Recent data show that lysosomal functions are transcriptionally regulated by transcription factor EB (TFEB) through the induction of genes involved in lysosomal biogenesis and exocytosis. Given these observations, we investigated the roles of TFEB and lysosomes in intracellular Ca2+ homeostasis. We studied the effect of transient modulation of TFEB expression in HeLa cells by measuring the cytosolic Ca2+ response after capacitative Ca2+ entry activation and Ca2+ dynamics in the endoplasmic reticulum (ER) and directly in lysosomes. Our observations show that transient TFEB overexpression significantly reduces cytosolic Ca2+ levels under a capacitative influx model and ER re-uptake of calcium, increasing the lysosomal Ca2+ buffering capacity. Moreover, lysosomal destruction or damage abolishes these TFEB-dependent effects in both the cytosol and ER. These results suggest a possible Ca2+ buffering role for lysosomes and shed new light on lysosomal functions during intracellular Ca2+ homeostasis.Lysosomes are membrane-bound organelles mainly involved in catabolic processes. In addition, lysosomes can expel their contents outside of the cell via lysosomal exocytosis. Some of the key steps involved in these important cellular processes, such as vesicular fusion and trafficking, require calcium (Ca It is now common knowledge that a variety of extracellular stimuli ranging from the binding of hormones, neurotransmitters, and growth factors to phenomena such as cell-cell interactions occur through diverse mechanisms  to induce increases in cytoplasmic Ca2+ concentrations ([Ca2+]c) that exhibit defined amplitudes and kinetics12Over the past two decades, our understanding of how extracellular signals are conveyed to eukaryotic cells by increasing the intracellular Ca2+ gradient exists across the plasma membrane (PM) (approximately 70 to 90 mV), but the [Ca2+]c is less than 1/10,000 that of the extracellular milieu. However, eukaryotic cells can store Ca2+ in many organelles and can mobilize the ion in response to endogenous and extracellular stimuli. The major intracellular Ca2+ storage unit is the ER 2+ level among its sub-regions. Upon stimulation with agonists such as histamine or ATP, the ER rapidly releases Ca2+ through the inositol 1,4,5-trisphosphate receptor (IP3R), thereby generating transient waves in the cytoplasm and mitochondria to promote cell activities42+ depletion, the luminal sensor protein STIM1 oligomerizes on the ER membrane and migrates to sites of ER/PM interaction to activate the highly Ca2+-selective ORAI channels located on the PM62+ store is replenished via the sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) pump89102+ entry or store-operated Ca2+ entry (SOCE). Similar to the ER82+ stores with a free Ca2+ concentration of ~0.4–0.6 mM122+ concentration (~100 nM). Although the depletion of lysosomal Ca2+ stores does not induce extracellular Ca2+ entry via SOCE, capacitative Ca2+ entry induced by ER Ca2+ release might contribute to the accumulation of Ca2+ inside lysosomes2+ in lysosomal function is supported by the well-established paradigm of its role in organelle and PM fusion2+ signaling center2+ release from the lysosome has been shown to be required for late endosome-lysosome fusion9182+-releasing factors such as NAADP2+-dependent functional coupling between the ER and lysosomesIn eukaryotic cells, a large electrochemical Ca2+ channel in the lysosome, Mucolipin 1 or TRP channel 1 (MCOLN1 or TRPML1), as well as lysosomal Ca2+ sensors such as the C2 domain-containing synaptotagmin VII, are also required for many of these functions9192+ content caused by mutations of the human TRPML1 gene is considered to be the primary pathogenic cause underlying some lysosomal storage diseases and common neurodegenerative diseases13The principal CaThe identification of transcription factor EB (TFEB)26303132During lysosomal exocytosis, TFEB induces both the docking and fusion of lysosomes with the PM in a process that requires the lysosomal calcium channel TRPML12+ homeostasis with the goal of characterizing Ca2+ dynamics in both the cytosol and ER upon the induction of SOCE.Based on these observations, we investigated the contribution of TFEB expression and lysosomes to intracellular Ca2+ homeostasis, we first evaluated the effects manipulating its expression. We transiently overexpressed TFEB (TFEB-3xflag) for 48 h in human cervical carcinoma (HeLa) cells (To investigate the role of TFEB and lysosomes in intracellular Caa) cells , revealiet al.As previously demonstrated by Sardiello −04 ± 4.13E−05 pixel/pixel2 for control vs. 2.04E−04 ± 2.14E−05 pixel/pixel2 for TFEB).It has been suggested that TFEB overexpression increases the pool of lysosomes proximal to the PM and promotes their fusion with the PM2+ homeostasis, particularly for phenomena occurring near the PM. We took advantage of aequorin technology2+ entering the cell across the PM via SOCE in the cell models previously described. To induce capacitative Ca2+ influx via the activation of SOCE, HeLa cells were pre-treated with a SERCA blocker  for 15 min in a Ca2+ free medium (KRB/EGTA) to encourage the depletion of ER Ca2+ stores. When the release of stored ER Ca2+ was complete, Ca2+ was added to the KRB perfusion. A major increase in [Ca2+]c due to Ca2+ influx through plasma membrane channels was observed when the perfusing buffer was supplemented with 1 mM CaCl2. Under these conditions, TFEB overexpression did not significantly influence SOCE (cytosolic Ca2+ uptake in control HeLa cells = 114.53 ± 3.97 a.u.c. vs. TFEB-overexpressing cells = 111.47 ± 3.66 a.u.c.)  and the discharge of the aequorin probe due to the high Ca2+ affinity that has been ascribed to lysosomes2+]c values obtained under these conditions were largely artifactual, as reported previously2+] of 50 μM.Next, we considered the possibility that an increased number of lysosomes and their proximity to the PM might contribute to intracellular Ca a.u.c.) . However2+]c followed by a gradual decline, providing evidence for the presence of an abundance of SOCE in HeLa cells .Samples transfected with the TFEB construct displayed a consistent and significant reduction of cytosolic Ca2+ influx using a different aequorin probe targeting the cytosolic side of the PM (PM-Aeq)2+ stores, Ca2+ (CaCl2 50 μM) was added to the KRB perfusion (2+ accumulation in close proximity to the PM compared with the control cells (PM Ca2+ uptake in control HeLa cells = 1410.30 ± 59.05 a.u.c. vs. TFEB-overexpressing cells = 1122.78 ± 42.22 a.u.c.).To confirm that the presence of lysosomes in the proximity of the PM might modulate SOCE, we measured the Caerfusion . In our Overall, these data suggest a specific role for TFEB in regulating SOCE, likely due to increased lysosomal localization at the PM.To determine whether the TFEB-dependent effects on SOCE are strictly related to altered lysosomal activity, we used different pharmacological approaches that compromise lysosomal structure and function through different modes of action.2+ stores14383940The first compound was the membrane-permeable di-peptide glycyl-L-phenylalanine 2-naphthylamide (GPN), which causes selective osmotic lysis of the lysosomal membrane triggered by cleavage of the lysosomal enzyme cathepsin CFollowing treatment with GPN, LysoTracker RED staining was abolished in both pcDNA3-transfected cells (mean LysoTracker RED intensity in untreated pcDNA3 HeLa cells = 27264.18 ± 1364.86 a.u. vs. LysoTracker RED intensity in GPN-treated cells = 1924.26 ± 95.69 a.u.) and TFEB-overexpressing HeLa cells (mean LysoTracker RED intensity in untreated TFEB HeLa cells = 26359.23 ± 1056.64 a.u. vs. LysoTracker RED intensity in GPN-treated cells = 1472.63 ± 100.17 a.u.), without affecting the distribution of LAMP1-GFP , indicat2+ accumulation in HeLa cells compared with the untreated condition (cytosolic Ca2+ uptake in control HeLa cells with vehicle = 17.16 ± 1.47 a.u.c. vs. control cells with GPN = 14.58 ± 1.42 a.u.c.) (2+ uptake of TFEB-overexpressing HeLa cells treated with a vehicle = 9.21 ± 1.01 a.u.c. vs. TFEB cells with GPN = 15.09 ± 1.20 a.u.c.).Activation of SOCE after pre-treatment with GPN (200 μM) did not affect cytosolic Ca a.u.c.) . Convers2+ ionophores and by membrane wounding2 vs. single object area of Vac-1-treated HeLa cells = 4.80 ± 0.37 μm2) or the TFEB construct (single object area of untreated TFEB cells = 2.42 ± 0.22 μm2 vs. single object area of Vac-1-treated HeLa cells = 4.58 ± 0.41 μm2).The second compound, vacuolin-1 (Vac-1), promotes the alteration of lysosomal morphology, resulting in the fusion of acidic organelles without affecting the pH of the lysosomal lumen2+ influx experiments on HeLa cells transiently transfected with empty vector (pcDNA3) or TFEB after pretreatment with Vac-1 (10 μM for 1 h) or DMSO (vehicle). Under experimental conditions including only vehicle, we observed maintenance of the differences in capacitative Ca2+ influx between control and TFEB-overexpressing samples .Then, we performed capacitative Ca samples . In cont−04 ± 3.14E−05 pixel/pixel2 for control siRNA cells vs. 8.67E−04 ± 2.23E−05 pixel/pixel2 for TFEB siRNA HeLa cells).To determine whether the effects of altered lysosome activity on SOCE are mediated by TFEB-dependent alteration of the lysosomal compartment, we transiently inhibited the expression of the transcription factor in HeLa cells for 72 h see . To vali2+ entering the cell across the PM via SOCE employing the same experimental setting used for TFEB-overexpressing HeLa cells. After induction of capacitative Ca2+ influx, Ca2+ (CaCl2 50 μM) was added to the KRB perfusion, and we observed that TFEB-silenced cells (see 2+ uptake compared with the control samples (cytosolic Ca2+ uptake in control siRNA HeLa cells = 17.63 ± 0.78 a.u.c. vs. TFEB siRNA cells = 25.68 ± 1.87 