individually. The absence of the N-terminal 63 amino acids in Orai1β limits the interaction with different partners, including AC8, AKAP79, or caveolin, as well as serine phosphorylation at residues 27, 31 and 34, present in Orai1α. These differences have been proposed to underlie the distinct channel inactivation properties, mobility profiles, and NFAT activation mechanisms. The presence of Orai1α and Oraiβ in a given cell type provides an additional tool to generate Ca2+ signals appropriate to the intensity of agonist stimulation. Orai1 is the key pore-forming protein of the CRAC channels, which mediate the prototypical and best-characterized store-operated current


However, the effects of long-term Ca2+ deficiency in orphan crops such as Tef [(Eragrostis tef) (Zucc.) Trotter], which accumulate high levels of Ca in the grains, remained unknown. Tef is a staple crop for nearly 70 million people in East Africa, particularly in Ethiopia and Eritrea. It is one of the most nutrient-dense grains, and is also more resistant to marginal soils and climatic conditions than main cereals like corn, wheat, and rice. In this study, tef plants were grown in a hydroponic solution containing optimum (1 mM) or low (0.01 mM) Ca2+, and plant growth parameters and whole-genome transcriptome were analyzed. Ca+2-deficient plants exhibited leaf necrosis, leaf curling, and growth stunting symptoms. Ca2+ deficiency significantly decreased root and shoot Ca, potassium (K), and copper content in both root and shoots. At the same time, it greatly increased root iron (Fe) content, suggesting the role of Ca2+ in the uptake and/or translocation of these minerals. Transcriptomic analysis using RNA-seq revealed that members of Ca2+ channels, including the cyclic nucleotide-gated channels and glutamate receptor-like channels, Ca2+-transporters, Ca2+-binding proteins and Ca2+-dependent protein kinases were differentially regulated by Ca+2 treatment. Moreover, several Fe/metal transporters, including members of vacuolar Fe transporters, yellow stripe-like, natural resistance-associated macrophage protein, and oligo-peptide transporters, were differentially regulated between shoot and root in response to Ca2+ treatment. Taken together, our findings suggest that Ca2+ deficiency affects plant growth and mineral accumulation by regulating the transcriptomes of several transporters and signaling genes.Calcium (Ca It is a structural component of the cell wall and membranes1 where it binds to an acidic group of the membrane lipids and crosslinks pectin2. Ca+2 also acts as a ubiquitous second messenger in environmental stress signaling3. Several environmental stimuli lead to the release of stored Ca2+, which binds to calmodulin. The Ca2+-calmodulin complex recruits several kinases and phosphatases that regulate transcription-dependent cellular processes4. Plant cells also possess other Ca2+-regulated proteins, including Ca+2 dependent protein kinase (CDPKs), calcineurin B-like (CBL) proteins, CBL interacting protein kinases (CIPKs), CDPK-related kinases (CRKs), etc., which have been well characterized6.Calcium (Ca2+ is taken up from the soil solution by roots and translocated to the shoot via the xylem4. Ca+2 enters plant cells through Ca2+ channels4. Plant glutamate receptor-like (GLR) channels, which are analogs to the mammalian ionotropic glutamate receptors (iGluRs), are implicated in Ca+2 sensing, signaling, and long-distance root to shoot Ca2+ wave propagation during wounding8. Several members of the AtGLRs have been characterized10. The role of AtGLR3.1 in C2+ sensing in Arabidopsis shoot has been reported11. Another class of Ca2+ channels known as cyclic nucleotide-gated channels (CNGCs) has also been implicated in Ca2+ transport13. Cellular Ca2+ concentration is tightly regulated to allow signaling role, control Ca2+ translocation to the shoot and prevent cation toxicity4. Antiporters, uniporters and Ca2+-ATPases localized in all major plant cellular membranes and rapidly expel Ca2+ from the cytosol. The Arabidopsis genome encodes five different Ca2+ efflux systems, called autoinhibited Ca2+-ATPases (ACAs), ER-type Ca2+ATPases (ECAs), and P1-ATPases (HMA1), the mitochondrial calcium uniporter complex (MCUC) and Ca2+exchangers (CAX)15. Similarly, Ca2+/Cation-exchangers have also been reported to play a role in cation homeostasis in plants17.In plants, Ca2+ deficiency is not commonly observed in most soils due to its relative abundance and regular liming practice18. However, developing tissues may experience Ca2+ deficiency due to lack of Ca2+ remobilization from older tissues, under conditions of low transpiration, in sandy soils untreated with lime (CaCO3 or Ca (OH)2), and inhibition of Ca2+ uptake due to competition from other cations18. Plants exposed to Ca2+ deficiency may experience necrosis of the meristems, shoot and root growth stunting4 and leaf carling. Ca2+-deficiency increases the level of reactive oxygen species, causing oxidative stress in high Ca-demanding plant species such as cabbage20 and broccoli21, Ca+2 treatment has been shown to enhance the antioxidant system and decrease oxidative stress in plants grown under high temperature22, drought23, salinity24 and heavy metals25.Ca+-deficiency26. The CBL1/9—CIPK23 has been implicated in the regulation of K+27 and Fe homeostasis (41). Similarly, CBL1/CBL9-CIPK11 interaction has been involved in regulating the bHLH transcription factor FIT, a crucial regulator of Arabidopsis Fe acquisition28. Fe deprivation has been linked to alteration of Ca2+ signatures evoked by exogenous ATP29, suggesting crosstalk between Ca2+ and Fe homeostasis. However, detailed studies on the interconnection between Ca2+ and Fe nutrition are missing. Recently, studies of factors modulating Ca2+-iron homeostasis are emerging30.Calcium-sensing networks, including the calcineurin B-like proteins (CBL)-CBL-interacting protein kinase (CIPK), has been implicated in plant abiotic stresses, including K32 and some other countries including the United Sates where it is grown primarily for animal feed33. Recently, tef has been getting increased attention in many laboratories due to its health benefits and potential roles in sustainable agriculture34. Its mineral nutrient composition has been studied35. Tef is one of the orphan crops reported to have higher mineral contents; mainly, the Ca2+ content of tef is higher than other crops such as sorghum, barley, wheat, and rice36. The Ca2+ content in the plant varies from 0.1 to 5% of the shoot dry weight4 and the average seed Ca2+ content may vary between 981 and 1811 mg/Kg in tef as compared to 33 mg/Kg in maize, 153 mg/Kg in rice and 533 mg/Kg in wheat35.Tef is an important orphan crop in the Horn of Africa2+ contents were higher than in other major crops such as wheat, rice, and maize37. Low Ca2+ consumption in the diet has been related to several disorders in humans, many of which can have significant health repercussions over time. Because most staple food grains are low in Ca2+, tef, which is an orphan crop with high Ca2+ content, has enormous promise as a nutritional security crop. However, orphan crops’ physiological and transcriptomic response to Ca2+ nutrition has not been studied before.Similarly, in millets such as finger millet, the Ca2+ deficiency and analyzed the physiological and transcriptomic responses. Our findings revealed that prolonged low Ca2+ treatment decreased shoot and root biomass, Ca, K, and copper (Cu) content while significantly suppressing Fe translocation from the roots to the shoot. Transcriptomic analysis using RNA-seq revealed that some members of Ca2+ transporters, including CNGC, GLR and CaUP, signaling genes such as CBP and CDPK and genes implicated in Fe homeostasis such as YSL, OPT, VIT and NRAMP are differentially regulated by Ca2+ treatment or between roots and shoots.This study exposed tef plants to long-term Ca2+ deficiency in tef, plants were grown in a modified Hoagland’s hydroponic solution supplemented with predetermined low (0.01 mM) or optimum (1 mM) Ca2+ for one month, and the plants were analyzed. As shown in Fig. 2+ exhibited typical Ca2+ deficiency symptoms, including leaf tip necrosis and growth stunting, compared to plants grown at optimum Ca2+ level . Plants grown in low Ca2+ media accumulated 0.5 g/Kg Ca+2 in the shoot (LCS) as compared to plants grown at optimum Ca+2 level (ConS), which accumulated 3 g/Kg Ca+2  and optimum Ca+2 (ConR). At the same time, there was a small but statistically significant difference between LCS and ConR , while in shoots, a small but significant decrease in Cu content was observed (0.01 g/Kg in ConS to 0.01 g/kg in LCS) . Complementary DNA libraries were generated from four independent biological replicates and sequenced using the paired-end sequenced using Illumina Hiseq 4000. A total of 476.5 million raw reads with an average of 30 million reads were obtained for each sample The total clean reads of the 16 libraries were aligned to the tef draft genome sequence38. On average 85.7% of the reads were uniquely aligned to the reference genome .To determine the integrity of RNA samples, we performed principal component analysis (PCA). The PCA shows no mark variation among most RNA samples from the same Cance Fig.  and mine2+ conditions. A large number of genes (4.654) were upregulated in roots and shoots of low Ca2+ relative to control, of which 366 and 4009 genes were uniquely upregulated in LCR and LCS relative to ConR and ConS, respectively. A total of 279 genes were commonly upregulated in LCR and LCS. We also detected 1279 DEGs that were downregulated in LCR (179) and LCS (1132) compared to ConR and ConS, respectively. A total of 32 genes were commonly downregulated in LCR and LCS as compared to ConR and ConS, respectively. Ca2+ deficiency also induced significant transcriptomic changes between roots and shoots. A total of 7045 DEGs were upregulated in roots, of which 4833 DEGs were upregulated in LCR relative to LCS, 6670 DEGs were upregulated in ConR relative to ConS, and 4458 DEGs were commonly upregulated in both LCR and ConR. Similarly, 7430 DEGs were downregulated in roots, of which 6,192 DEGs were downregulated in LCR relative to LCS, 6834 DEGs were downregulated in ConR relative to ConS, and 5596 DEGs were commonly downregulated in both LCR and ConR relative to LCS and ConS, respectively. Taken together, the number of transcripts that were upregulated in response to Ca2+ deficiency in both roots and shoots were higher than those whose transcript abundance was downregulated. Moreover, the number of DEGs that were upregulated in ConR versus ConS were higher than those DEGs that were upregulated in LCR versus LCS  enrichment analysis using the AgriGo database to determine the potential function of DEGs identified in our analysis. In the GO analysis, the DEGs were grouped into molecular function, cellular components, and biological process categories Fig. A for the2+ transport in plants. This study identified transcriptomic changes in some families of Ca2+ transporters, including GLRs, CNGCs, Ca2+ uniporters, cations/Ca2+ exchangers, and Ca2+-transporting ATPases. Our analysis identified Ca2+ transporters including Ca2+ uniporters, cation/Ca2+ exchangers and Ca2+ transporting ATPase , CNGC2 (R2 = 9626) and CaE1 (R2 = 9626), while a positive but weak correlation was observed for CNGC17 (R2 = 0.1689). These findings validate the RNA-seq data.To the RNA-seq data, we performed quantitative RT-PCR (qPCR) to analyze the expression of a few DEGs, including +2 in grains than other cereals43. However, the effect of prolonged Ca2+ deficiency on their physiology and transcriptome remained unknown. In