a.u.c.). Overall, these data suggest that the effect of TFEB activity on SOCE is strictly dependent on both lysosomal activity and the PM-specific positioning of lysosomes.Subsequently, we assessed the level of cytosolic Caells see  displaye2+ in the ER, and it represents the main path for the maintenance of Ca2+ levels in the ER during stimulation.As seen above, the capacitative influx is activated in response to the depletion of intracellular Ca2+ dynamics in response to stimuli. Reticular Ca2+ measurements were performed using the genetically encoded fluorescence resonance energy transfer (FRET)-based probe D1ER chameleon, which is targeted to the ER via a KDEL retention sequence2+-binding domain that, once Ca2+ is bound, allows the highly efficient excitation transfer from a donor cyan fluorescent protein to an acceptor yellow fluorescent protein. The degree of FRET provides a ratiometric indicator of the Ca2+ level within the ER2+ (50 μM) at the start of the experiment, we measured the baseline FRET ratio, which corresponds to the resting Ca2+ level  cells (2+ emptying with ionomycin (3 μM) and EGTA (600 μM) (see Methods), the cells were perfused with Ca2+ at an extracellular concentration of 50 μM, and the maximum basal level of accumulated Ca2+ was measured (2+ levels in TFEB-overexpressing cells compared with control cells (ER Ca2+ concentration in control HeLa cells = 223.96 ± 3.40 μM vs. TFEB-overexpressing HeLa cells 230.75 ± 4.31 μM). However, TFEB-overexpressing cells showed a significant reduction in the Ca2+ re-uptake rate (2+ re-uptake rate in HeLa control cells = 0.83 ± 0.08 R/Rmin × 100 vs. TFEB-overexpressing cells = 0.44 ± 0.05 R/Rmin × 100), supporting the notion that TFEB-mediated SOCE inhibition could reduce Ca2+ refilling within the ER.HeLa cells transiently overexpressing TFEB did not display any difference in ER Ca3) cells   .GPN treatment (200 μM for 30 min) minimized the effects of TFEB overexpression on the ER Can × 100) . Moreoven × 100) , we obse2+ re-uptake due to the Ca2+ buffering capacity of PM-located lysosomes.Taken together, these results confirm the effect of TFEB overexpression on the rate of ER Ca2+ re-uptake (2+ re-uptake compared to control cells (Ca2+ re-uptake rate in HeLa control cells = 0.07 ± 0.01 R/Rmin × 100 vs. TFEB-silenced cells = 0.12 ± 0.02 R/Rmin × 100), supporting a specific role for TFEB in regulating SOCE.To verify the effect of TFEB on SOCE modulation, we performed analogous FRET experiments in TFEB-depleted cells and measured the rate of ER Cae-uptake . Down-re2+ regulation and to exclude a direct effect of TFEB overexpression on SOCE molecular machinery, we first examined the protein expression levels of two main modulators of the capacitative Ca2+ influx pathway: STIM1 and ORAI1. 2+ emptying from the ER under the same experimental conditions used for cytosolic Ca2+ measurements and assessed the number of ORAI1 clusters using the recombinant reporter ORAI1-EYFP. After treatment with thapsigargin; (200 nM) in Ca2+-free medium (KRB/EGTA), the number of clusters of ORAI1 increased considerably  using the recombinant reporter STIM1-ECFP; again, we did not observe significant differences between TFEB-expressing and control HeLa cells see   using an aequorin fused with the full-length cathepsin D protein, which specifically targets the probe to lysosomes2+]Lys. To induce capacitative Ca2+ influx via SOCE activation, HeLa cells were treated with ionomycin (3 μM) for 1 h in a Ca2+-free medium (KRB/EGTA) during aequorin reconstitution with coelenterazine to encourage depletion of ER and lysosomal Ca2+ stores. When the release of stored Ca2+ was complete, Ca2+ (CaCl2 50 μM) was added to the KRB perfusion. In our experimental setting, HeLa cells overexpressing TFEB (2+ compared with control cells ([Ca2+]Lys in control HeLa cells = 168.80 ± 6.59 μM vs. TFEB-overexpressing HeLa cells 194.17 ± 5.80 μM). Accordingly, we detected a significant reduction in [Ca2+]Lys in TFEB-silenced cells compared to siRNA control cells ([Ca2+]Lys in siRNA control HeLa cells = 175.26 ± 11.58 μM vs. siRNA-TFEB HeLa cells 134.91 ± 11.26 μM)  , corrobo2+ concentration in HeLa cells transiently transfected with empty vector (pcDNA3) or TFEB after pretreatment with Vac-1 (10 μM for 1 h) or DMSO (vehicle). Under experimental conditions including only the vehicle, we observed maintenance of the differences in lysosomal Ca2+ between control and TFEB-overexpressing samples , without altering the lysosomal Ca2+ levels of control cells . Overall, these data suggest that the effect of TFEB activity on SOCE and on lysosomal Ca2+ refilling is strictly dependent on both lysosomal activity and the PM-specific positioning of lysosomes.As previously observed, Vac-1 treatment affected the capacity of lysosomes to interact with other organellesples see . Importaet al.2+ release via MCOLN1 controls the activity of the phosphatase calcineurin during starvation. This pathway activates TFEB, thus promoting its nuclear translocation and lysosomal compartment expansionAs previously demonstrated by Medina 2+ entering via SOCE in pcDNA3 and TFEB-overexpressing HeLa cells under the same condition described above. Control cells showed no significant differences in SOCE either during starvation  or after CsA treatment . Intriguingly, TFEB-mediated reduction of SOCE (cytosolic Ca2+ uptake by fed pcDNA3 HeLa cells treated with DMSO = 21.32 ± 1.65 a.u.c. vs. fed TFEB HeLa cells treated with DMSO = 13.95 ± 1.60 a.u.c.) was amplified by starvation (fed TFEB HeLa cells treated with DMSO = 13.95 ± 1.60 a.u.c. vs. starved TFEB HeLa cells treated with DMSO = 3.74 ± 1.35 a.u.c.), and this effect was completely prevented by CsA treatment during starvation (starved TFEB HeLa cells treated with DMSO = 3.74 ± 1.35 a.u.c. vs starved TFEB-overexpressing HeLa cells treated with CsA = 14.35 ± 0.78 a.u.c.). These data indicate that increasing the nuclear localization of TFEB through starvation further stimulates lysosomal biogenesisTo validate our experimental setup, we first performed immunofluorescence staining of HeLa cells overexpressing TFEB before and after 6 h of starvation. Calcineurin activity was inhibited with cyclosporin A (CsA) to prevent TFEB translocation to the nucleus. As expected, analysis of the TFEB distribution revealed a nuclear-enriched pattern upon starvation   disruption of lysosomal Ca2+ uptake does not affect SOCE. The results of the present study confirm these findings; alteration of the lysosomal compartment by different pharmacological approaches did not compromise SOCE in control cells   see . Althoug2+ entry through the re-distribution of the lysosomal compartment to the PM. These findings not only describe TFEB as a new regulator of intracellular Ca2+ homeostasis but also propose lysosomal control of SOCE as an important aspect of lysosomal function and cellular health.In conclusion, we provide new evidence for the role of TFEB in shaping extracellular CaThe following chemicals were used: di-peptide glycyl-L-phenylalanine 2-naphtylamide , vacuolin 1 , thapsigargin (Sigma-Aldrich), ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid , cyclosporin a .2 Corning flasks. All cells were maintained at 37 °C under 90% relative humidity with 5% CO2. Before transfection, cells were seeded onto 13 mm glass coverslips for intracellular Ca2+ measurements with aequorin and onto 24 mm glass coverslips for microscopic analysis. Cells were grown to 70% confluence and then transfected for 12 h at 37 °C for transient plasmid overexpression. Transfection was performed via the standard Ca2+phosphate procedureHeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum , 2 mM L-glutamine, and 100 U/ml penicillin (EuroClone), and 100 mg/ml streptomycin (EuroClone) in 75 cm2+ measurements with aequorin and onto 24 mm glass coverslips for microscopic analysis. siRNAs were transfected with Lipofectamine RNAiMAX (Invitrogen) using a reverse transfection protocol. After 24 h, the cells were transfected for 12 h at 37 °C to achieve transient plasmid overexpression, via the standard Ca2+ phosphate procedureFor siRNA experiments, cells were seed onto 13 mm glass coverslips for intracellular Ca2+ entry current in the experiments with cyt-Aeq, at 48 h post-transfection, the coverslips were incubated with 5 μM coelenterazine (Fluka) for 1.5 h in Krebs–Ringer modified buffer -1-piperazineethanesulfonic acid [HEPES], pH 7.4, at 37 °C) supplemented with 1 mM CaCl2. For the PM-Aeq experiments, the coverslips were incubated in KRB supplemented with 5 μM coelenterazine and 100 μM EGTA for 1 h.The probes used in these experiments were chimeric aequorins targeted to the cytosol (cyt-Aeq), PM (PM-Aeq), ER (ER-Aeq) and lysosomes (lys-Aeq). To evaluate the capacitative Ca2+ emptying, cells were incubated with 200 nM thapsigargin (SERCA inhibitor) and 1 mM EGTA in Ca2+-free KRB for 15 min at 4 °C. A coverslip with transfected cells was subsequently placed in a perfused thermostat-controlled chamber located in close proximity to a low-noise photomultiplier with a built-in amplifier/discriminator. After 30″ of perfusion with KRB supplemented with 100 μM EGTA, Ca2+ was added to KRB, as specified in the figure legends. All experiments were terminated by lysing cells with X-100 Triton in a hypotonic Ca2+-containing solution (10 mM CaCl2 in H2O), thus discharging the remaining aequorin pool.Then, to induce ER Ca2+] of the intracellular Ca2+ stores was first reduced by incubating the cells for 45 min at 4 °C in KRB supplemented with 5 μM coelenterazine N, the Ca2+ ionophore ionomycin, and 600 μM EGTA. After incubation, the cells were extensively washed with KRB supplemented with 2% bovine serum albumin and 2 mM EGTA before luminescence measurements were initiated. After 180″ of perfusion with KRB supplemented with 100 μM EGTA, 50 μM Ca2+ was added to KRB, as specified in the figure legends. For the ER-Aeq experiments, the agonist (histamine at 100 μM) was added to the same medium after a Ca2+ plateau was reached.To reconstitute ER-Aeq and lys-Aeq with a high efficiency, the luminal  values offline, using a computer algorithm based on the Ca2+ response curve of wild-type aequorin. Further experimental details were previously describedThe output of the discriminator was captured with a Thorn EMI photon-counting board and stored in an IBM-compatible computer for further analyses. The aequorin luminescence data were calibrated into [CaHeLa cells expressing lysosomal-targeted GFP (LAMP1-GFP) were stained with LysoTracker RED (Invitrogen) and treated with GPN, Vac1 or vehicle alone, as indicated in the text and figures. For all treated cells, digital images were acquired in a 37 °C thermostat-controlled incubation chamber with a confocal microscope (Zeiss LSM510), using a 40 × 1.4 NA Plan-Apochromat oil-immersion objective. The acquired images were subsequently analyzed using the open source software Fiji.2+ dynamics were measured using single-cell Ca2+ imaging and the Ca2+-sensitive FRET (fluorescence resonance energy transfer)-based chameleon protein D1ERexcitation = 430 nm and λemission = 470 and 535 nm every 2 s using a Zeiss Axiovert 200 M inverted microscope equipped with a C-Apochromat 40x/1.2 W CORR objective and a cooled CCD camera (Photometrics). CFP emission and yellow fluorescent protein (YFP) FRET emission were alternately collected at 470 and 535 nm, respectively. The FRET signal was normalized to the CFP emission intensity, and changes in ER Ca2+ were expressed as the ratio of the emissions at 535 and 470 nm. Cells were perfused with KRB with 50 μM Ca2+ at the beginning of the experiment, and the baseline of the FRET ratio was measured, which corresponds to resting Ca2+. Then, 100 μM histamine was added to the perfusion, thus stimulating Ca2+ release from the ER through IP3Rs (Rmin). Finally, the agonist as washed out using KRB with 50 μM Ca2+, and the rate of Ca2+ re-uptake in the ER was measured. Cells were pre-treated with GPN, Vac1 or DMSO (vehicle), as previously described for cytosolic Ca2+ measurement with aequorin and indicated in the text and figures.Luminal CaStatistical analyses were performed using an unpaired two-tailed t-test (two groups) or one-way ANOVA with Tukey correction (for groups of three or more). For grouped analyses, two-way ANOVA was performed; p < 0.05 was considered significant. All data are reported as the means ± SEs.How to cite this article: Sbano, L. et al. TFEB-mediated increase in peripheral lysosomes regulates store-operated calcium entry. Sci. Rep.7, 40797; doi: 10.1038/srep40797 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Eleusine coracana (L.) Gaertn.], an orphan crop has an immense potential as a nutritional security crop due to its exceptionally high calcium content. Understanding the existing genetic variation as well as molecular mechanisms underlying the uptake, transport, accumulation of calcium ions (Ca2+) in grains is of utmost importance for development of calcium bio-fortified crops. In this review, we have discussed molecular mechanisms involved in calcium accumulation and transport thoroughly, emphasized the role of molecular breeding, functional genomics and transgenic approaches to understand the intricate mechanism of calcium nutrition in finger millet. The objective is to provide a comprehensive up to date account of molecular mechanisms regulating calcium nutrition and highlight the significance of bio-fortification through identification of potential candidate genes and regulatory elements from finger millet to alleviate calcium malnutrition. Hence, finger millet could be used as a model system for explaining the mechanism of elevated calcium (Ca2+) accumulation in its grains and could pave way for development of nutraceuticals or designer crops.Calcium is an essential macronutrient for plants and animals and plays an indispensable role in structure and signaling. Low dietary intake of calcium in humans has been epidemiologically linked to various diseases which can have serious health consequences over time. Major staple food-grains are poor source of calcium, however, finger millet [ Mineral malnutrition is affecting one half of the world’s population. People have no access to a variety of minerals such as iron, calcium, zinc, magnesium, and copper. Most of the staple food crops such as rice, wheat and maize which constitute the major part of the diet of people are often deficient in these macro/micronutrients, thus insufficient to meet the daily needs . Deficie2+) in pregnant women is predominantly important for fetal skeleton development, increasing birth weight prenatal hypertension and avoidance of pre-clamsia  content in grains/edible portion of crop plants include conventional breeding methodologies, marker assisted selection, transgenic technology and biofortification. Conventional plant breeding which aims in improving the target traits mainly by introgression from donor lines through hybridization has number of drawbacks such as the time required for breeding of cultivars, linkage drag, and absence of sufficient genetic variability in the primary gene pool with respect to a particular trait  that interfere with the absorption or utilization of these nutrients in humans . Thus, ectively, .Figure 1 are being currently practiced. Through plant breeding approaches one could select genotypes with low concentration of anti-nutrients or alternatively molecular biologists alter genes in staple crops so as to reduce or completely eliminate these anti-nutrients. However, doing so is associated with many risk factors and should be done with utmost care because many of the anti-nutrients are major plant metabolites that might play important roles in plant abiotic stress resistance, plant metabolism and in resistance of plant to crop pests or pathogens  absorption we need to consume food sources that contain types of calcium that are easily digested, assimilated and absorbed. Because of the calcium-magnesium ratio in dairy products, our body is not capable of absorbing the calcium  content. It is not only an excellent source of dietary calcium (Ca2+) but also an excellent model to explore the genetic control and molecular mechanisms contributing to high grain calcium (Ca2+) content.Finger millet is a potential staple crop cultivated mostly in Eastern and Central Africa and India. It ranks fourth in importance among millets in the world after sorghum, pearl millet, foxtail millet and commonly referred as n millet . Nutritin millet . Finger n millet , dietaryn millet , and a rn millet  and ironn millet . Ravindrn millet , 293–390n millet ; 50–300 n millet . Furthern millet  and 489 n millet  has beenn millet . Besidesn millet . Thus, fThe millet is also well known for its health benefits such as hypocholesterolemic, hypoglycaemic and anti-ulcerative properties . Further2+) is normally acquired by root from the soil solution in free ionic form. The outer layers of root, the epidermal cells and their elongated projection (root hairs) get in touch with the soil solution where Ca2+ are present in milli-molar range. Through a variety of Ca2+ permeable channels, which includes cyclic nucleotide gated channels (CNGCs), glutamate receptor like (GLR) proteins, two-pore channels (TPCs) and mechano-sensitive Ca-permeable channels (MSCCs) calcium (Ca2+) enters inside the root epidermal cells, where it moves both apoplastically and symplastically to the cortex and then to the stele. Further, it may reach xylem either apoplastically through extracellular spaces or symplastically, by entering root cells and moving from cell to cell through plasmodesmata.Calcium  of xylem walls and intensity of transpiration is the major factor for Ca2+ transportation  delivery and distribution in leaves is mostly dependent on the pathway of water flow to and through the leaves . Understanding the role of these genes and the availability of the calcium in rhizosphere might help in development of calcium biofortified plants. CAX1 gene in roots of high calcium genotype GPHCPB45 in addition to two more genes (TPC1 and ATPase) in most of the tissues at vegetative growth and developing spikes except flag leaf. Higher expression of CAX1 in roots suggests that calcium uptake is taking place at high rate probably under the regulation of calmodulin independent pathway as the expression of calmodulin is invariably low in all vegetative tissues (CAX1 was observed to be low in root tissue as compared to those in other tissues as reported earlier (2+) accumulation in GPHCPB45 genotype. Interestingly, the expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf tissue indicating that 14-3-3 might interact with the CAX1 for calcium content regulation in leaves. Further in the developing spike (S1 – S4 stage), all the transporters exhibited an increased expression with higher expression in GPHCPB45 and hence correspond to the higher calcium (Ca2+) content  in comparison to GPHCPB1 in the developing spikes. All the regulatory proteins also exhibited expression patterns similar to the transporter genes in developing spike with generally a higher expression in GPHCPB45 genotype. CaM exhibited a similar expression pattern as Ca2+ATPase gene indicating that this CaM isoform might be specific to developing spike and activating the Ca2+ ATPase through binding to the CBD domain , Calcinurin- B like protein kinases (CIPKs), calmodulin dependent protein kinases (CaMKs), calcium/calmodulin dependent protein kinases (CCaMKs) and transporters genes viz., Cachannels . Calcium tissues . The exp earlier . This su content . Higher D domain . CAM1 waof water . The res2+) in the two genotypes of finger