the present study, we examined the response of tef to prolonger Ca2+ deficiency based on agronomic, physiological, and transcriptome changes in plants grown in hydroponics solution under optimum and Ca2+ deficient conditions.The Chloridoideae subfamily of Poaceae, including tef and finger millet, accumulate higher levels of Ca2+ deficient conditions showed typical deficiency symptoms in shoots, including leaf curling, tip necrosis, and growth stunting 1. Several Ca2+-permeable channels have been implicated in the uptake of Ca2+ from the soil solution to plant roots48. These include the GLRs, CNGCs, two-pore channels (TPCs), mechanosensitive-like channels (MSLs) etc.49. Ca2+ pumps are involved in maintaining homeostasis by controlling Ca2+ efflux from the cytosol to organelles and/or to the apoplast50 . In our dataset, we identified several DEGs including Ca2+ uniporters (CaUPs), cation/Ca2+ exchangers (CaE), Ca2+-transporting ATPases (CaTA) etc. 56.When plants undergo certain environmental stresses, Ca2+ transport against its concentration gradient and enhance the influx of monovalent cations 57. For example, overexpression of the apple cation/Ca2+ exchanger MdCCX1 in transgenic Arabidopsis and apple root cells has been shown to decrease Na+ accumulation and ROS levels and increase salt tolerance in transgenic cells56. In this study, Ca2+ deficiency decreased Ca and K content in roots. Therefore, upregulation of CaE1 in LCR and LCS could be a mechanism by which roots enhance K+ or Ca2+ acquisition from the external solution or facilitate their distribution between tissues. Transcripts of CaTA9-1, CaTA5-1, CATA9, and CaUP6 are downregulated by Ca2+ deficiency in the shoots. The CaTA5 was the only gene among the transporters that was downregulated by Ca2+ deficiency in roots. CaTA5 is a homolog of the plasma membrane Ca2+-transporting ATPases (PMCAs), which are high-affinity Ca2+-pumps that export Ca2+ ions from the cytosol into the extracellular solution58. CaTA5 may be a functional Ca2+-pump, and its downregulation under Ca2+ deficiency could be a strategy to suppress the cytosolic Ca2+ export to the extracellular solution. The autoinhibited Ca2+-ATPase ACA13 has been implicated in successful pollination in the Brassicaceae by exporting pollen to compatible pollen tubes59.The CaCA superfamily has been reported to facilitate Ca2+ and control plants. In Arabidopsis, Oligopeptide Transporter 1(AtOPT1) is a major nitrate/oligopeptide transporter family60. The Arabidopsis OPT3 is a phloem-specific iron transporter61 and its rice counterpart, OsOPT7, has been implicated in Fe loading into the phloem, the long-distance Fe transport, and loading to seeds63. OPT4 is involved in oligopeptide transport64. Furthermore, Arabidopsis Oligopeptide Transporter 6 (AtOPT6) was reported to transport glutathione65 and a possible connection between prolonged Ca2+ deficiency, redox status, and increased iron transport is a likely scenario as suggested in a recent review28. In this study, transcript levels of several transporters such as OPT1, EtNRAMP5, EtVIT5 and EtYSL2 were upregulated by several folds  across the root cell membrane, while the rice OsYSL2 is implicated in the long-distance transport of Fe associated with a PS precursor, nicotianamine (NA)68. The role of YSL2 in Fe uptake and distribution has been studied previously in maize69 and Arabidopsis71. The NRAMPs such as AtNRAMP3 and AtNRAMP4 function in transporting Fe out of vacuoles under Fe-limited conditions, while members of the VIT family mediate Fe transport into vacuoles72. For example, overexpression of the wheat VIT2 under the control of an endosperm-specific promoter increases Fe content in white flour fractions of wheat73. Some of these transporters are not specific to Fe but also transport other minerals, including Zn, Cd, Cu, and Mn74. Taken together, differential regulation of several mineral transporters in response to Ca nutrition suggests the involvement of Ca2+-signaling in the homeostasis of these minerals. The decrease in root to shoot Fe translocation in this study due to Ca2+ deficiency , promoting Fe uptake in Arabidopsis. Ca2+ promotes the interaction of C2-Domain Protein Enhanced Bending1(EHB1) and the root Fe-regulated transporter IRT1, inhibiting Fe acquisition in Arabidopsis76. However, further studies are needed to understand the crosstalk between Fe and Ca nutrition in Ca and Fe-rich cereals such as tef and finger millet.This study also identified various Fe/metal transporters, including OPTs, VITs, NRAMPs, and YSLs. All these transporters proteins were differentially expressed in the roots and shoots of low Ca2+-transporting channels were differentially expressed by Ca treatment. Our findings showed that transcripts of the CNGC, including CNGC2, CNGC4, and CNGC4-2, were more abundant in the shoots regardless of the Ca treatment 9, it has been shown that GLR1 encodes a non-selective ion channel that is permeable to Ca2+. Upregulation of GLR2.8 and GLR2.9 in roots compared to shoots in this study may suggest their involvement in Ca2+ uptake in the roots. Upregulation of GLR2.8 in LCR compared to ConR may suggest its role in Ca2+ acquisition under Ca2+-deficient conditions. However, the GLRs which were upregulated in the shoots under both high and low Ca2+, such as GLR3.4-1, GLR2.4, and GLR3.1, may play a role in Ca2+ distribution in the shoots, which remains to be studied in the future.Our study also revealed that several Caent Fig. B. This m2+ deficiency severely inhibits the growth of tef plants by disturbing the homeostasis of some minerals, including Ca, K, Fe, Mn, and Cu. Furthermore, our transcriptomic analysis revealed various genes, including those implicated in Ca2+-signaling, Ca2+- transporters, and Ca2+-permeable channels; Fe and metal transports which were differentially regulated