millet. Studies on finger millet CAX1 and ATPase might offer further insights in understanding their auto-inhibition and strategic regulation. However, higher transcript abundance does not always corroborate with higher gene product abundance and activity. There are many post translational modifications and other bottle necks that may alter the fate of transcriptional activity. The results of transcriptome needs further validation using knock in and knock out approaches. Thus, finger millet plant might as well befall as a model system for better understanding of the underlying genetic control and molecular physiological mechanisms contributing to high grain calcium. Figure 2 depicts the role of potential transporters and regulatory genes involved in calcium (Ca2+) transport from source to sink in finger millet.Briefly, the differential expression of genes shows differential spatial and temporal accumulation of calcium  routes  and maternal tissues (seed coat and nucellar cell) with no direct vascular connection between them . General) routes . The cal) routes , but fin) routes . Therefo2+) transport and accumulation, a hypothetical pathway of seed calcium accumulation is proposed in Figure 3. In this model, it is proposed that Ca2+ from xylem passes through the pedicel tissues (Xylem transfer cell and pigmented strand) and then enters the maternal tissue (seed coat) with the help of transporters. It gets pumped toward the apoplast from seed coat, where it is absorbed by aleurone and then in endodermal cell. So, prior to uptake by the embryo, Ca2+ are deposited in the seed-coat apoplastic space abutting the surface of the embryo  and calcium phytates in the embryo of mature seed has been reported to be an important factor for high grain calcium in plants . There is good evidence that Ca uptake from the apoplast is mediated by activity of Ca channels, which would be the most energetically efficient mechanisms because they utilize the large inward directed electrochemical potential gradient for Ca (2+ channels in the cell membrane toward the xylem (funiculus) and Ca2+-ATPases toward endosperm might be responsible to pump Ca2+ in the seed . Once Ca2+ enters inside the seed it might get stored inside cell organelles viz., vacuole, ER etc. Vacuole is reported as the major reservoir of Ca2+ and up to 80% or more of the cellular volume of plant cells is occupied by vacuole  in seeds  in the tonoplast membrane has been reported for the uptake and storage of Ca2+ in vacuole of leaf cell  isolated from finger millet, CAX1 showed strong expression in the later stages of spike development indicating its role in accumulating high amounts of calcium in seeds  are the main Ca transporting proteins that pump Ca inside seed. Based on the structural and expression analysis of the calcium transporter genes and available data in literature, a hypothetical model for accumulation of calcium in the cereal grains was proposed , 2012. Tin seeds . Therefo2+ transporters including CAX-1, CAX-3 and calcium sensor protein such as CIPK-24 and CaM in high seed calcium finger millet genotype (GPHCPB45). Transcript profiling (qPCR) study of such identified candidate genes indicated that Ca2+ transporters including CAX-1, CAX-3 and calcium sensor proteins such as CIPK-8 and CaM are highly expressed in high seed calcium finger millet genotype (GPHCPB45) than GPHCPB1. Based on expression analysis and insilico interaction studies a speculative model is shown in Figure 5 which shows tri-partite interaction of calcium transport and sensor genes leading to activation of transport machinery that pumps calcium from cytosol to vacuole in developing seeds in finger millet. It is suggested that SOS3/CBL4 and SOS2/CBL10 strongly interact with CIPK-24 in either vegetative or developing spikes stage. Such SOS/CBL complexes bind and activate CIPK-24 kinase activity by relieving self-inhibitory folding within the regulatory and kinase domain of the CIPK-24 protein  content (450 mg/100 g) which is 10–30 times higher than wheat and rice (2+) content indicating that variation in calcium content is also genetically controlled (Table 2). In order to identify the markers associated with high grain calcium trait, 146 genic SSR markers were assessed for cross species transferability across a diverse panel of grass species. The average transferability of genic SSR markers from sorghum to other grasses was highest (73.2%) followed by rice (63.4%) with an overall average of 68.3% which establishes the importance of these major crops as a useful resource of genomic information for minor crops. The genic SSR primers (69.7%) failed to detect variations across the finger millet germplasm, indicating that the mineral transport and storage machinery remain conserved in plants and even SSR variations in them remain suppressed during the course of evolution (2+) content on linkage group 3 and 8 respectively have been identified  sensing, transport and accumulation mechanisms across genotypes differing in their grain calcium (Ca2+) content with the use of high-throughput transcriptome sequencing (2+) accumulation, 2+) content variation in two contrasting finger millet genotypes differing in grain calcium (Ca2+) content . The differential expression analysis on the basis of FPKM value resulted in 24 genes highly expressed in GPHCPB45 genotype and 11 were highly expressed in GPHCPB1 genes (2+)  at four stages of spike development. Significant correlation between the expression of selected Ca2+ sensor genes and amount of exogenous Ca2+ supply was observed  in seed, expressions of 5 each calcineurin and calreticulin genes of finger millet were studied during grain filling. EcCRT1, which shows high expression in the S1 stage of seed development, was partially cloned, sequenced and its full length cloning is under progress  are the main Ca2+ transporting proteins that pump Ca2+ inside seed  sensors and transporters was carried out to identify the differentially expressed genes during developing spikes of finger millet and can serve as potential candidate genes having role in seed calcium accumulation (2+ Exchanger (CAX), an important Ca2+ transporter that pump Ca2+ inside vacuoles were developed, which resulted in significant Ca2+ increase but deleterious effects like mineral imbalance, stunted plant growth and other structural changes were also seen (2+) accumulator at low external calcium (Ca2+) concentrations, whereas GPHCPB1 was found to be more responsive to increased external calcium (Ca2+) concentration (E. coracana) represents the most extensive expressed sequence resource available for finger millet to date  sensor protein such as CIPK-24 and CaM are highly expressed in high seed Ca2+ finger millet genotype. In order to understand the role and interrelationship expression of these genes in high seed calcium (Ca2+) accumulation, comparative tissue wide transcript profiling study of potential candidate genes, viz. CAX-1, CAX-3, CIPK-24 and CaM at vegetative stage and developing spikes stage have shown mostly higher expression in high grain calcium (Ca2+) genotype. The up-regulation of CAX-1 in vegetative tissues and developing spikes and CAX-3 in developing spikes provide most plausible clue explaining the accumulation of high calcium (Ca2+) in finger millet. The genetic information generated from these studies can be deployed in calcium (Ca2+) biofortification program by using genetic engineering or marker assisted selection strategies. Breeding for high grain Ca2+ in finger millet can be accelerated by using the closely linked markers to identified genes (EcCIPKs). This is just beginning, and it needs further studies in cloning and validation of the above genes using a set of different contrasting genotypes.Through peptide mass finger printing, two Caet seeds . Followiprogress . The mecide seed . The actmulation . Howevermulation , 2016. Tlso seen . GPHCPB4ntration . Compreh to date . AnalysiRecently, an effort has been also made to study the genetic make of diverse world finger millet collection and population structure analysis of 113 finger millet accessions using genotyping by sequencing (GBS) . This reConsidering the examination of mineral/micronutrients and their complex networks, ionomics or ionome profiling has emerged as a striking area that facilitates the genome-scale understanding of dynamics of elemental accumulation in the living systems . In the ).Metabolomics is another area to better explore the chemical components which are present in food grains, how they are synthesized and the genetic and environmental factors that are involved in determining food composition and stability. Metabolomics is much more than just a diagnostics tool  and can Robert and Jennifer, 20102+) content. The major aim of calcium biofortification strategy is to increase calcium (Ca2+) content in edible parts of crops without adversely affecting plant growth or increasing the plant requirement for additional inputs, i.e., calcium (Ca2+) fertilization or increased water use (2+) in edible part. The first approach is to enhance calcium (Ca2+) storage capacity through expression of calcium (Ca2+) transporter proteins viz., Ca2+ ATPase and Ca2+/H+ antiporters. Ca2+ ATPase requires energy for calcium (Ca2+) transport in the form of ATP while Ca2+/H+ antiporters is activated by proton motive force. Lower energy demands of transport through Ca2+/H+ antiporters make them a good candidate for Ca2+ increase  gene was first over expressed in tobacco plant under the control of cauliflower mosaic virus 35S promoter and 100% increase in calcium content was observed  in Arabidopsis and tobacco (2+/H+ antiport activity as well as increased Ca2+ accumulation (upto 50%). Later several transgenic event were attempted by expression of different CAX proteins viz., AtsCAX1 in carrot (AtCAX4 in tomato (AtsCAX2B in potato (AtsCAX2a in tomato (AtsCAX2b in bottle guard (2+) increase were reported. However, tissue Ca2+ increase was negatively co-related with Ca2+ deficiency symptoms. Interestingly, over expression of AtCAX4 or modified AtsCAX2B have shown weaker Ca2+ transport and fewer deficiency symptoms , Mn (magnese) modification of the region could be used to increase specificity  accumulation by over expression of calcium (Ca2+) channel proteins. Calcium (Ca2+) channels in plasma membrane have been reported in all known cell types (2+) across the PM is passive  and is driven by Ca2+ channels (LCT1) was expressed in tobacco under the control of CaMV 35S promoter. LCT1-transformed plants expressed a phenotype distinct from controls only under conditions of low calcium, they grew significantly better and had slightly higher shoot calcium concentration  content in plant was by over expression of calcium (Ca2+) binding protein. The use of calcium (Ca2+) binding proteins might be very efficient way to increase calcium (Ca2+) accumulation in tissues. The over expression of Zea mays calreticulin  in Arabidopsis resulted in bioavailable calcium (Ca2+) increase in plant (2+) content was measured for induced transformants compared to controls. The use of calcium binding protein as candidate for Ca2+ biofortification is good but compared to CAX transporter very less increase of Ca2+ has been reported  or reduced (cod6) calcium oxalate crystals were identified. In this report 10% reduction in calcium(Ca2+) content but 23% increase in calcium(Ca2+) absorbed by mice in feeding trial was observed (2+) biofortification in plants, maximum calcium(Ca2+) increase (up to 300% time) has been reported in potato over expressing modified Arabidopsis AtsCAX1 protein but the bioavailability of calcium(Ca2+) was found with calreticulin gene (Table 3).The fourth approach was through mutagenesis. Model forage crop observed . Out of 2+) accumulation in finger millet once identified through these approaches can be validated by over expressing them through transgenics and subsequently the candidate genes may possibly be used to genetically modify crops and help them increase their grain calcium content. Genome wide variant detection in crops like finger millet is a preliminary step toward linking genotypic variation and phenotypes. The conversion of these genetic variants  into genetic markers is particularly important in agronomically valuable crops to allow for effective marker assisted selection strategies, map based gene cloning, whole genome fingerprinting, association studies and population based analyses. Toward these goals, in the absence of the genome sequence of finger millet an increasing number of large scale genetic variant discovery initiatives are being taken in conjunction with NGS platforms, allowing for drastically quicker and cheaper variant discovery, and leading toward a far more comprehensive view of the genome or transcriptome.Hi- throughput technologies of genomics, transcriptomics, proteomics, metabolomics and ionomics have shifted the focus from single gene research to a holistic understanding of gene function. None of the methods used in isolation provides enough information to infer function of an unknown gene, instead combined data from different functional genomics tools bring us close to this goal. Potential genes involved in high grain calcium  content. Potential candidate genes responsible for high grain calcium (Ca2+) accumulation isolated from finger millet can help in improving other cereal crops through functional genomics and molecular breeding approaches and pave way for the development of designer crops.Genomics information has not only helped in efficient understanding of structural and functional aspects of many plant genomes but also has provided a feasible platform for manipulation of genomes for crop improvement. The near-future completion of genome sequencing project of finger millet will help in determining the function of every gene and ultimately how genes interact to form the basis of complex traits such as calcium nutrition. Finger millet is hence, no more called as a AK conceptualized the manuscript. DS, GJ, and US wrote the manuscript. DS and SS assisted, edited and updated the manuscript. AK contributed critically in revising the draft and updating the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer TB and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


To determine the causal relationship between circadian clock gene expression and [Ca2+]i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca2+]i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca2+ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca2+]i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca2+]i both exhibited cell autonomous circadian rhythms, but [Ca2+]i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca2+]i rhythms. As predicted by the model, PER2 and [Ca2+]i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca2+]i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca2+]i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca2+]i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network.Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca However, what drives these [Ca2+]i rhythms is not well understood. To test how rhythmic circadian clock gene expression and the SCN neuronal network influence [Ca2+]i rhythms, we monitored [Ca2+]i and PER2 (a rhythmic clock gene) in individual SCN neurons in dispersed and slice cultures by fluorescence and bioluminescence imaging, respectively. We also tested the effects of blocking neuronal firing or knocking out Bmal1, an essential clock gene. We found that the [Ca2+]i rhythm requires intact clock gene rhythms, and that it does not require the SCN neuronal network (so is cell autonomous) but is reinforced and modulated by the neuronal network.Intracellular calcium  and Cryptochrome (Cry1 and Cry2) gene transcription by a BMAL1/CLOCK heterodimer, and delayed inhibition of this process by complexes containing PER and CRY proteins . Circadian rhythm generation within SCN neurons and other cells depends on a core transcription-translation negative feedback loop (TTFL). This core loop involves activation of proteins . Null mubehavior  or singlbehavior .2+, neuronal firing rate, and neuropeptide release  the TTFL molecular clock, (2) spontaneous action potentials within single SCN neurons, and (3) the synchronized neuronal network coupled by neurotransmitters. To monitor [Ca2+]i and clock gene expression rhythms simultaneously in individual SCN neurons, the genetically encoded Ca2+ reporter GCaMP3 was introduced by adeno-associated virus (AAV) to SCN slices or dispersed cells cultured from PER2::LUC knock-in reporter mice, which express a fusion of PER2 and firefly luciferase in all cells  knock-in mice was described previously . This line was created by crossing Bmal1-floxed mice -cre mice . Genotyping was performed by multiplex PCR as described in a previous report  throughout gestation and from birth until used for experiments. Mouse studies were conducted in accordance with regulations of the Institutional Animal Care and Use Committee at University of California, San Diego.Bmal1–/– mice were anesthetized by ketamine  and decapitated. The hypothalamus block was dissected from the whole brain, and coronal slices were cut by a tissue chopper (Stoelting) to a thickness of 250 µm. Each SCN slice was cultured on a Millicell-CM membrane insert  in explant medium with low sodium bicarbonate (EML) i  for SCN cells exhibiting various patterns of [Ca2+]i. Shown at left are cells in dispersed cultures (A-E), including a cell with a sinusoidal [Ca2+]i rhythm (A), a cell with a [Ca2+]i rhythm showing a secondary peak (B), an initially non-rhythmic cell with spontaneous recovery of both PER2 and [Ca2+]i rhythms (C), and cells in which the [Ca2+]i rhythm became weaker (D) or stronger (E) during TTX. Shown at right are cells in SCN slice cultures (F-J), including a cell with a sinusoidal [Ca2+]i rhythm (F), a cell with a [Ca2+]i rhythm showing a secondary peak (G), a cell with an unusually phased [Ca2+]i rhythm peaking after PER2 (H), a cell in which TTX had no discernible effect on the [Ca2+]i rhythm (I), and a cell in which the [Ca2+]i rhythm was weaker during TTX (J). Download Figure 1-2, EPS file.Additional plots of PER2  and [Ca10.1523/ENEURO.0160-17.2017.f3-1Figure 3-12+]i rhythm in dispersed SCN cells. (A) PER2 and [Ca2+]i patterns of a representative cell in a dispersed cell culture. Relative levels of PER2  and [Ca2+]i  are shown. Time 0 is start of imaging. (B) Comparison of average RI values for PER2 rhythms (black bars) and [Ca2+]i rhythms (green bars) for cells before and during 100 μM ryanodine application. n.s. p > 0.05, mixed effect model. Download Figure 3-1, EPS file.Effects of ryanodine on PER2 and i rhythms was calculated as the peak of cross-correlation for linear-detrended time series using xcorr in Matlab. The time lag associated with this peak was used to estimate the phase difference between the PER2 and [Ca2+]i rhythms of each cell. To test for statistical differences of RI and coherence between SCN slice cultures and dispersed cell cultures, Fisher z-transformation followed by hierarchical (mixed effect) model  model  was applt) model .2+]i for each cell-like region were calculated after detrending using a discrete wavelet transform.After removing cosmic rays by a running minimum algorithm in MetaMorph, FL and BL stack files were analyzed using ImageJ and Matlab scripts using methods described previously . Briefly2+]i) and ryanodine storage calcium concentrations , Bmal1 mRNA transcription , and Bmal1 mRNA degradation , as described previously (2+]i (distributed evenly between 0 and 1). All other parameter