by Ca2+ deficiency and/or between root and shoot tissues, suggesting their involvement in Ca2+ homeostasis. Heterologous expression and loss-of-function mutations studies of the DEGs identified in this study would elucidate their physiological functions in tef.In conclusion, we found that CaEragrostis tef) accessions obtained from the USDA-GRIN germplasm collection43. We used the accession with the highest seed Ca (1811 mg/kg) content in this study. Tef seeds were sterilized according to a method previously published with slight modifications80. Briefly, seeds were soaked in 70% ethanol for one minute, followed by soaking in 1% NaOCl solution containing 0.1% Tween-20 for 10 min with vortexing every 1 min. Subsequently, the seeds were washed with autoclaved high purity water (18.2 MΩ (megaohms)) five times. The seeds were then transferred to a petri-dish with a sterile wet filter paper disc. The plates were incubated in a growth chamber  for six days under 16 h/8 h day-night cycles at 28 °C. The seedlings were transferred to a modified Hoagland hydroponic solution81 in which the Ca(NO3)2 was replaced with NH4NO3 to investigate the effect of Ca2+. Plants were grown in the media containing either 1 mM Ca2+ (optimum or control) or 10 µM (low Ca or Ca-deficient). The nutrient solution was continuously aerated and was renewed every 4 days.We recently performed seed mineral profiling of diverse tef .One-month-old tef plants grown in 1/4th-strength Hoagland’s hydroponics solution was analyzed. Roots and shoots were separated and rinsed three times with Milli-Q (18.2 MΩ) water. Before weight measurements, the tissues were oven dried at 65 °C overnight and ground to fine powder using a Waring Laboratory spice blender. Briefly, 500 mg of ground tissue was digested using concentrated HNOFor RNA extraction, root and shoot samples were ground into powder under liquid nitrogen using a mortar and pestle. Total RNA was isolated using the GeneJET RNA purification kit following manufacturer’s procedure . The quality and quantity of the RNA were determined using a Nanodrop . mRNA-seq libraries were prepared using NEBNext® Ultra II RNA Library Prep Kit for Illumina  according to the manufacturer’s instructions. For each library, 500 ng total RNA was used. The mRNA-seq libraries were sequenced on an Illumina Hiseq platform using the 150 bp paired-end sequencing strategy.sickle with the options − q 12 and − l 15 38. All default parameters were used except –very-sensitive and –summary-file options passed to the hisat2 aligner83. The number of reads mapped to a genome feature was determined using htseq-count command line script from the HTSeq python package84(-s reverse -a 10 -t exon -I Parent -m union) and count data was imported to the R statistical computing environment for further analysis42. Initially, genes that have no count in at least 4 of the replicate samples were excluded from the analysis, and the remaining 28,796 features were used for downstream analysis. Data normalization based on dispersion and differential analysis were conducted using the DESeq2 package85. We fitted a generalized linear model (~ Treatment) between the expression count. Finally, differential expression analysis was performed between ConR verus LCR, ConS versus LCS, Con R versus ConS, and LCR versus LCS, and differential expressions were identified by applying multiple testing correction using Benjamin-Hockberg procedure86.Data analysis for RNA-Seq was performed by the Bioinformatics Core associated with the Center for Human Health and Environment at North Carolina State University. Quality control of read data was briefly evaluated with FastQC, and alignment trimming was performed using GLR3.1, 5′-tcatcttccatgctgccaaaggc-3′ and 5′-tctccagttgggccattgtgtc-3′ for CNGC2, 5′-gcgactcgaggtgtgaatgaagag-3′ and 5′-aatggaacccgccgaacaagac-3′ for CNGC17, and 5′-tgctgcaattccagagggcttg-3′ and 5′-tcacagttgtgcaacccaatgtc-3′ were used to amplify CaE1. The internal control, eukaryotic elongation factor (EF1A) was used as a housekeeping gene and amplified using sense 5′-catcaacatcgtggtcatcg and antisense 5′-gatgcctccaagcttgtaga-3′ primers. Gene expression in the untreated samples were used as a calibrator, and blank (without template) was used as a reference control. The qPCR parameters were as follows: 950C for 30 s, 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and a final incubation at 72 °C for 5 min followed by melting curve analysis. Relative expression levels based on fold changes were calculated using the ΔΔCT method87 available with the QuantStudio3 software  and in MS-Excel .To validate the RNA-seq data for representative genes, quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed using a QuantStudio 3 real-time PCR system and the PowerUP™ SYBR Green pre-formulated 2 × master mix . Total RNA was treated with DNase I  to remove contaminating genomic DNA before first-strand cDNA synthesis. First-strand cDNA was synthesized using 5 µg of total RNA and the SuperScript® III First-Strand Synthesis System  following the manufacturer’s instructions. First-strand cDNA was used in the real-time qRT-PCR reaction containing 2xSYBR Green Master Mix, 500 nM forward, and reverse gene-specific primers in a 20 uL reaction volume. Gene-specific sense and antisense primers 5′-tttggctcctgggactcctaac-3′ and 5′-ggccagattgcagtgtagagtttc-3′ for Eragrostis tef were annotated by comparing it to the genome sequences of Setaria italica (GCF_000263155.2), Oryza sativa88, and Sorghum bicolor (GCF_000003195.3). The CDS of E. tef were compared to S. italica, O. sativa, and S. bicolor using Blastx. A CDS was considered significant with e-value < 0.00001. Significant hits were first annotated by comparison to S. italica , then to O. sativa  and S. bicolor . A total of 94.6% of the CDS of E. tef had a significant hit to a CDS