values were preserved from previous modeling studies  from 0.2 to 1.0.For simulations in which cells were coupled together, the GABA network topology was designed  to confo2+]i rhythms are cell autonomous or require a rhythmic SCN neuronal network, we measured [Ca2+]i in SCN neurons dissociated from PER2::LUC mice and maintained in dispersed cultures. Both PER2 and [Ca2+]i dynamics were monitored by imaging in single neurons  had robust circadian rhythms of PER2 expression, as reported previously C, D. i rarely exceeded that for PER2  TTFL rhythms persisted at baseline levels even in the absence of [Ca2+]i oscillations  [Cllations .2+]i rhythmicity, we compared the rhythmicity of single SCN neurons oscillating independently in dispersed cell cultures to those oscillating synchronously in slice cultures. Because we sliced 250-μm coronal sections, the SCN was usually divided into two slices . Using RI as a measure of rhythm strength, single-cell PER2 and [Ca2+]i rhythms were both significantly stronger in slices than in dispersed cultures , suggesting higher similarity between PER2 and [Ca2+]i waveforms or improved rhythmicity. Peaks of [Ca2+]i preceded peaks of PER2 by an average of 4.8 ± 2.6 h .To examine the effects of the SCN network on PER2 and [Cao slices . The sli for 4 d A–C. Singcultures D. In SCN2+]i activation in the SCN and how the neuronal network may influence those patterns, peak times of PER2 and [Ca2+]i across the SCN were computationally detected in five SCN slices. Cell-like regions exhibiting significant rhythmicity in either PER2 or [Ca2+]i are depicted as small circles. Spatiotemporal patterns of PER2 and [Ca2+]i peaks and phase difference between PER2 and [Ca2+]i peaks in all five slices are shown in 2+]i and PER2 phases generally showed a similar spatial pattern  or caudal (n = 3) slices.A minority of cells in the dorsomedial region expressed very different phase relationships, including i rhythms requires a rhythmic, synchronized SCN network, we compared effects of TTX  on rhythmicity in slice and dispersed cultures. Both PER2 and [Ca2+]i rhythmicity were analyzed for 69 SCN neurons in 5 slices and 45 SCN neurons in 3 dispersed cell cultures. Cultures were imaged in control medium for 3–4 d, then in medium containing 1 µM TTX for 3.0 d, and finally in fresh control medium for 3.8 d.To determine whether reinforcement of PER2 or [Ca2+]i rhythms: e.g., no effect or loss of rhythmicity. On average, TTX decreased [Ca2+]i RI to 60% of the value before TTX, and refreshing medium resulted in recovery to 95% of the pre-TTX control value , but no significant difference was observed in either PER2 or [Ca2+]i RI  are cell autonomous and do not require a synchronized network  network , (3) do  network , (4) hav network , and (5) network .2+]i rhythms in independently oscillating dispersed SCN neurons, as shown here  are necessary for these processes. Our data suggest that [Ca2+]i oscillation works as a clock-controlled messenger to regulate circadian cellular functions rather than as a clock component to generate circadian rhythms.Many dispersed SCN neurons did not show [Caneration . However2+]i rhythms, but especially the latter, were greatly strengthened in SCN slice cultures relative to dispersed cultures, suggesting that the SCN neuronal network amplifies [Ca2+]i rhythms. Coherence between PER2 and [Ca2+]i rhythms was also improved in slice cultures, suggesting that control of PER2 over [Ca2+]i is stronger in slices than in dispersed cells. We cannot, however, rule out the possibility that improved rhythmicity in SCN slices was at least partly artifactual, owing to scattered light from neighboring cells smoothing the rhythm wave form and reducing noise. This concern could be addressed by future experiments restricting [Ca2+]i reporter expression to only a few cells per SCN slice, e.g., by gene gun  have been observed to generate stochastic PER2 oscillations with variable periods overlapping the circadian range (2+]i buffering by GCaMP3 (It has been reported that circadian rhythms of ileotides . In somean range . We did y GCaMP3 .2+ rhythms depend on Gq signaling (2+ stores (2+]i release in the mouse SCN and to be necessary for rat behavioral rhythm output (2+]i or PER2. Differences in experimental conditions, such as developmental stage and reporters, might have led to different [Ca2+]i responses, but, our report is consistent with previous work in finding that ryanodine does not affect PER2 rhythms (2+]i release from intracellular stores is completely unknown. SCN microarray studies reveal circadian oscillation of several interesting Ca2+ related genes (e.g., calreticulin and calumenin, which are Ca2+-binding proteins located in ER, and NCX1, which is a sodium/calcium exchanger expressed in plasma membrane and mitochondria; 2+ regulatory proteins.Studies in SCN slices have found that Caignaling  and ryan+ stores , but not+ stores . Ryanodim output . In this rhythms . Possibl rhythms , via cAM rhythms . However2+]i rhythms in dispersed SCN neurons, SCN network reinforcement of both PER2 and [Ca2+]i rhythms and modulation of the phase relationship between the two rhythms, and dependence of SCN [Ca2+]i rhythms on the transcriptional circadian clock but not on neuronal firing within individual cells. How the transcriptional circadian clock drives [Ca2+]i rhythms, and how SCN neuronal networks modulate [Ca2+]i rhythm phase relative to PER2, are intriguing questions for future study. Much work remains to be done to elucidate the complete mechanism of the SCN circadian clock, including cytosolic signaling pathways and intercellular networks.In summary, in this study, we demonstrate autonomy of circadian [Ca


We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C–7°C–30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states.Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca Indeed, in contrast to voltage-gated Ca2+ channels that are typical for excitable tissues, SOCE are widely spread in all eukaryotes from yeast to humans and, thereby, represent the primordial Ca2+ entry pathway [2+ sensor, and Orai1 as a functional membrane component of store-operated channels (SOC), was a key step in defining the mechanism of SOCE [2+ stores and within the temperature range characterizing transitions between torpid and active states.Alternatively, hibernators may evolutionary conserve another mechanism capable of increasing cytoplasmic [Ca pathway . Discove of SOCE ,37. Addi of SOCE –41. In p of SOCE –45. HoweSpermophilus undulatus, were approved by the Biological Safety and Ethics Committee (Institute of Cellular Biophysics) in accord with Directive 2010/63/EU of the European Parliament. Ground squirrels were live trapped during August—September in Yakutia, where this species is abandoned and unprotected, by trained personnel of the laboratory of Ecology and Ecosystem Resilience of North . Animals were shipped by air to Pushchino, the Moscow region, housed in a specially equipped vivarium and fed ad libitum. In October, prior to the beginning of endogenous hibernating cycle. the temperature in the vivarium was kept at ~4°C with no light to prepare animals for hibernation. This study implemented two experimental groups of active ground squirrels: 1) active winter animals  included animals taken between bouts of hibernation (interbout), during 12–24 h after spontaneous arousing; 2) summer group (June–July) included animals that were removed from the cold room in April and kept at room temperature. The averaged rectal temperature of animals in the both experimental groups was 36.5 ± 0.5°C. All surgery was performed under sodium pentobarbital anesthesia (50 mg/kg i.p.), and all efforts were made to minimize suffering.This study did not involve endangered or protected species and all animal procedures performed with male Sprague-Dawley rats  and ground squirrels, 2/5% CO2) Tyrode solution containing (in mM): NaCl, 135; KCl, 4; MgCl2, 1; CaCl2, 1.8; NaHCO3, 13.2; KH2PO4, 1.8; glucose, 11; (pH 7.4) as previously described [2) were mounted horizontally in a temperature controlled chamber (30°C), and stretched to a length at which tension of contraction is maximal. Isometric force of contraction was measured using a SI-H KG series force transducer (WPI Germany GmbH). Stimuli were applied using bipolar Ag-AgCl electrodes by square-wave pulses of 5 ms duration and amplitude set at 25% above the excitation threshold. Prior to each experiment, muscle preparations were stimulated at 0.3 Hz for 1 h until complete mechanical stabilization. All experiments were performed using a customized heating/cooling temperature controller engineered based on a Peltier thermocouple with feedback control to the targeted temperature.Isolations of right ventricle papillary muscles were performed from hearts of anesthetized animals. Measurements of the isometric force of contraction of rat and ground squirrel papillary muscles were performed in oxygenated  using a qPCRmix-HS SYBR+LowRox kit (Evrogen) with the forward (F) and reverse (R) primers indicated in T) was determined by the 7000 System SDS software . Transcript levels were quantified by the 2-ΔΔCT method taking into consideration the identified amplification efficiencies implementing REST 2005 software  specialized for these purposes [Total RNA was extracted from papillary muscles of rats and winter active (interbout) ground squirrels using ExtractRNA reagent . Treatment of RNA samples with DNAase was followed by chloroform extraction and subsequent precipitation in 96% ethanol. RNA pellets were washed three times with 70% ethanol and dissolved in RNase-free water. Synthesis of cDNA was carried out with the commercially available reverse transcription MMLV RT kit  using an oliogo(dT)purposes ,48.g for 5 minutes. Proteins were separated by denaturing 