of these three genomes, and the corresponding gene annotations were used.Coding sequences (CDS) of 89. Signature genes from the RNAseq analysis were used as an INPUT, and significantly enriched pathways and/or families were selected based on molecular function, cellular structure, and biological process category.Go analysis was performed using the agriGO90. After the significant F-tests, the Tukey’s multiple comparisons92 were used to separate the means (P ≤ 0.05). Values represent mean ± SD of six (Figs. Treatments were replicated four times, and two independent experiments were conducted. Data were analyzed by the one-way ANOVA using the PROC GLM procedure in SASix Figs. B,C and fTef is primarily cultivated in East Africa and is becoming popular in several countries including South Africa, Australia, and the U.S. The tef accessions used in this study was obtained from Dr. John Cushman at the University of Nevada Reno. All methods are performed in accordance with relevant guidelines and regulations.Supplementary Information 1.Supplementary Information 2.


CaM) have been linked to two cardiac arrhythmia diseases, Long QT Syndrome 14 (LQT14) and Catecholaminergic Polymorphic Ventricular Tachycardia Type 4 (CPVT4), with varying degrees of severity. Functional characterization of the CaM mutants most strongly associated with LQT14 show a clear disruption of the calcium-dependent inactivation (CDI) of the L-Type calcium channel (LCC). CPVT4 mutants on the other hand are associated with changes in their affinity to the ryanodine receptor. In clinical studies, some variants have been associated with both CPVT4 and LQT15. This study uses simulations in a model for excitation–contraction coupling in the rat ventricular myocytes to understand how LQT14 variant might give the functional phenotype similar to CPVT4. Changing the CaM-dependent transition rate by a factor of 0.75 corresponding to the D96V variant and by a factor of 0.90 corresponding to the F142L or N98S variants, in a physiologically based stochastic model of the LCC prolonger, the action potential duration changed by a small amount in a cardiac myocyte but did not disrupt CICR at 1, 2, and 4 Hz. Under beta-adrenergic simulation abnormal excitation–contraction coupling was observed above 2 Hz pacing for the mutant CaM. The same conditions applied under beta-adrenergic stimulation led to the rapid onset of arrhythmia in the mutant CaM simulations. Simulations with the LQT14 mutations under the conditions of rapid pacing with beta-adrenergic stimulation drives the cardiac myocyte toward an arrhythmic state known as Ca2+ overload. These simulations provide a mechanistic link to a disease state for LQT14-associated mutations in CaM to yield a CPVT4 phenotype. The results show that small changes to the CaM-regulated inactivation of LCC promote arrhythmia and underscore the significance of CDI in proper heart function.Mutations in the calcium-sensing protein calmodulin ( CaM) mutants have been associated with cardiac arrhythmia in clinical and experimental studies T is the total CaM concentration, dhpr,nj (15%) was also added. 2+ in the subspaces are rest. Ca2+ sparks and Ca2+ quarks result in increases in the amount of Ca2+:CaM complex in the dyad. The amount of Ca2+:CaM complex in the subspace is greater during pacing at 8 Hz compared to pacing at 1 Hz pacing leading to increased Ca-dependent inactivation of the L-type channel. The endogenous buffer as lobes . For eacview see ). In L-t the CDI . The modier data . In addiCaM alter the dynamics of LCC inactivation depend on local changes to the way CaM interacts with the cytoplasmic domains of the channel. While different mutants can alter the mechanism in unique ways, the net impact on the LCC channel dynamics is to decrease the rate of CaM-dependent inactivation. On the scale of the compartmental myocyte model, the LCC interactions with CaM are modeled through the transition rate constant k24. Lowering the transition rate from O2 to C4 by 10% and 25% simulates mutations in CaM which only slightly impact channel function, allowing for the systematic impact on cell function to be studied. The wild type and two mutant CDI models were each simulated for 100 s using a current clamp protocol and determined that a steady train of transients were obtained after 20 s. Simulations were then run 6 times (each with a different seed for the pseudo random number generator) for the wild-type and two mutant CDI models for 30 s, at 1, 2, 4, and 6 Hz stimulation rate. The simulations all converged on a steady train of transients that overlapped. In the figures, data from a single simulation are shown.The specific mechanisms by which mutations in 24 might be compared the experimentally observed changes in the Ca2+ binding affinity of the CaM variants, a steady-state assumption was made about Ca2+ binding to CaM based on the simulations seen in Ca:CaM] is given byTo understand how the 10% and 25% reduction in k2+ is assumed to be 10 µM, the ratio of the [Ca:CaM]myo of the variant to the wildtype yields the factor modifying k24 shown in When dyadic Cahttps://web.jhu.edu/animalcare/procedures/rat.html last accessed 19 December 2022). However, in experimental studies included those that explore CPVT4 and LQTS14 isolated myocytes are stimulated at 1, 2, 3, 4, and other pacing rates. The simulations are performed at the rates used in experiments as well as physiological rates. Interestingly, simulations of wildtype rat ventricular myocytes paced at 8 Hz and observed electrical and Ca2+ alternans which are abolished when β-adrenergic stimulation is added [CaM variants have been performed in rodents, which is the reason this current study was performed in a model of rat ventricular myocyte EC coupling. Simulations at pacing rates 1 Hz and 2 Hz were performed because these stimulation