10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane . Primary rabbit polyclonal antibodies (Abcam) against Trpc3 (ab51560) and Trpc5 (ab63151), mouse polyclonal antibodies against Trpc6 (ab63038) and Trpc7 (ab93618) and goat primary antibody against actin  were diluted at 1:1000 and used to probe immunoreactive proteins. Counterstain was performed with horseradish peroxidase (HRP)-conjugated anti-rabbit , anti-mouse  or anti-goat  secondary antibodies, respectively. HRP signals were detected using 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate  and film-captured.Isolated papillary muscles from interbout and summer ground squirrels were lysed in hypotonic buffer containing NaCl 20 mM, Tris-HCl 20 mM (pH 7.4) and 1% Triton X-100, supplemented with proteinase inhibitors and centrifuged at 2,500∙2+ medium” containing (in mM): NaCl, 80; KCl, 10; KH2PO4, 1.2; MgSO4, 5; glucose, 20; taurine, 50; L-arginine, 1; HEPES, 10 (pH 7.2), as described previously [2+ medium supplemented with 200 μM CaCl2. Only rod-shaped cardiomyocytes with clear striations were used. Membrane currents in isolated cardiac myocytes were measured using the perforated mode of the whole-cell patch clamp technique, as described [2, 2; CaCl2, 1.8; TEA-Cl, 20; Glucose 1g/L; TRIS 10, pH 7.25 with HCl. In pipette and bath solutions cesium and TEA ions were used to diminish K+ currents. L-type Ca2+ currents were suppressed by 10 μM of nifedipine. Currents were measured using an Axopatch 200B amplifier . Cellular membrane resistance along with cell capacitance were defined online based on analysis of capacitive transient currents using the custom BioQuest software [Hearts were dissected from anesthetized animals, and retrogradely perfused with “low-Caeviously . Cardiomeviously  and storescribed . Membransoftware  and a L-Student’s t-test was used to compare continuous variables. P value of 0.05 was predetermined as determining statistically significant differences. All data are presented as mean ± standard error (S.E.). Results from RT-qPCR were presented using Whisker-Box plots, where the box area encompasses 50% of all observations, the dotted line represents the sample median and the whiskers shows 95% confidential intervals.2+ release from sarcoplasmic reticulum (SR), supported by SERCA, whereas at high stimulation frequencies the force of contraction is primarily controlled by Ca2+ influx via L-type Ca2+ channels [Initial contractions at 30°C in both cardiac papillary muscles (PM) from interbout ground squirrels and rats were designated as zero, baseline values, and isometric contraction forces were measured at reduced ambient temperatures in response to 0.1 Hz stimulus . During channels ,51–53. Wchannels ,49,51. T2+ levels, and normally should correlate well with contractile pause durations, during which SR Ca2+stores can be refilled by SERCA [2+ levels within SR, which, in turn, may be a consequence of relatively high spatial Na+ accumulation limiting removal of intracellular Ca2+ via reverse mode of NCX operation [ρ = 0.3, P = 0.68; ρ = 1.0, P = 0.017; ρ = 0.6, P = 0.35), and improved force of contraction at the stimulation frequencies below 0.3 Hz  of PM contractility can serve as a qualitative parameter of SR Caby SERCA ,54,55. Tperation ,50. Indew 0.3 Hz . Concominificant . These d3-fold increase in relative expression of Stim1, Trpc4, and Trpc5, as well as the 106-fold increase in Trpc7 mRNAs in ground squirrels compared to rat PM, indicating that SOCE may contribute to adaptive function of hibernating hearts. We did not find differences in the mRNA expression levels of all Trpc, Stim1 and Orai1 genes in the summer versus winter ground squirrel groups. However, while there was no a significant difference in the expression of Trpc5, Trpc6 and Trpc7 proteins between active winter and summer ground squirrels, Trpc3, which exhibited significantly lower protein levels in summer compared to interbout ground squirrels , a widely-used nonselective regulator of store-operated channels [2+ levels, in the presence of CPA, gradually eliminated the contractility in both rat and ground squirrel PM  inhibited whole-cell membrane current in ground squirrel cardiomyocytes , which is typical for non-selective SOCE measured in heterologous expression systems [2+ stores, indicating that the detected current could be induced by certain Trpc-Orai1-Stim1 channels capable of operating in store-independent mode [2+ entry to myocardial contractility is considered to be minimal [The impact of SOCE on Camyocytes . Subtrac systems ,60 or in systems ,61. Howeent mode ,40,62. C minimal ,51,53. Sr = -7.8 ± 4.2 mV, n = 3) similar to 2-APB-sensitive current in ground squirrels, yet with significantly reduced current density . In isolated PM from rats, Ca2+ entry provoked by 2-APB at concentrations exceeding 10 μM usually induced arrhythmia and sporadic contractile dysfunction, which was identified in all 5 performed experiments, whereas in PM from ground squirrels this agent reduced contractility without any proarrhythmic effects  through the plasma membrane [2+ sensor, Orai1, a functional membrane unit of SOC, and the canonical Trp isoforms are putative constituents of cardiac SOC [2+ stores [2+ homeostasis in cardyomyocytes has been associated with pathological cardiac remodeling [An established system that can be triggered by inhibition of SERCA due to depletion of SR Camembrane . Stim1, diac SOC ,36,38,45diac SOC . It has + stores . While mmodeling ,43. In fmodeling ,64–66. H6-fold increase in Trpc7 mRNA expression identified by qPCR in ground squirrels compared to rats, may not necessarily indicate its redundancy in the myocardium of hibernators, but rather may be due to the very low levels of these transcripts in rat tissue. Surprisingly, the expression levels of particular mRNA and Trpc proteins, except Trpc3 protein, were not different in hibernating (winter interbout) versus summer active ground squirrels, suggesting that the mobilization of SOC function may rely on signaling mechanisms. This suggestion is supported by our observation that PM of summer ground squirrels although can functionally withstand experimental hypothermia, lack characteristic potentiation of contractility at low temperatures as well as CPA-induced elevation of contractile function  that are highly selective to Ca2+ ions [ICRAC [ISOC) that are permissive to other cations [ISOC, since ICRAC would exhibit a reversal potential near the equilibrium potential for Ca2+, which under the experimental conditions used here was estimated within a range of +150 to +170 mV [2+ stores, such principle of their operation may not be applicable to all conditions in which Stim1 clustering results from the store depletion by inositol-1,4,5-trisphosphate-induced Ca2+ release or by inhibition of SERCA [2+ from intracellular stores that, under certain conditions, may reduce SR Ca2+ levels to the threshold of SOC activation. However, it is not evident that Ca2+ stores in ground squirrels PM are significantly depleted during excitation [2+ channels would prevent RyR-mediated Ca2+ release from SR. Thus, we are tempted to suggest that in ground squirrel cardiomyoctes, the overexpression of Stim1 may provoke creation of punctae [2+ entry in some cells [2+ store depletion [2+ entry via voltage-independent ion channels may elicit ectopic electrical activity leading to ventricular fibrillation and persistent or paroxysmal tachycardia [2+ controlling mechanism that is still in use by hibernators.Unfortunately, in the present study we cannot specify the exact composition of SOC in ground squirrel myocardium, which would require future extensive investigations implementing, for example, gene silencing techniques. However, in cardiomyocytes isolated from hearts of hibernating ground squirrels, testing using the whole-cell mode of the patch-clamp technique revealed the non-selective SOC activity without drug-induced depletion of intracellular Ca+ stores ,38,41. Aa2+ ions –37. In cs [ICRAC  and in c cations ,70,71. I +170 mV . Despiteof SERCA ,72. Cardcitation ,73, as s punctae . Our obsme cells  and prodme cells ,76. Indeepletion . This dahycardia ,76. Thushycardia ,43,63,642+ homeostasis and myocardial contractility in hibernating ground squirrels. Permissive for Ca2+ ions, SOC can serve as a scaffold for local signaling complexes that directly sense the proximal Ca2+ emerging from the channel [2+ in specific microdomains to reload particular SR compartments that are distinct from the greater SR involved in regulating contractility [+/Ca2+ permeability ratio (PCa/PNa) between 1 and 1/10 [+ load in cardiomyocytes. This mechanism can contribute to the spatial Na+ accumulation, which can be sufficient to drive Ca2+ entry via reverse mode of NCX [2+ [Since the physiological significance of SOCE remains elusive, especially in excitable cells, such as cardiomyocytes, we can only outline their possible contribution to Ca channel ,78,79. Iactility . On the and 1/10 ,81, at pe of NCX ,83, sugge of NCX ,46,84. F NCX [2+ , which iDespite the absence of a clear understanding of the proposed mechanisms for SOC-dependent control of contractile function in hibernating ground squirrel PM, direct testing of SOC inhibition during a cooling-reheating cycle revealed the clear contribution of 2-APB-sensitive components to hypothermic adaptation of cardiac contractility. We conclude that, in contrast to non-hibernating species, hibernating animals evolutionary retain the SOCE-dependent mechanism to secure adaptation of cardiac contractility during transitions in and out of hibernating states. Further investigation of this phenomenon may open a new pathway to comprehension of the protective mechanism governing cold tolerance in the myocardium of hibernating animals.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.


Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+]i, in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane, are unclear. We investigated the effects on diastolic [Ca2+]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo‐3 in voltage clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+]i. This increase of [Ca2+]i was larger when SR function was impaired either by making the ryanodine receptor leaky (with caffeine or ryanodine) or by decreasing sarco/endoplasmic reticulum Ca‐ATPase activity with thapsigargin. The increase of diastolic [Ca2+]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient, such that there was no change of time‐averaged [Ca2+]i. Time‐averaged [Ca2+]i was increased by β‐adrenergic stimulation with isoprenaline and increased in a saturating manner with increased stimulation frequency; average [Ca2+]i was a linear function of Ca entry per unit time. Diastolic and time‐averaged [Ca2+]i were decreased by decreasing the L‐type Ca current (with 50 μm cadmium chloride). We conclude that diastolic [Ca2+]i is controlled by the balance between Ca entry and efflux during systole. Furthermore, manoeuvres that decrease the amplitude of the Ca transient (without decreasing Ca influx) will therefore increase diastolic [Ca2+]i. This identifies a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+]i.The intracellular Ca concentration ([Ca 2+]i) must increase during systole to activate contraction and then fall, during diastole, to allow the myofilaments to relax and the heart to refill with blood.For the heart to function as a pump, intracellular calcium concentration ([Ca2+]i in rat ventricular myocytes.The present study investigates the control of diastolic [Ca2+]i is increased by manoeuvres that decrease sarcoplasmic reticulum function. This is accompanied by a decrease of systolic [Ca2+]i such that the time‐averaged [Ca2+]i remains constant.We show that diastolic [Ca2+]i is controlled by the balance between Ca2+ entry and Ca2+ efflux during systole.We report that diastolic [Ca2+]i.The results of the present study identify a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca Although the mechanisms that control the amplitude of the systolic rise of [Ca2+]i are well understood    Act, 1986 and Directive 2010/63/EU of the European Parliament. The experiments were approved by the University of Manchester Ethical Review Board. Male Wistar rats (weighing ∼200–250 g) were killed by stunning and cervical dislocation. Single ventricular myocytes were isolated by digestion with collagenase and protease as described previously (Eisner m) 135 NaCl, 11.1 glucose, 1 CaCl2, 10 Hepes, 1 MgCl2 and 4 KCL. Then, 4‐aminopyridine (5 mm) and BaCl2 (0.1 mm) were added to inhibit K+ currents and the solution was titrated to pH 7.4 using NaOH. Probenecid (2 mm) was added to reduce loss of indicator from the myocytes. Micropipettes (< 5 MΩ) were filled with a solution consisting of (in mm): 125 KCH3O3S, 12 KCl, 10 NaCl, 10 Hepes, 5 MgCl2 and 0.1 EGTA; titrated to pH 7.2 with KOH; and a final concentration of amphotericin B of 240 g ml−1. Cells were voltage clamped with the perforated patch clamp technique using the discontinuous switch clamp mode (frequency 1–2 kHz and gain 0.3–0.7 nA mV−1) of an Axoclamp 2A voltage clamp amplifier . Cells were voltage clamped and stimulated at a range of frequencies (0.2 to 3 Hz) with a 40 mV, 100 ms duration pulse from a holding potential of −40 mV. All experiments were performed at room temperature.Isolated myocytes were superfused with a solution (control) consisting of (in mm for 10 min) and allowed to de‐esterify before use. An aliquot was then placed in a superfusion chamber mounted on the stage of an inverted fluorescence microscope. To measure changes of [Ca2+]i, at the end of each experiment, the maximum fluorescence (Fmax) was measured by damaging the cell with the patch pipette. The dissociation constant of Fluo‐3 (Kd) was taken as 864 nm , R&D Systems  or Fisher Scientific . Caffeine was added as required. Ryanodine and thapsigargin were both stored as 1 mF test. P < 0.05 was considered statistically significant.Data are reported as the mean ± SEM where applicable for descriptive analysis. Statistical comparisons were made using two‐way ANOVA. Regression lines were compared with an A show the effect of periods of stimulation at 2 Hz. Under basal conditions, the diastolic level of [Ca2+]i during the 2 Hz stimulation was only slightly greater than that during rest. The subsequent addition of caffeine (1 mm) to increase RyR leak decreased the amplitude of the Ca transient and modestly elevated diastolic [Ca2+]i. The effects of β‐adrenergic stimulation with isoprenaline (ISO) (1 μm) were then investigated on these phenomena. In agreement with previous work, ISO increased the amplitude of the systolic Ca transient  show values normalized to those obtained at a slow stimulation rate (0.5 Hz) in the absence of both caffeine and ISO. It is clear that the effects of ISO on both systolic and diastolic [Ca2+]i are greatly affected by the presence of caffeine. For example, in the absence of caffeine, ISO greatly increases the amplitude of the Ca transient , whereas there is no effect on diastolic [Ca2+]i . By contrast, in the presence of caffeine, ISO had a much smaller effect on the amplitude of the Ca transient  but markedly increased diastolic [Ca2+]i .The first series of experiments examined the effect of increased RyR leak on the response to an increased stimulation rate. The grey traces in Fig. caffeine  mm to incaffeine  mm to incaffeine  mm to in2+]i at the same time as decreasing the systolic rise of [Ca2+]i. Accordingly, we next investigated the effect of caffeine on time‐averaged [Ca2+]i as shown by the blue trace in Fig. A. It is clear that average [Ca2+]i is: (i) increased by stimulation; (ii) increased by ISO; and (iii) unaffected by caffeine in both control and ISO. These observations are confirmed by the mean data of Fig. D, which show that the average [Ca2+]i does not significantly change upon addition of caffeine . A similar finding is seen in the presence of ISO . Finally, these data also show that ISO increases average [Ca2+]i in both the presence and absence of caffeine (P < 0.001).The above data show that caffeine increases diastolic [Ca2+]i were more prominent in the presence of ISO, all subsequent experiments were performed in the presence of ISO. The experiment shown in Fig. A shows the data obtained in the presence and absence of caffeine. As the frequency of stimulation increased, the Ca transient amplitude decreased slightly and diastolic [Ca2+]i increased. The decrease in Ca transient amplitude was accompanied by (and is presumably at least in part caused by) a decrease in the amplitude of the L‐type Ca current  . Average [Ca2+]i did, however, increase in a saturating manner with an increasing frequency of stimulation. Figure B sheds light on this saturation of average Ca. The Ca influx via the L‐type Ca current on each pulse decreased with an increasing stimulation rate  as a result of increasing inactivation  was a saturating function of frequency and therefore paralleled the frequency dependence of average [Ca2+]i. Data obtained in the presence of caffeine  were identical to those in its absence . The correlation between Ca entry per unit time and average [Ca2+]i is emphasized by Fig. D, which shows a clear linear relationship between these two parameters that is not statistically different  in the absence and presence of caffeine.The above observations of the frequency dependence of average Ca+i are emA, the application of thapsigargin decreased systolic [Ca2+]i and increased diastolic [Ca2+]i during stimulation at 0.5 Hz. On increasing the stimulation rate to 2 Hz, there was a more marked increase of diastolic and decrease of systolic [Ca2+]i. When stimulation was stopped, [Ca2+]i declined to a level similar to the original diastolic one. The mean data of Fig. B show the effects of thapsigargin at 0.5 and 2 Hz. . Thapsigargin decreased the amplitude and increased diastolic [Ca2+]i at the same time as having no effect on average [Ca2+]i.The purpose of the experiments shown in Figs A, the application of ryanodine decreased systolic [Ca2+]i and increased diastolic [Ca2+]i. The dark line shows that average [Ca2+]i remained constant during this period. When stimulation was stopped, [Ca2+]i declined to a level similar to the original diastolic one. Subsequent stimulation at 2 Hz increased average [Ca2+]i. Ryanodine was reapplied to increase its effect. When stimulation was recommenced at 0.5 Hz, the Ca transient was considerably smaller than that observed previously at this frequency. The average [Ca2+]i was, however, almost identical. Increasing the stimulation rate to 2 Hz resulted in a smaller Ca transient than that seen at 2 Hz before. Once again, the average Ca was unaffected. These data were interrupted by exposures to caffeine plus 2,3‐butanedione monoxime to release Ca from the SR and thereby obtain an estimate of the fall of SR Ca content. The mean data of Fig. B confirm that ryanodine decreases the amplitude of the Ca transient  and increases diastolic [Ca2+]i , whereas there is no effect on average [Ca2+]i .In the experiment shown in Fig. 2+]i depends on a balance between Ca influx and efflux. If this is the case, diastolic [Ca2+]i would be expected to be decreased by reducing Ca influx. In the experiment shown in Fig. A, caffeine had been added when stimulating at either 0.5 or 3 Hz. In agreement with the results reported above, the rise of diastolic [Ca2+]i was greater at the higher frequency. Addition of cadmium chloride (50 μm) to decrease the L‐type Ca current was observed to decrease diastolic [Ca2+]i at both stimulation rates . On average, cadmium decreased the L‐type Ca current to 33 ± 5% (n = 5 cells). Figure C shows the mean data obtained from five cells (at 0.5 Hz). One‐way ANOVA showed that cadmium decreased average and diastolic [Ca2+]i, as well as the amplitude of the Ca transient . Specifically, cadmium decreased the average [Ca2+]i to 69.6 ± 1.7%.The above experiments suggest that the level of diastolic [Ca2+]i in rat ventricular myocytes. In agreement with previous work, we find that increasing stimulation frequency increases diastolic [Ca2+]i  this is accompanied by a decrease of systolic [Ca2+]i, such that the time‐averaged [Ca2+]i remains constant; (iii) time‐averaged [Ca2+]i is increased by β‐adrenergic stimulation and is a saturating function of frequency reflecting a frequency‐dependent decrease of Ca influx per beat; and (iv) diastolic [Ca2+]i is regulated by the difference between the Ca influx and the systolic efflux on each beat.The present study investigates the control of diastolic [Ca2+]i . This may be because inhibition of SERCA greatly slows the decay of the Ca transient. At low rates, the increased duration of the Ca transient compensates for the decreased amplitude in maintaining efflux such that diastolic [Ca2+]i does not increase. This is impossible at high rates because the decay of the transient is interrupted by the next stimulus.Previous work has shown that ryanodine and thapsigargin increase diastolic [Ca+]i Figs , 2 and 4gin Fig. B. This 2+]i such that average [Ca2+]i is maintained constant when SR function is altered? We suggest that the answer resides in the mechanisms that maintain Ca flux balance. In the steady‐state, during each cycle of stimulation, the influx mediated by the Ca current must be precisely equal to the Ca efflux, largely via sodium‐calcium exchange (NCX) . It is also possible that an increase of Ca leak from the SR, possibly via a Ca/calmodulin‐dependent kinase II (CaMKII) mechanism  increased Ca influx per unit time ; (ii) decreased systolic efflux; and (iii) decreased diastolic time for efflux to occur in. Ca flux balance can therefore only be established with an increase of diastolic [Ca2+]i. If SR function is depressed, increased frequency produces a larger decrease of systolic [Ca2+]i and therefore a larger rise of diastolic [Ca2+]i would be expected. In agreement with these predictions, we find that increasing frequency increases diastolic [Ca2+]i and this increase is potentiated by increasing SR leak or decreasing SERCA activity. Average [Ca2+]i is a saturating function of frequency  presumably because the Ca entry per unit time also saturates with frequency as a result of increased inactivation of the L‐type Ca current , . It should be noted that, if the L‐type Ca current did not decrease at higher frequencies, the rise of diastolic [Ca2+]i would be even greater.An increase of stimulation frequency will increase Ca influx per unit time, thereby requiring an increase of average Ca+i to maiD. It is clear that, even with zero influx through the L‐type Ca current, [Ca2+]i has a finite value. This has been accounted for by a background Ca entry  may have on NCX activity. [Na+]i will be increased by an increase of stimulation rate and as a consequence of the increased NCX activity in response to the Ca loading produced by β‐adrenergic stimulation. It will be decreased as a consequence of phosphorylation of phospholemman and stimulation of the sodium pump  a component activated by the diastolic level of [Ca2+]i and (ii) an additional component that occurs during the systolic Ca transient .