rates are often used in experiments. Simulations at 4 Hz represent the physiological basal stimulation rate in the living rat. The experiments were repeated in a model of beta-adrenergic stimulation at 6 Hz pacing to reflect the functional remodeling which occurs as a result of increased cardiac stress. In this state, the calcium current through the LCC and SERCA channels is increased by a factor of 1.2 and 1.3, respectively. Beta-adrenergic stimulation was also performed at 1, 2, and 4 Hz to show the enhancement in Ca2+ loading compared to control simulations that is sometimes studies in experiments.Living rats have heart rates of 330 to 480 beats per minute which translates to 5.5 to 8 Hz (is added . It is p+-Ca2+ exchanger (NCX) channels (Incx), which play an important role in restoring the cell to its resting potential. Together, these variables can be used to investigate the underlying mechanism leading to an arrhythmia prone phenotype.Several important emergent measures of cellular phenotype are simulated by the model and arise from the independent regulation of calcium channels in CICR. The cyclic increase in calcium concentration in the bulk compartments of the myoplasm is determined by the diffusion of calcium from each of the 20,000 dyad subspace volumes. The subsequent loading of calcium from the bulk cellular volume to the NSR occurs through SERCA channels, and the concentration is also reported by the simulation. Additionally, the flow of potassium, sodium, and calcium ions contribute to the transmembrane voltage and impact the magnitude and duration of the action potential. Additionally, the individual flow through each CRU can be used to determine the independent contributions of the LCC and RYR2 dynamics. When summed, they provide a measure of the contribution to the transmembrane calcium current by the LCC and flux through RYR2 channels. Additional contributions to the calcium current come from the bidirectional Na2+). The myoplasmic calcium indirectly links all the release sites through modulation of the subspace (Ca2+). Thus, the fraction 10% was selected so that the amount of calcium release does not change much in the bulk myoplasm of the system so that these CRUs are assumed to experience the same bulk myoplasmic calcium. Model code is available in the The system of ordinary differential equations comprising the model is solved using the explicit Euler method. The simulation uses no-flux boundary conditions based upon the assumption that there was not a significant gradient across cells at the border of the simulation. The adaptive timestep (10–100 nanoseconds) is required for numerical stability and is also necessary to capture the fast and stochastic gating of DHPR (dihydropyridine receptor) and RyR2 channels. Each release site uses a different sequence of pseudo-random numbers to control channel gating. These Monte Carlo simulations are computed on Fermi-GPGPU cards, with pseudo-random numbers derived from the Saru pseudo-random number generator on GPGPU provided by Asfar et al. . When th2+ spark data for the 20,000 Ca2+ release units for analysis. To address this, for the Ca2+ spark analysis, data from the last second of each simulation were retained for 10% of the 20,000 Ca2+ release units (2000 CRUs). The data were processed to calculate spark amplitudes, spark duration, spark frequency, and spark time to peak using a program created in Fortran. For this calculation a spark was considered to start with the dyadic space Ca2+ rose above 25 µM. A spark was assumed to terminate when the dyadic space Ca2+ subsequently fell below 5 µM. Microsoft Excel was used to calculate the means for these properties and the two-sided student’s t-test comparing the means under the assumption of different variances and sample sizes. To ensure that the simulations were consistent each simulation was run 6 times with different starting seed values for the pseudo random number generator. Simulations were compared to make sure that the results were similar across simulations. A representative simulation was chosen to display in the figures. The model generates an intractable amount of CaCaM-dependent inactivation of LCC plays an important role controlling the amount of Ca2+ used to initiate CICR and ensuring the channel is closed prior to the ensuing action potential. The impact of even a slight decrease in the CaM-dependent transition probability can be seen in the simulation results in The 2+ is seen in both mutants, and the relative increase over the wild-type concentrations is magnified by pacing  but Ca2+ alternans developing with the reduction in CDI.The increase in the peak concentration of cellular Cay pacing . An addiz pacing C than thz pacing A. Still,The simulation results for the cardiac myocytes show the mutations impacting the CDI have a similar impact on the NSR calcium dynamics . At the Despite the changes in calcium loading in both the bulk cell and NSR, the overall impact on the action potential is minimal in the simulations A,B. WithCaM mutations on the action potential is less pronounced. In the simulation for a given cell type (wild type and mutant), the difference in action potential from beat to beta is negligible after 20 s because all simulations are able to achieve a new homeostasis. This is further demonstrated by the uniformity of the cyclic peaks in the final second of simulations. Still, the simulated mutations do push the simulations toward instability, as oscillations in the peak cellular calcium begin to occur for the more severe mutant under 4 Hz pacing. In this condition, the cell has assumed a state on the verge of arrhythmia, indicating a possible threshold of diminished CDI function that is tolerated by the cardiac myocyte. On a longer timescale, a 30 s simulation at 4 Hz pacing is shown in 2+ from the CRU. Known as a Ca2+ spark, the Ca2+ release can trigger larger changes in the ionic homeostasis and promotes arrhythmia when occurring spontaneously at the wrong time, and the impact of the small Ca2+ release is compounded when sparks increase in frequency, duration, and rate. As seen in CaM inactivation leads to an increase in spark frequency over the wild-type model. 2+ spark properties. The spark duration during pacing ranges between 89.6–707.9 sparks per ms which translates to up 100,000’s of Ca2+ sparks per second. With such a large sample size the standard deviation will be small and the difference of the means will show statistical significance [p < 0.001 level. Hence, statistical significance is not shown. At 4 Hz the average Ca2+ spark duration is increased significantly with the mutants and increases pacing frequency  characteristic of CaAn advantage of the computational model is that it allows the underlying mechanism of the arrhythmia to be probed in detail. The calcium current through the LCC was totaled across all 20,000 CRUs to produce the plots in 2+ flux from the SR. The total calcium flux through all the RYR2 channels in the 20,000 independent CRUs is provided in 2+ release is increased in the CDI mutants while the duration of release remains the same. The increased window of calcium current through the LCC initiates CICR in an increased number of RYR2 channels but does not impact the transition to a closed state. However, when beta-adrenergic stimulation is applied the increased loading in the SR elongates the Ca2+ release into the dyad subspace. During pacing, these small changes accumulate and ultimately interrupt the myocyte’s ability to regulate the transmembrane voltage through the Na+-Ca2+ exchanger.The increase in open probability of the RYR2 channels causes a small increase in the Ca+-Ca2+ exchanger in the cell membrane is shown in +-Ca2+ exchanger current. This reversal in flow is necessary to restore the action potential in the cell membrane. However, the accumulated impact of the increased CICR drives up the calcium concentration in the myoplasm and reduces gradient responsible for driving the ion the exchange. As seen in the 1 Hz simulations, the impact of the CDI mutation on is to prolong the time restore the Na+-Ca2+ exchanger current to the diastolic level. In the beta-adrenergic stimulated myocytes, the increase is magnified, and the effect accumulates in the more severe mutant and is accelerated by pacing. Once the cumulative impact reaches a threshold, the cell is no longer able to reverse current in response to the action potential. The ability to maintain the transmembrane potential is lost and the rhythmic pacing of the cell deteriorates into arrhythmia. The current through the Na+-Ca2+ exchanger. Once the calcium concentration becomes too large, the channels are not able to efficiently reverse direction to export sodium. The mechanism feeds into itself, as the increased flow through an individual CRU promotes larger releases through loading of calcium into the NSR, until the cell reaches a state where the regulatory mechanism is overcome. Arrhythmias associated with CPVT4 typically have altered RYR2 function but can also occur by diminished function of other channels which influence the concentration of ions in the cardiac myocyte. The movement of calcium through the cellular compartments creates an interconnected relationship between channel dynamics and underscored the difficulty in identifying the mechanism associated with a disease phenotype only detectable at the tissue level.The overall result of a small change to the rate of CDI leads to the rapid formation of arrhythmia, as the high cellular concentration of calcium interrupts the ability to regulate the transmembrane voltage through the Na2+ dynamics result in a normal wave train during 1 Hz, 2 Hz, and 4 Hz pacing rates. A pacing rate of 1 Hz and 2 Hz are often used in experimental studies and a 4 Hz pacing rate is the physiological heart rate of rat. Rapid pacing at 6 Hz resulted in abnormal Ca2+ dynamics and action potential dynamics in the form of alternans which is precipitated by high level of Ca2+ in the ventricular myocyte.The simulation study presented here explored how mutations to N98S and D96V that result in reduced CDI corresponding can lead to abnormal EC coupling and therefore arrhythmia. As the CDI was reduced by a factor of 0.9 (N98S) and 0.75 (D96V) in the variants the action potential duration increased a small amount. This is consistent with clinical findings that the QT prolongation in patients with the N98S variant is small . and Ca22+ transients and action potentials with prolonged duration in the mutant simulations. Pacing at 4 Hz under beta adrenergic stimulation produced alternans in both the Ca2+ transient and action potentials. Pacing at 6 Hz, resulted in a very irregular train of action potentials and the underlying Ca2+ dynamics. Under the rapid pacing simulations Ca2+ levels were significantly elevated in the SR and myoplasm consistent with Ca2+ overload. For comparison, in a mouse knock-in model the N98S mutant ventricular myocytes showed that beta adrenergic stimulation induced a delay in repolarization and increase L-type Ca2+ current in optical and electrical measurement of membrane potential in isolated hearts compared to control [Under beta adrenergic stimulation, pacing at 1 Hz and 2 Hz showed a steady train of Ca control .2+ spark frequency, amplitude, and duration. These findings are supported by other studies. Wescott and co-workers also observed in increase in Ca2+ spark duration in their simulations of CPVT when arrhythmia was observed [2+ wave incidence, and suppressed CPVT symptoms in mice and humans [2 spark frequency has also been observed in atrial fibrillation in human atrial myocytes by Hove-Madsen and co-workers [Underlying the arrhythmic behavior was an increase in Caobserved . In exped humans ,51. Macqd humans . This in-workers .CaM with these two arrhythmic cardiac diseases raises questions about how complex, emergent disease states are classified [CaM-dependent inactivation of LCC and are easy to diagnose using an ECG. However, other mutations in structurally similar locations in CaM manifest as the harder to detect CPVT4. Since CaM interacts with a range of other proteins in the cell such as the Na+ channel and SK channel, even those involved in regulating EC coupling and CICR, it’s possible that some of the CaM mutants’ function normally in LCC inactivation and the CPVT4 state arises through other interactions [CaM interactions with the Na+ channel [Calmodulin variants have been associated with long QT syndrome 14 (LQT14), catecholaminergic polymorphic ventricular tachycardia type 4 (CPVT4), and idiopathic ventricular fibrillation (IVF), however, arrhythmogenesis has been attributed mainly to either prolonged repolarization (LQT14 phenotype) or increase calcium release from the SR (CPVT phenotype) ,54. The ractions ,56,57. E channel .CaM associated with LQT14 shown in CaM inactivation of LCC would clearly alter the electrical signature on the tissue scale and make the increased chances for arrhythmia easily identifiable. In fact, and experimental optical mapping study in heart by Němec and co-workers suggested that the abnormal Ca2+ dynamics associated with CPVT, can also be associated with the T-wave lability found in LQT [2+ dynamics is the Ca2+ overload in the myocyte for both LQT and CPVT4 [CaM mutations, the deviation from the wild-type action potential would be difficult to detect using available diagnostic methods, and a mutation which only fractionally decreases the CaM-dependent LCC transition to a closed state would avoid detection in the unstimulated heart. While the phenotypic impact of less severe CaM mutations is not as easily detectable, the altered dynamics of the cardiac myocyte increase the susceptibility to arrhythmia.The prolonged action potential of guinea-pig ventricular myocytes expressing mutant d in LQT . The undnd CPVT4 ,60. For 2+ binding affinity changes for the different CaM variants has been measured experimentally by others  and the Caers see ,31,61). ,61. 2+ b2+ overload is the mechanism behind the overlapping phenotypes of the arrhythmia and provide a way of differentiating how they arise. The simulations show that even a small change to the rate of CaM regulated inactivation of the LCC promotes an arrhythmia prone state. Increased calcium loading in the bulk myoplasm, and SR is exaggerated through pacing, as is the spark rate and intensity. The altered ionic homeostasis increases the likelihood of disrupting the EC coupling cycle by a mechanism similar to CPVT4, despite the originating from disrupted function associated with LQT14. A greater understanding of conditions which give rise to complex genetic diseases such as these in enhanced through modeling the emergent impact of altering individual functional interactions. The simulations clarify that CaCaM with Ca2+ and the L-type Ca2+ channel. The two lobes of CaM each have two Ca2+ binding sites and both of the are involved in CDI [CaM variants associated with LQT14 affect Ca2+ binding sites in both lobes. The current model does not differentiate between the contributions of the two lobes and their combined contributions to CDI. This has been left for future studies. The experimental literature gives a wide range of values of the Ca2+ binding affinity of CaM ranging from 2.5 to 150 µM when interacting with the L-type Ca2+ channel and from 0.03 to 4.0 µM when interacting with the RyR2 [CaM seems to respond to the high Ca2+ inside the CRU during a Ca2+ spark (>100 µM) as well as the bulk myoplasmic Ca2+ levels seen in the myoplasm (1 µM). The varied values observed experimentally might all be valid with the value depending on the experimental (or cellular) environment of CaM. Understanding these complex issues will require integration of new experimental and modeling studies.In the cardiac ventricular myocyte CDI is a complex phenomenon conferred by the interactions of d in CDI . CaM varthe RyR2 ,31,32,332+-dependent inactivation by calmodulin that demonstrated the binding and unbinding of calmodulin from the L-type Ca2+ channel as a mechanism of complex decoding of the Ca2+ signal [2+-dependent activation on excitation–contraction coupling, Limpitikul et al. developed a bi-lobal model of Ca2+-dependent inactivation in the cardiac ventricular myocyte of the L-type channel based on experimental data to characterize the effects of the D130G variant [Kr (LQT2). Grandi and co-workers studied the compound mutations involving the slowly and rapidly activating delayed-rectifier potassium currents (IKs and IKr) and the fast sodium current (INa). Previous computational modeling studies have studied the mechanisms LQT14. Tadross et al. developed a model of Ca+ signal . Lai and+ signal . The pre variant . They sh variant ,66,67,682+ transients due to loss of Ca2+-dependent inactivation. These changes are potentiated with beta adrenergic stimulation and are accompanied by the development of Ca2+ overload and alternans which is seen during CPVT4. The increased RyR opening due to the increased myocyte Ca2+ contributes to this phenotype. Taken together, this confluence of phenotypic features can cause the LQT14 variants causing a change in Ca2+-dependent inactivation to appear as if they are causing a CPVT4 phenotype. Our computational model of the rat ventricular myocyte demonstrates the changes in the features of excitation–contraction coupling in myocytes with wild-type and variant calmodulin. With variants that are attributed to causing LQT14, the myocytes have increase action potential duration and large Ca